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系統識別號 U0026-0812200910425420
論文名稱(中文) 探討MSP/RON路徑在膀胱癌細胞癌化過程的影響
論文名稱(英文) The involvement of macrophage stimulating protein (MSP)/RON pathway in the progression of human bladder cancer
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 91
學期 2
出版年 92
研究生(中文) 林彥儒
學號 s4690401
學位類別 碩士
語文別 中文
口試日期 2003-07-23
論文頁數 88頁
口試委員 指導教授-劉校生
指導教授-周楠華
口試委員-陳鴻震
口試委員-賴明德
關鍵字(中) 膀胱癌
關鍵字(英) MSP
c-met
RTK
RON
學科別分類
中文摘要 RON基因所控管的蛋白質產物,是具有酪胺酸酵素活性的生長因子接受體,被歸類為一種原致癌基因(proto-oncogene)。其配合基 (ligand)為巨噬細胞刺激蛋白質(macrophage stimulating protein, MSP)。MSP/RON之間的交互作用會影響細胞的各種生物現象,包括細胞分化、細胞移動、型態改變以及細胞增生。這些作用在癌症的癌化過程中,可以扮演一種促進癌化的角色。本研究主要是想了解MSP/RON相關之傳導路徑,以及在膀胱癌的癌化過程中所扮演角色。從臨床膀胱癌檢體的統計分析,發現在侵犯性膀胱癌檢體以及上泌尿道移行上皮癌的檢體中,RON之表現和膀胱癌的惡化因子有顯著的相關性,另外有2.8%的檢體顯示出RON蛋白異常分佈在細胞質中的現象。顯見RON對於膀胱癌的發生,的確扮演重要的角色,很值得進一步的釐清。
本研究使用十株不同細胞株,其中有一株正常的膀胱上皮細胞株,以及九株不同癌化等級的膀胱癌細胞株。比較RON蛋白表現及mRNA表現的分析,我們發現RON被表現在許多的腫瘤細胞中(包括RT4, TSGH8301, TCCSUP, UB37),其中以UB37的表現量最高。另外在UB40及UB47則有分子量異常的RON蛋白質表現。其中TSGH8301在MSP刺激後RON會產生大量磷酸化的現象。而UB37細胞似乎能透過自體分泌(autocrine)MSP和RON產生交互作用。除此之外,本研究也在八個臨床膀胱癌病患的尿液檢體中以及UB37的細胞培養液中發現MSP蛋白的存在,這樣的結果顯示在膀胱癌癌化過程中RON和MSP之間的關係可能是以自體分泌或是旁分泌(paracrine)的方式存在。為了進一步了解RON活化之後,所影響的生物現象。我們構築一個可受四環素調控表現的RON質體且能夠成功的在UB09細胞株中表現。結果發現RON在大量表現的情況下,的確會使細胞的生長速率、移動速率及抗細胞凋亡(anti-apoptosis)能力增強。另外以PI3K的抑制藥物wortamanin抑制PI3K活性結果發現RON所造成的細胞凋亡抑制的現象消失了。之後以報導基因(reporter gene) 研究RON可能的下游傳導路徑,結果發現當RON活化時,可能會透過PI3K-Akt活化NFkB,是否是這樣的路徑造成上述生物現象的改變,仍然須要進一步的釐清。
針對UB47細胞株,透過cDNA序列分析,在transmembrane domain前近N端處,發現有147bp核酸被刪除的現象,證實UB47所表現的RON確實為一種突變型的蛋白,另外我們也發現這個突變型的蛋白會異常的累積在細胞質中且不需MSP刺激即會產生大量磷酸化的現象。我們也利用SSCP方式分析RON的kinase domain,結果我們並沒有發現點突變的情形。我們在細胞株上所發現的現象可以解釋臨床上我們所看到的異常的RON蛋白累積的現象。另外我們也意外的發現RON蛋白的入核現象,且似乎不受MSP刺激的控制。這樣的結果或許意謂著RON入核之後也可能扮演著特殊的角色,不過這個假說尚須更深入的研究才能證實。
總結,在本研究中我們證實了RON和膀胱癌臨床發生的相關性。MSP/RON路徑在膀胱癌中可能會透過自體分泌及旁分泌的機制來進行作用。此外RON基因表現時的確會加強癌細胞的各種生物現象。而我們也發現PI3K-Akt路徑的活化和膀胱癌細胞抗凋亡的能力有關,且當其活化之後NFkB也跟著被活化,是否這樣的路徑調控了RON 所影響的生物現象則必須進一步的釐清。除此之外突變型的RON蛋白及RON入核的現象也是首次在膀胱癌中被探討。透過了解膀胱癌中MSP/RON機制,期望能夠對未來癌症的治療上有所助益。
英文摘要 RON is a transmembrane receptor tyrosine kinase encoded by RON proto-oncogene. The specific ligand of RON is macrophage-stimulating protein (MSP). The interaction of MSP and RON mediates a number of biological properties, including cell morphology, motility and proliferation. The major goal of this study is to reveal the possible MSP/RON signaling pathways in the progression of bladder carcinogenesis. By using immunostaing, we initially analyzed the correlation between RON expression and biological indicators of human bladder cancer. We found a 42.9 % and 33% expression rates for invasive bladder cancer and upper urinary tract transitional cell carcinoma, respectively. Expression of RON showed a significant association with non-papillary growth and multiple tumors of bladder cancer. In addtion, aberrant localization of RON protein in the cytosol of cancer cells was detected in about 3% of both bladder and upper tract cancer samples. Therefore, multifunctional mechanisms for RON activation seem to be involved in the progression of bladder cancer.
A total of nine bladder cancer and one immortalized uroepithelial cell line were employed in this study. Expression of wild-typed RON protein in many cancer cell lines (such as RT4, TSGH8301, TCCSUP, and UB37) were detected; while aberrant pattern of RON protein was observed in UB40 and UB47 cell lines. TSGH8301 cells showed a MSP-dependent phosphorylation of RON; but UB37 cells exhibited an autocrine MSP/RON interaction. Besides, MSP protein could be detected in the urine of bladder cancer patients (n = 8), as well as in the supernatant of UB37 cells. Taken together, both autocrine and paracrine of MSP/RON signaling appears to exist in human bladder cancer. As for biological effects of RON activation, an inducible construct of full-length RON cDNA was successfully expressed in UB09 cells. Over- expression of RON was proven to enhance the cell growth, migration, and anti-apoptosis. By using PI3K specific inhibitor (wortmannin), we confirmed that PI3K-Akt pathway involves in the RON-related anti-apoptosis. The transient reporter gene assay also confirmed the activation of NFkB by PI3K-Akt pathway.
UB47 cells were proven to have a 147bp deletion at the extracelluar domain near transmembrane region as described in the gastric cancer. The aberrant RON protein in UB47 cells accumulated in the cytoplasm, and exhibited a MSP-independent phosphorylation. But, there was no evidence of mutation in the kinase domain screened by SSCP. The results essentially support the clinical relevance of aberrant RON expression in human bladder cancer. Interestingly, we also observed a MSP-independent nuclear localization of RON protein, but the underlying mechanism and the clinical significance remain to be clarified.
Taken together, we support the clinical importance of RON in the progression of bladder carcinogenesis and the effect of MSP/RON may act through autocrine or paracrine pathway. Moreover, mutant RON is first discovered in the bladder cancer and both wild type and mutant RON could translocate into nucleus which has nerver been discussed before. We also confirmed that PI3K-Akt pathway and NFκB activation involve in the RON-related biological response, but the underlying mechanism and the clinical significance remain to be clarified. The understandings may provide a molecular basis in developing gene-targeting therapy for human bladder cancer.
論文目次 中文摘要 ..............................................................1
英文摘要 ..............................................................3
致謝 ..............................................................5
目錄 ..............................................................6
圖表目錄 ..............................................................8
縮寫及符號.............................................................10
一、緒論
I. 膀胱癌致病機轉....................................................12
II. 酪胺酸磷酸激酶14
III. MSP/RON 對於膀胱癌細胞的影響....................................15
IV. RNAi的原理及應用.................................................17
二、材料和方法
I. 菌種與細胞株......................................................19
II. 細菌培養及細胞培養...............................................19
III. 質體及其製備....................................................21
IV. 膀胱癌細胞株之RNA製備............................................23
V. 反轉錄聚合酵素連鎖反應............................................24
VI. 蛋白質電泳及西方墨...............................................27
VII. 免疫沈澱法......................................................31
VIII. RON下游報導基因分析............................................32
IX. Acridine Orange染色..............................................33
X. Time lapse recording..............................................34
XI. Subcellular Fraction of proteins extraction......................35
XII. 免疫螢光染色36
XIII. 電穿孔(electroporation)........................................37
XIV. 單股多型性聚合酶連鎖反應(SSCP)..................................38
三、結果
I. 探討RON在膀胱癌中所扮演的角色.....................................40
1. RON表現和膀胱癌發生之關係.......................................40
2. 膀胱癌細胞株RON蛋白質表現.......................................40
3. 巨噬細胞刺激蛋白質(macrophage stimulating protein, MSP)
和RON之間的交互關係..............................................41
a. 自體分泌(autocrine)及旁分泌(paracrine)........................41
b. 不同程度的RON表現特性經MSP刺激後的磷酸化影響41
II. 建立可誘導RON表現之膀胱癌細胞短暫分析模式42
III. RON基因表現後對細胞生物現象之影響分析...........................43
1. RON基因過量表現會增加細胞生長速率...............................43
2. RON過量表現使膀胱癌細胞產生抗細胞淍亡的能力.....................43
3. RON過量表現造成細胞移動特性增加.................................43
4. RON透過PI3K-Akt活化NFκB.........................................44
IV. 突變型RON蛋白功能分析及RON入核現象之探討.........................45
1. 異常分子量之RON及transmembrane domain的基因刪除..................45
2. 突變型RON在細胞質中累積的現象及RON入核特性之分析.................45
V. RON RNA interference..............................................46
四、討論...............................................................48
五、參考文獻...........................................................54
六、圖表...............................................................63
自述...................................................................88
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系統識別號 U0026-0812200911431876
論文名稱(中文) 可攜帶型姿勢監測器的研發
論文名稱(英文) Development of a Portable Posture Monitoring Device
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 93
學期 2
出版年 94
研究生(中文) 黃暐勳
學號 P8692111
學位類別 碩士
語文別 中文
口試日期 2005-07-14
論文頁數 56頁
口試委員 口試委員-蔡明世
召集委員-陳若佟
口試委員-張冠諒
指導教授-鍾高基
口試委員-陳守義
指導教授-張志涵
關鍵字(中) 姿勢不良
脊椎曲線
姿勢矯正
MSP430F169微控制器
聲音回饋
彎曲感測器
關鍵字(英) audio-feedback
flex sensor
abnormal posture
spinal curve
posture correct
MSP430F169 microcontroller
學科別分類
中文摘要   姿勢為身體各肢段在空間中相對位置。臨床上,凡是會增加關節承受應力的姿勢統稱為不良姿勢;而正確的姿勢能降低關節所承受應力。本研究針對常見之頸、背不良姿勢進行探討。當脊椎長時間維持在不正常的曲線下,易引發頸、背骨骼肌肉系統問題,現代人往往因工作或娛樂上的需求,必須長時間維持在固定的姿勢如久坐、久站,很少有機會改變姿勢讓肌肉骨骼重新舒展,且部份正常人亦經常習慣性不自覺的處於不良姿勢下。本研究目的為發展一可攜帶式的姿勢監測器,以聲音回饋訊號幫助習慣性姿勢不良使用者主動地維持在適當的姿勢。

  本研究發展之姿勢監測器包括軟式Flex sensor及自行製作的Data logger。Data logger配合Flex sensor監測使用者姿勢變化時,所造成的脊椎曲線變化,當使用者姿勢不良,超過設定的臨界值時,發出警告音提醒使用者調整其姿勢,進而達到姿勢監測、訓練目的。Data logger採用低功耗元件,包括MSP430F169微控制器、運算放大器、升壓型電源供應、RS-232通訊介面IC、蜂鳴器,已初步符合一般可攜帶式裝置標準,系統並可記錄使用者使用期間的姿勢變化。目前可攜帶型姿勢監測器,能提供有姿勢不良習慣之正常人長時間使用,以偵測並提醒改善其不良姿勢習慣。現階段姿勢監測器搭配Flex sensor能偵測、比較使用者每次貼附使用時,由sensor反映出之不同電壓值,以代表使用者於不同姿勢下形成之脊椎曲線,而非絕對的脊椎曲線角度值,因此需讓使用者每次開始使用前,自行提供設定一欲維持之姿勢給姿勢監測器,做為參考比較標準。若未來搭配能提供絕對脊椎曲線角度值且同時保有Flex sensor軟式高彈性塑膠薄片材質且低成本特性之感測器,則本系統可提供之臨床應用將更廣泛。
英文摘要  The posture is defined as the relative orientations among body segments. Clinically, a good posture could decrease the joint stress, while a faulty posture would increase the joint stress. The continual abnormal spinal curves induced by prolonged faulty neck and back postures might cause the postural pain syndrome, postural dysfunction and even musculoskeletal system disorders. Moreover a number of healthy person maintain his/her own body in abnormal posture habitually and unconsciously. The aim of this study was to develop a portable posture monitoring device by applying an audio-feedback signal to promote the active correction of the abnormal posture and to prevent continue deteriorated.

 The device includes a self-developed microcontroller based data logger powered by batteries and using the flexible sensors (FLX-01) on the spine to monitor the user posture during daily activities. This system used a pre-set angle threshold, provided by the subject under an appropriate posture, to remind the subject adjusting to a suitable posture by audio warning once the threshold is violated. The data logger adopts low power components, including MSP430F169 microcontroller, operation-amplifier IC, step-up power supply module, RS-232 interface IC and buzzer. The power consumption and size of the data logger can fit in the portable instrument specification. The posture information could be stored in the nonvolatile memory and linked with a recorded time stamp. Presently, the developed system can detect the subject’s postural change and remind the subject adjusting to a suitable posture by audio warning. This system could be used to progressively correct abnormal postural for subject with habit faulty posture. The use of flex sensor reflects the value of voltage change related to the relative differences of spinal curve between various postures not the quantitative absolute spinal angle. In order to overcome this limitation, in this study, subject is required to provide an appropriate posture as a reference prior to each usage. In the future, a senor could measure the real angle of spinal cure, along with the flexible, thin and low cost features of the sensor used in this study, could provide more useful information for the clinical applications.
論文目次 中文摘要 I
Abstract II
誌謝 IV
目錄 V
圖目錄 VIII
表目錄 XI


第一章 緒論 1
1.1 研究背景 2
1.1.1 理想的姿勢 2
1.1.2 脊椎之功能性解剖 3
1.1.3 常見的不良姿勢外觀及易引發之問題 6
1.1.4 目前臨床上的解決方法 9
1.2 文獻回顧 9
1.3 研究目的及特定目標 13
1.3.1 研究目的 13
1.3.2 特定目標 13

第二章 材料與方法 14
2.1 Sensor特性 15
2.1.1 Flex sensor特性測試實驗(一) 16
2.1.1 Flex sensor特性測試實驗(二) 17
2.2 不良姿勢判定標準 19
2.3 Data logger硬體架構 21
2.3.1 MSP430F169微控制器 25
2.3.2 電源供應部份 29
2.3.3 類比姿勢訊號處理 32
2.3.4 姿勢資料儲存 34
2.3.5 Real-time clock 36
2.3.6 PC端通訊介面 36
2.3.7 LCD顯示電路 36
2.4 微處理器韌體程式 37

第三章 結果與討論 40
3.1 姿勢監測器實際人體偵測驗證 41
3.2 Data logger電量消耗測試 45
3.3 討論 47

第四章 結論與未來展望 49
4.1 結論 49
4.2 未來展望 49

參考文獻 51
附錄 54
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[14] Wong M.S., Mak A.F., “Effectiveness and biomechanics of spinal orthoses in the treatment AIS” Prosthet Orthot Int. 24:148-162, 2000

[15] Szeto G.P., Straker L., Raine S., “A field comparison of neck and shoulder postures in symptomatic and asymptomatic office workers” Appl Ergon. 33(1):75-84, 2002

[16] AIC1638 1-Cell, 3-Pin, Step-Up DC/DC Converter Data sheet, Analog Integrations Corporation

[17] MSP430F169 Mixed Signal Microcontroller Data sheet
(Rev. D) SLAS368D Texas Instruments, 2005

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Texas Instruments ,2005

[19] 韓毅雄, “骨骼肌肉系統之生物力學” 華杏出版股份有限公司,219-243, 1983

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------------------------------------------------------------------------ 第 3 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200913373095
論文名稱(中文) 小型飛機數位航電平臺
論文名稱(英文) Small Aircraft Digital Avionics Platform
校院名稱 成功大學
系所名稱(中) 航空太空工程學系碩博士班
系所名稱(英) Department of Aeronautics & Astronautics
學年度 95
學期 1
出版年 96
研究生(中文) 李璟輝
學號 P4693141
學位類別 碩士
語文別 英文
口試日期 2007-01-21
論文頁數 70頁
口試委員 口試委員-詹劭勳
指導教授-林清一
口試委員-楊介仙
關鍵字(中) 自主性
航電平臺
關鍵字(英) Accelerometer
Gyro
MSP430
INS
GPS
學科別分類
中文摘要 於GPS 與INS 的信號整合中,GPS 提供系統絕對位置與角度資訊,其長期穩定性佳,但容易受地形因素與天候影響,可能使GPS 功能降低。INS 之自主性(Autonomous)較高且短期穩定性佳,但是本身的定位誤差會隨著時間累積。GPS 與INS 的整合,可以彌補兩者的缺點,成為一個更佳的導航系統。相較於一般用於單晶片上之儲存裝置,MMC 記憶卡具有較大儲存空間,可記錄較大的數據資料量,配已適當的運算軟體,可以做為後續應用的基礎。本文利用微處理器設計一個獨立的航電平台,將GPS、INS 與資料儲存整合為一體,可以用在小型飛機或無人操作的飛行載具上。本文從系統的設計、軟硬體制作、以及實際的測試,完成一個小型的航電平台。
英文摘要 In the integration of GPS and INS, the GPS provides a good positioning and heading for good long period reliability, but it might be affected by the environment and whether to degrade its function capability. The INS is an autonomous system of good short period stability, but possesses significant time drift. The integration of GPS and INS compensates both defects and becomes a good navigation system. Comparing to many storage devices in microcontrollers, the memory size of Multimedia Card is larger than others that can be used to store long period data recording. By appropriate algorithm, the data in the memory card can be reconstructed for further analysis. This thesis adopts microprocessors to construct an independent avionic platform using GPS and INS integration for small aircrafts or unmanned aerial vehicles. System design, implement and test are completely demonstrated on an avionics platform.
論文目次 ABSTRACT......................................... I
摘要..............................................II
誌謝............................................. III
CONTENTS......................................... IV
LIST OF ILLUSTRATIONS ........................... VI
LIST OF TABLES ...................................VIII
LIST OF ACRONYMS................................. IX
CHAPTER I INTRODUCTION ...........................1
1-1 Background and Motivation ....................1
1-2 Benefit of Integrating INS and GPS [2] .......3
1-3 Thesis Outline ...............................4
CHAPTER II INTRODUCTIONS OF INS AND GPS ..........6
2-1 Designs of Inertial Navigation System.........6
2-1-1 Gimbaled Systems............................7
2-1-2 Strapdown Systems ..........................8
2-2 Principle of Inertial Sensor .................9
2-2-1 Principle of Accelerometers ................9
2-2-2 Principle of Gyroscopes ....................11
2-3 Introduction of Global Positioning System.....13
2-3-1 Overview of GPS ............................13
2-3-2 GPS data....................................15
2-4 GPS-INS integration...........................16
CHAPTER III SYSTEM ARCHITECTURE ..................19
3-1 Introduction of the Platform..................20
3-2 Introduction of MSP430 .......................21
3-3 Inertial Measurement Sensor ..................24
3-3-1 Gyroscope ADXRS300 .........................25
3-3-2 Accelerometer ADXL311 ......................27
3-4 GPS Receiver..................................28
3-5 Multimedia Card...............................29
3-6 Introduction of File Allocation Table (FAT16) 30
3-6-1 Overview of a FAT16 File System ............31
3-6-2 Structure of a FAT16 File System............32
CHAPTER IV EXPERIMENTS AND VERIFICATIONS..........36
4-1 Testing Strategy .............................37
4-2 Unit Test ....................................38
4-2-1 ADC Output Test ............................38
4-2-2 Gyroscope and Accelerometer ................40
4-2-3 GPS Receiver Test ..........................42
4-3 Interface Communication ......................45
4-3-1 MSP430F149 and GPS test ....................45
4-3-2 MSP430 and MMC test ........................47
4-4 System Integration Test ......................49
4-5 Algorithm and data mapped.....................54
CHAPTER V DISSCUTIONS ............................61
CHAPTER VI CONCLUSIONS ...........................66
REFERENCES........................................67
VITA..............................................70
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[2]. A. Soloview, Investigation into performance enhancement of integrated Global Positioning Inertial Navigation Systems by frequency domain Implementation of inertial computational procedures, College of Engineering and Technology, Ohio University, 1998. Ph.D. dissertation.
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系統識別號 U0026-0812200914015647
論文名稱(中文) 應用於醫療保健器材之無線生理安全監控系統之設計與研製
論文名稱(英文) The Development of Wireless Physiological Monitoring Security System applied to Medical and Health Care Devices
校院名稱 成功大學
系所名稱(中) 電機工程學系碩博士班
系所名稱(英) Department of Electrical Engineering
學年度 95
學期 2
出版年 96
研究生(中文) 陳永鑫
學號 n2694198
學位類別 碩士
語文別 中文
口試日期 2007-07-26
論文頁數 50頁
口試委員 指導教授-戴政祺
口試委員-毛齊武
口試委員-林志隆
口試委員-陳天送
關鍵字(中) MSP430
醫療保健器材
無線傳輸
生理安全監控
關鍵字(英) physiological monitoring
Medical and health care devices
wireless
MPS430
學科別分類
中文摘要 本論文之主要目的為設計與研發一套以MSP430省電型微處理器為核心,結合人體體溫、血氧飽和度、心跳率等生理訊號量測,以智慧型醫療保健器材安全系統的概念,發展一套「應用於醫療保健器材之無線生理安全監控系統」,以無線方式,隨時將人體生理訊號傳送至醫療保健器材安全監控系統,達到安全、良好之健康管理。本系統之主要功能與特色包括:(1)以MSP430省電型微處理器為發展核心;(2)反射式血氧飽和度量測功能;(3)數位式體溫量測功能;(4)心跳次數量測功能;(5)生理訊號整合至無線傳輸模組之功能;(6)接收端安全控制警示系統…等。本研究已經達到初步的體溫、心跳、與血氧量測,並可無線傳輸信號。未來將可供醫療保健器材廠商提升產品功能,並進一步提升國內醫療保健器材水準。
英文摘要 In this research we develop a wireless physiological monitoring security system for medical and health care applications. This system is based on the ultra-low power consumption microcontroller MSP430TM from Texas Instruments. The developed system can be used to measure physiological signals, such as temperature of human body, oxygen saturation, heart rate, etc. The features of the system include: (1)Adopting MSP430 low power microcontroller as the core of the system; (2)Reflective-type oxygen saturation measurement; (3)Digital body temperature measurement; (4)Heart rate measurement; (5)Integration of physiological monitoring system and wireless transmission module; (6) Alarm and control at receiving end. This research has already completed preliminary body temperature, heart rate and oxygen saturation measurements as well as data transmission by wireless. The achievements in this research are useful for the developer of medical and health care devices and can be used to enhance the functions of said equipments.
論文目次 中文摘要 I
英文摘要 II
目 錄 V
表 目 錄 VII
圖 目 錄 VIII
第 一 章 緒論 1
1-1 研究動機與目的 1
1­2 文獻回顧 1
1-3 本文架構 6
第 二 章 生理訊號量測 7
2-1 人體體溫量測 7
2-2 TMP75數位IC式溫度感測器 10
2-3 血氧飽和度量測 11
2-4 心跳率 13
第 三 章 系統架構與設計 14
3-1 省電型微處理器 MSP430FG439 16
3-2 無線傳送/接收模組 18
3-3 編解碼IC 20
3-4 發射端模組電路設計 20
3-4-1 TMP75溫度感測 23
3-4-2 血氧飽和度與心跳率感測 25
3-5 接收端模組 29
第 四 章 實驗結果與討論 32
4-1 TMP75省電量測設計量測 32
4-2 體溫量測記錄 34
4-3 無線傳送/接收模組電路測試 37
4-4 接收模組電路測試 40
第 五 章 結論與未來展望 44
5-1 結論 44
5-2 未來展望 44
參考文獻 46
自 述 50
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系統識別號 U0026-0812200915081666
論文名稱(中文) 探討物理性刺激對於骨質疏鬆症之影響
論文名稱(英文) Investigate the Effect of Physical Stimulation on Osteoporosis Using Animal Models
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 張昱婷
學號 p8696403
學位類別 碩士
語文別 中文
口試日期 2009-05-13
論文頁數 114頁
口試委員 指導教授-鍾高基
口試委員-王國照
口試委員-蔡明世
口試委員-陳若佟
口試委員-邱毓賢
關鍵字(中) 單一脈衝式電磁場
連續脈衝式電磁場
全身性振動
骨質疏鬆
關鍵字(英) HMSP-EMF
PEMF
Osteoporosis
Whole body vibration
學科別分類
中文摘要 隨著時代與科技的進步,國民的生活習慣日趨改變,導致骨質疏鬆症罹患率升高。全球超過50歲以上的女性罹患骨質疏鬆症的機率為1/3,男性為1/5。在台灣,超過60歲的女性罹患骨質疏鬆症的比例超過16% [1]。治療骨質疏鬆症的方法多為服用鈣片、賀爾蒙或藥物,但藥物易產生副作用或是價格昂貴。因此發展使用外在的物理性刺激治療骨質疏鬆症將成為趨勢。本研究目的為探討物理性刺激對於骨質疏鬆的效應。本實驗之特定目標:1) 應用骨質疏鬆SD rats模型,探討振動刺激對於骨質的影響;2) 應用骨質疏鬆BALB/C mice模型,探討兩種不同磁刺激對於骨質的影響。

實驗主要分為振動刺激與磁刺激兩部份:Phase A - 32隻八週大的SD rats,隨機分為四組:control組有8隻健康之大鼠;normal + WBV組8隻健康大鼠使用WBV刺激;osteoporosis組有8隻骨質疏鬆症大鼠及osteoporosis + WBV組使用WBV刺激8隻骨質疏鬆症大鼠。應用0.26 ~ 0.73G之全身性振動儀於骨質疏鬆SD rats模型,實驗進行時間為4星期,將大鼠犧牲取出脛骨,使用微電腦斷層攝影掃描脛骨頭,並計算骨密度、骨髓腔內骨頭體積百分比、骨小樑厚度、骨小樑間距、骨小樑數量等五種骨型態學參數,使用one-way ANOVA進行四組分析比較,選取α=0.05。Phase B - 37隻八週大的BALB/C mice,隨機分為六組:Control組有6隻健康小鼠;Normal + PEMF組有6隻健康小鼠,進行連續脈衝式電磁場(PEMF)刺激;Normal + HMSP-EMF組有5隻健康小鼠,進行單一脈衝式電磁場(HMSP-EMF)刺激;Osteoporosis組有6隻骨質疏鬆小鼠; Osteoporosis + PEMF組有7隻骨質疏鬆小鼠,使用PEMF刺激; Osteoporosis + PEMF組有7隻骨質疏鬆小鼠,使用HMSP-EMF刺激。應用PEMF與HMSP-EMF於骨質疏鬆小鼠模型,在磁刺激4, 6, 8, 10, 12星期時,使用micro-CT活體掃描脛骨頭計算分析,使用Two - way ANOVA進行磁刺激方式與刺激時間的分析比較,選取α=0.05;12星期後將小鼠犧牲取出脛骨頭,使用微電腦斷層攝影掃描脛骨頭,並計算分析骨密度、骨髓腔內骨頭體積百分比、骨小樑厚度、骨小樑間距、骨小樑數量等五種骨型態學參數,使用one-way ANOVA進行六組分析比較,選取α=0.05。

Phase A的結果顯示結果顯示:Control組、Normal + WBV組的BMD、骨頭體積百分比及骨小樑間距在統計上比Osteoporosis組、Osteoporosis + WBV組有顯著差異。Osteoporosis + WBV組與Osteoporosis組比較結果顯示:BMD、骨頭體積百分比有增加的趨勢,在統計上沒有顯著的差異。未來實驗將延長刺激時間並減少振動頻率,相信必能改善骨質流失的現象。

Phase B的結果顯示Osteoporosis + PEMF組及Osteoporosis + HMSP-EMF組的骨質密度在統計上顯著大於Osteoporosis組(p<0.05);使用磁刺激6, 8, 10, 12週的小鼠其骨質密度在統計上顯著大於治療0, 4週(p<0.05)。使用單一脈衝式電磁場刺激6週後骨質已有顯著的增生,長時間使用也可維持其骨質密度。小鼠犧牲後所得到的五個型態學參數結果顯示:Osteoporosis + HMSP-EMF組的骨質密度、骨頭體積百分比與骨小樑數量在統計上有顯著大於Osteoporosis組(p<0.05);Osteoporosis + PEMF組與Osteoporosis組的骨小樑厚度在統計上顯著小於Normal + HMSP-EMF組與Control組(p<0.05)。Osteoporosis + PEMF組與Osteoporosis組的骨小樑間隙在統計上顯著大於Normal + PEMF組及Control組(p<0.05)。單一脈衝式電磁場刺激時間一天僅須3分鐘,6週後有改善骨質流失的效果;連續脈衝式電磁場一天需要進行8小時的刺激,治療時間須長達12星期,治療效果有限。

本研究發現HMSP-EMF刺激可促進骨質疏鬆症的治療效果。未來可探討其缺失並發展改良,包括以下幾點:Phase A - 利用不同的頻率強度對於骨質的增生進行探討與分析,並延長實驗刺激的時間,以得到最佳療效。Phase B - 設計不同的骨質疏鬆動物模型(例如:脊椎等部位損傷),應用脈衝式電磁場於各不同型態之骨質疏鬆模型進行探討與分析。
英文摘要 The change of human living style due to advanced technological development has led to a dramatic increasing incidence and prevalence of osteoporosis recently. In the worldwide, 1/3 of the female population with above 50 years old suffer from osteoporosis, the prevalence was about 1/5 for the male with ages above 50 years old. In Taiwan, 56% of the female with above 60 years old suffer from osteoporosis [1]. The conventional treatment for osteoporosis were to take calcium, hormone or drug medication, but these medication were either high cost or often producing side effects on the user. The physical stimulation including ultrasound, electrical, electromagnetic field, and vibration and so on were likely to enhance the bone formation. This study was to investigate the effect of physical stimulation on osteoporosis in osteoporotic animal models. More specifically, the research aims were to: 1) investigate the effect of whole body vibration (WBV) on osteoporotic SD rats model, and 2) investigate the effect of pulsed electromagnetic field (PEMF) and high magnetic single pulsed electromagnetic field (HMSP-EMF) on osteoporotic BALB/C mice model.

The experiment was divided into two phases: Phase A – a total of 32 SD rats with 8 weeks old were randomly assigned to 4 groups including: control group with 8 normal rats, normal + WBV group with 8 normal rats plus WBV, osteoporosis group with 8 osteoporotic rats, and osteoporosis + WBV group with 8 osteoporotic rats plus WBV. The dosage of WBV was increased from 0.26G to 0.73G for 4 weeks. After 4 weeks, the rats of all groups were sacrificed and the tibia were took out for micro-CT scanning to measure bone mineral density (BMD), percent bone volume, trabecular thickness, trabecular separation, and trabecular number. One-way ANOVA was used to analyze the bone parameter and compare all 4 groups with α=0.05. Phase B – a total of 37 BALB/B mice with 8 weeks old were randomly assigned to 6 groups including: control group with 6 normal mice, normal + PEMF group with 6 normal mice plus PEMF, normal + HMSP-EMF group with 5 normal mice plus HMSP-EMF, osteoporosis group with 6 osteoporotic mice, osteoporosis + PEMF group with 7 osteoporotic mice plus PEMF, osteoporosis + HMSP-EMF group with 7 osteoporotic mice plus HMSP-EMF. The dosage of PEMF was 1.8 mT and the dosage of HMSP-EMF was 0.8 T for 12 weeks stimulation. On 4, 6, 8, 10, 12 weeks, all 6 groups were scanned by micro-CT to measure the BMD of tibia. Two-way ANOVA was used to analyze the treatment type and treatment time on the BMD with α=0.05. After 12 weeks, the mice of all groups were sacrificed and the tibia of all mice were took out for micro-CT scanning to measure BMD, percent bone volume, trabecular thickness, trabecular separation, and trabecular number. One-way ANOVA was used to analyze the bone parameter and compare all 6 groups with α=0.05.

The results of Phase A show the BMD and percent bone volume of control and normal + WBV groups are significantly large than those of osteoporosis and osteoporosis + WBV groups. To compare the osteoporosis group, the BMD and percent bone volume of osteoporosis + WBV group have an increasing tendency, but no significantly difference. The insignificant improvement of bone mass is probably due to only 4 weeks vibration training. Future study will increase the vibration training time and decrease training gravity for improved osteoporosis treatment.

The results of Phase B show that the BMD of osteoporosis + PEMF and osteoporosis + HMSP-EMF groups is significantly large than that of osteoporosis group (p<0.05); the BMD of both groups treated at 6, 8, 10, 12 weeks is significantly large than that of both groups treated at 0, 4 weeks (p<0.05). The HMSP-EMF treatment shows that the BMD of osteoporotic mice has significantly improved on 6 weeks treatment and then maintain the same quality after later treatment. The BMD, percent bone volume, and trabecular number of osteoporosis + HMSP-EMF group are significantly large than those of osteoporosis group after scarification (p<0.05); the trabecular thickness of osteoporosis + PEMF and osteoporosis groups is significantly smaller than that of normal + HMSP-EMF and control groups (p<0.05); the trabecular separation of osteoporosis + PEMF and osteoporosis groups is significantly large than that of normal + PEMF and control groups (p<0.05). The efficacy of HMSP-EMF stimulation is validated through 3 minutes stimulation per day to improve the bone quality after 6 weeks treatment. In contrast, the application of PEMF is limited by 8 hours stimulation per day for 12 weeks.

The findings of this study are that the HMSP-EMF stimulation is likely to improve bone mass for osteoporosis treatment. Future research is recommended to include the following: Phase A – to investigate different frequencies and amplitude and treatment duration on bone quality for osteoporotic animal model; Phase B – to design different osteoporotic animal models, such as spinal cord injury, in order to study the treatment effect of different EMF on bone quality.
論文目次 中文摘要I
AbstractIII
誌謝V
目錄VII
表目錄X
圖目錄XII

第一章 緒論1

1.1 骨質疏鬆症2
1.1.1 全球骨質疏鬆症的盛行率2
1.1.2 骨質疏鬆症病因學4
1.1.3 骨質疏鬆症臨床診斷與治療方式6
1.2 骨的功能性解剖及生理機轉8
1.3 磁刺激對於骨質的物理效應16
1.3.1 磁的基礎理論16
1.3.2 電磁波的物理特性19
1.3.3 磁刺激的生物效應21
1.3.4 磁刺激應用於生物效應的文獻探討23
1.4 振動刺激對於骨質的物理效應26
1.4.1 振動刺激的基礎理論26
1.4.2 振動刺激應用於骨頭的文獻探討28
1.5 微電腦斷層掃描29
1.5.1 微電腦斷層的原理30
1.5.2 微電腦斷層掃描應用骨質型態之診斷評估31
1.6 研究動機與目的32

第二章 系統設計與材料方法33

2.1振動刺激對於骨質的影響34
2.1.1 動物模型34
2.1.2 儀器設備34
2.1.3 實驗設計與實驗流程36
2.1.4 統計分析37
2.2磁刺激對於骨質的影響38
2.2.1 動物模型38
2.2.2 儀器設備39
2.2.3 實驗設計與實驗流程42
2.2.4 統計分析45

第三章 結果與討論46

3.1全身性振動系統刺激骨質增生的效應46
3.1.1全身性振動刺激對於骨質密度改變的結果48
3.1.2全身性振動刺激對於骨頭體積百分比改變的結果49
3.1.3 全身性振動刺激對於骨小樑間隙改變的結果50
3.1.4全身性振動刺激對於骨小樑間隙改變的結果51
3.1.5全身性振動刺激對於骨小樑數量改變的結果52
3.1.6 全身性振動刺激對於骨質改變結果之討論53
3.2電磁場系統刺激骨質增生的效應54
3.2.1磁刺激及刺激時間對於骨質密度改變之結果55
3.2.2 磁刺激與刺激時間對於骨質密度改變之討論58
3.2.3 小鼠犧牲後觀察磁刺激對於骨質密度改變的結果59
3.2.4 磁刺激對於骨質改變結果之討論66
3.2.5 微電腦斷層掃描影像與傳統切片圖片之比較66

第四章 結論67

4.1 結論67
4.2 未來展望68

附錄69

附錄A….69
附錄B74
附錄C75

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------------------------------------------------------------------------ 第 6 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200915264561
論文名稱(中文) 探討微小球蛋白MSP58調控細胞週期和細胞老化的分子機制
論文名稱(英文) Molecular Mechanism of Cell Cycle Control and Senescence by the 58-kDa Microspherule Protein (MSP58)
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 許哲嘉
學號 s2696407
學位類別 碩士
語文別 中文
口試日期 2009-07-24
論文頁數 86頁
口試委員 指導教授-張文昌
指導教授-林鼎晏
口試委員-郭毓良
口試委員-蔣輯武
關鍵字(中) 細胞老化
MSP58
關鍵字(英) MSP58
senescence
學科別分類
中文摘要 中文摘要

58-kDa微小球蛋白(MSP58)是一個分子量大小約為58-kDa的核仁蛋白,已經被報導和許多蛋白質有交互作用,比如核仁蛋白p120、Mi-2b、RNA結合蛋白FMRP和轉錄因子Stra13以及Daxx。 這些蛋白質交互作用暗示著MSP58在細胞核與核仁中扮演基因轉錄調控的功能。 除此之外,MSP58也被報導在中心體(centrosome)被偵測到以及會與中心體蛋白Nde l和DIPA有蛋白質交互作用。 另外,研究指出MSP58的鳥類同源基因,TOJ3,可透過v-Jun的調控而增加其表現並且具有細胞轉型的活性。 最近研究更提到MSP58可能擔任一個致癌基因(Oncogene)的角色並且因為與腫瘤抑制因子PTEN的交互作用因而抑制其細胞轉型的能力。 然而人類MSP58的一個異構物(isoform),MCRS2,發現會與端粒酶抑制蛋白LPTX/PinX1以及端粒酶催化次單位hTERT有蛋白質交互作用。 本實驗室初步實驗結果顯示,過度表現MSP58基因會依賴不同細胞的形式而對細胞增生速率有不同影響。 持續大量表現MSP58在人類子宮頸上皮細胞株HeLa中,會增加細胞增生的速率; 而以基因剔除方法將MSP58剔除掉後,會抑制HeLa細胞增生的速率。 但是在人類纖維肉瘤細胞株HT1080中,持續大量表現MSP58基因則會誘導細胞走向類似細胞老化的生長抑制過程,並觀察到細胞週期中,S時期和G2時期的進行受到延遲以及抑制了端粒酶的活性。 這些結果暗示出MSP58蛋白質的功能可能具有調控細胞老化的能力。
英文摘要 英文摘要

The 58-kDa microspherule protein (MSP58) has been reported to interact with several proteins, such as nucleolus proteins p120, Mi-2; RNA-binding protein FMRP and transcription factors STRA13, Daxx. These findings suggest that MSP58 may have functions of transcriptional regulation in the nucleus and nucleolus. In addition, MSP58 can be detected on the centrosome and interacted with centrosomal proteins Nde1 and DIPA. A study of the quail homologue of MSP58, TOJ3, showed that this protein can be up-regulated by v-Jun and exhibits cell transformating activity. Recently, a study revealed that MSP58 behaves as an oncogene and that its transformation activity can be inhibited by physical interaction with PTEN tumor suppressor. Moreover, a splice isoform of human MSP58, MCRS2, binds the LPTX/PinX1 telomerase inhibitor and the catalytic hTERT subunit of telomerase. In the preliminary studies, we show that overexpression of MSP58 gene alter cell proliferation rate in a cell type dependent manner. Stably transfection of MSP58 promotes cell growth whereas knockdown MSP58 inhibits cell proliferation in HeLa cells. Unexpectedly, forced expression of MSP58 induces senescence-like growth arrest , delays S and G2 progression and reduces telomerase activity in HT1080 cells. These observations provides a previously uncharacterized biological function of MSP58 in its ability to regulate cellular senescence.
論文目次 目錄
中文摘要……………………………………………………………………...I
英文摘要……………………………………………………………………..II
誌謝………………………………………………………………………….III
目錄…………………………………………………………………………..V
圖目錄…………………………………………………………………...…VII
附錄目錄…………………………………………………………………..VIII
縮寫指引……………………………………………………………………IX
第一章 緒論………………………………………………………………….1
第一節 Microspherule Protein 58 (MSP58)之介紹……………………1
第二節 細胞老化與其訊息傳遞路徑………………………………....3
第三節 細胞老化與癌症的關係………………………………………4
第四節 SWI/SNF染色質重組複合體和癌症………………………...5
第五節 研究動機與目的……………………………………………....6
第二章 實驗方法…………………………………………………………....8
第一節 細胞培養……………..………………………………………..8
第二節 質體之建構…………………………………………………....9
第三節 持續表現MSP58蛋白質細胞株之製備…………………….13
第四節 全細胞液之抽取與蛋白質定量……………………………...15
第五節 硫酸十二酯鈉聚丙烯醯胺凝膠電泳法(SDS-PAGE)/西方點墨
法(Western blot)………………………………………………17
第六節 免疫螢光染色(Immunofluorescence)………………………..24
第七節 端粒重複序列放大反應(Telomere Repeat Amplification Protocol, TRAP assay)………………………………………27
第八節 Senescence associated -galatosidase(SA--gal)之活性試驗..32
第九節 免疫沉澱法(Immunoprecipitation)…………………………...33
第十節 染色質免疫沉澱分析(Chromatin immunoprecipitation assay,
ChIP assay)…………………………………………………34
第十一節 全量的RNA抽取………………………………………...39
第十二節 及時定量反轉錄-聚合酶連鎖反應(Quantitative real-time
RT-PCR)………………………………………………….40
第十三節 報告基因分析法(reporter assay)………………………….42
第三章 實驗結果…………………………………………………………..44
第一節 過度表現MSP58蛋白質誘導HT1080細胞老化…………….44
第二節 MSP58所誘導細胞老化過程是透過改變細胞週期中相關因子表現……………………………………………………………...45
第三節 MSP58調控p53和p21的轉錄活性…………………………..46
第四節 利用酵母菌雙雜交技術找出MSP58新穎的結合蛋白質…….47
第五節 MSP58結合在p21啟動子上…………………………………..48
第六節 MSP58結合至p21啟動子上需要p53存在…………………..49
第七節 過度表現MSP58在SW-13細胞中影響其造成
細胞扁平的能力………………………………………………...50
第八節 MSP58抑制E2F1所調控的啟動子轉錄活性…………………51
第九節 MSP58影響DNA損傷訊息傳遞路徑………………………….52
第四章 討論………………………………………………………………...54
第五章 參考文獻…………………………………………………………...61
附圖………………………………………………………………………….68
附錄………………………………………………………………………….79
自述………………………………………………………………………….86
參考文獻 參考文獻

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系統識別號 U0026-0812200915292489
論文名稱(中文) 利用智慧型手機建立孕婦生理參數監視系統
論文名稱(英文) Development of a Wireless Physiological Monitoring System for Gravida Based on SmartPhone
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 李易晏
學號 p8696109
學位類別 碩士
語文別 中文
口試日期 2009-07-10
論文頁數 54頁
口試委員 指導教授-陳天送
口試委員-陳琮琳
口試委員-陳培展
口試委員-陳世中
關鍵字(中) 生理監控
MSP430
智慧型手機
藍芽
關鍵字(英) Bluetooth
Smart phone
Physiological monitoring
MSP430
學科別分類
中文摘要 臨床的產檢中,子宮收縮信號和胎兒心跳率是非常重要的生理資訊;醫護人員可以藉由測量這些參數,來了解孕婦與胎兒的狀況。異常的數值變化,通常是早產或流產的警訊。但醫護人員卻只能在產檢時,藉由儀器才能了解孕婦的子宮收縮情況,也因此無法於生理參數異常時,第一時間內提供孕婦必要之協助。智慧型手機不但具有傳統PDA與GSM手機的功能,並提供藍芽傳輸、顯示介面與簡易的程式撰寫,另外長時間的待機與行動無線網路之功能,更適合用於生理參數之長期監控。有鑑於此,本研究希望建立一套可攜式長時監控孕婦子宮收縮系統,利用藍芽技術,並結合智慧型手機作長時監測。
系統主要架構可分為兩個部份;第一部份為生理訊號之擷取,利用單晶片(MSP430F169)分析處理,並透過藍芽模組,將數值變化傳送到手機上。第二部份為智慧型手機的即時訊號顯示、儲存孕婦的生理參數變化,並在參數持續異常時,經由手機撥號,在最短時間內聯絡醫護中心,以達到即時救護的功能。
英文摘要 Uterine contraction (UC) and fetal heart rate (FHR) are two important parameters to check the fetal status on prenatal diagnosis, the abnormal UC and FHR will cause premature delivery or miscarriage. Therefore, how to develop a long-term monitoring system of uterine contraction and FHR become an important issue on obstetrics and Gynecology.
The aim of this study is to set up a long-term monitoring system. A wireless portable device based on micro-controller (MSP430) is used to acquire the physiological signals and transmit them to the smart phone via the Bluetooth wireless technology. The acquired signals and waveform are displayed on smart phone’s screen and saved for further processing. If a possibility of premature delivery occurs, system will send an emergency call for medical help automatically, to provide medical service immediately. This system highly promotes the convenience of both the pregnant women and the medical personnel and further improves the quality of health care.
論文目次 目錄
中文摘要…………………………………………………………… I
英文摘要……………………………………………………………Ⅱ
誌謝……………………………………………………………… Ⅲ
目錄………………………………………………………………… Ⅳ
圖目錄………………………………………………………………Ⅶ
表目錄………………………………………………………………XI
第一章 緒論…………………………………………………………1
第1-1節 前言…………………………………………………………1
第1-2節 研究背景……………………………………………………2
第1-2-1節子宮收縮和早產陣痛……………………………………3
第1-2-2節 胎兒心跳率(FHR) ………………………………………3
第1-3節 臨床上之監測方法…………………………………………5
第1-4節 研究動機……………………………………………………7
第1-5節 文獻回顧……………………………………………………8
第1-6節 章節提要……………………………………………………12
第二章 系統原理…………………………………………………… 13
第2-1節 壓力感測器…………………………………………………13
第2-1-1節 壓電訊號轉換原理………………………………………14
第2-2節 心電圖的基本理論…………………………………………15
第2-3節 微控制器……………………………………………………17
第2-3-1節 發展環境與工具程式……………………………………19
第2-4節 無線傳輸技術介紹........................ .............................21
第2-4-1節 藍芽技術特色……………………………………………22
第2-5節 行動通信網路………………………………………………25
第2-6節 智慧型手機…………………………………………………26
2-6-1 PDA手機開發軟體簡介………………………………………27
2-6-2 Microsoft Visual Studio 2005 軟體介紹 ………………27
第2-7節 電源供應部份………………………………………………28
第三章 系統實現……………………………………………………30
第3-1節 系統架構……………………………………………………30
第3-2節 硬體電路……………………………………………………31
第3-2-1節放大器電路設計……………………………………………32
第3-2-2節濾波電路原理與設計………………………………………32
第3-2-3節校正電路……………………………………………………33
第3-3節系統軟體………………………………………………………34
第3-3-1節單晶片端類比數位轉換……………………………………35
第3-3-2節單晶片端壓力信號處理……………………………………35
第3-3-3節單晶片端ECG信號處理……………………………………36
第3-3-4節藍芽傳輸…………………………………………………37
第3-3-5節手機端接收程式設計……………………………………38
第3-3-6節伺服器端程式設計………………………………………39
第四章 實驗結果與討論……………………………………………41
第4-1節實作系統說明………………………………………………41
第4-1-1節硬體方面…………………………………………………41
第4-1-2節軟體方面…………………………………………………43
第4-2節系統驗證……………………………………………………46
第4-2-1節壓力系統測試……………………………………………46
第4-2-2節傳輸功能驗證……………………………………………48
第五章 結論與未來展望……………………………………………50
第5-1節 結論…………………………………………………………50
第5-2節 未來展望……………………………………………………50
參考文獻………………………………………………………………52
自述……………………………………………………………………54
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系統識別號 U0026-2607201015494100
論文名稱(中文) 新穎致癌基因 MSP58 之功能性探討
論文名稱(英文) Functional analysis of a novel candidate oncogene, 58-kDa microspherule protein (MSP58)
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 98
學期 2
出版年 99
研究生(中文) 王翠霙
學號 s2697103
學位類別 碩士
語文別 中文
口試日期 2010-07-16
論文頁數 59頁
口試委員 指導教授-張文昌
指導教授-林鼎晏
口試委員-王育民
關鍵字(中) 細胞老化
細胞凋亡
關鍵字(英) MSP58
apoptosis
senescence
學科別分類
中文摘要 58-kDa微小球蛋白(MSP58)已被報導參與在基因轉錄調控和細胞轉型(transformation)能力。在我們的研究中發現利用干擾RNA降低MSP58的表現會導致細胞分裂不正常進而誘導細胞凋亡(apoptosis)的發生,然而大量表現MSP58會導致類似細胞老化的生長停滯。研究持續大量表現MSP58的人類纖維肉瘤細胞株HT1080中發現細胞生長停滯是由於誘導pRB去磷酸化、CDK的抑制劑p21的表現上升以及端粒酶(telomerase)活性的抑制。進而我們利用干擾RNA在持續表現MSP58的HT1080細胞株中降低p21的表現會阻止MSP58 所誘導的細胞老化。此外,近來我們確認分析一些與MSP58有交互作用的新穎蛋白質如需ATP的染色質重建複體(chromatin remodeling complex)成員與參與端粒酶活性的結合蛋白。總結以上實驗我們提供了MSP58調控基因轉錄和細胞老化的新功能。
英文摘要 The nucleolar 58-kDa microspherule protein (MSP58) has been implicated in functional roles of genes transcriptional regulation and cellular transformation. In our studies, abrogation of the endogenous MSP58 function by small interfering RNA knockdown caused aneuploidy and apoptosis, whereas overexpression of MSP58 gene induced the senescence-like growth arrest in HT1080 cells . Studies in the MSP58 stably expressed clones in HT1080 cells revealed that the growth arrest may in part be accounted for by inducing hypophosphorylation of pRB, up-regulation cyclin-dependent kinase inhibitors p21 and telomerase activity inhibition. In addition, abrogation of the endogenous p21 function by RNA interferences in MSP58 stable clones prevents the induction of senescence. Recently, we identified and characterized some MSP58-interacting proteins that is comprised of a SWI/SNF chromatin remodeling complex sununit and novel telomerase regulators. These results provides a previously uncharacterized biological function of MSP58 in its ability to regulate gene expression and cellular senescence.
論文目次 中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VI
附錄目錄 VII
縮寫指引 VIII
第一章 緒論 1
第一節 Microspherule Protein 58, MSP58 1
第二節 細胞老化(Cell Senescence) 2
第三節 Brahma-related gene-1,BRG1 2
第四節 細胞凋亡(apoptosis) 3
第五節 端粒酶調控與細胞老化 3
第六節 研究動機和目的 4
第二章 實驗材料與方法 6
第一節 實驗材料 6
第二節 實驗方法 11
一、細胞培養 11
二、質體建構 12
三、持續抑制蛋白質細胞株之製備 16
四、全細胞液之抽取與蛋白質定量 17
五、西方墨點法(Western blot) 18
六、免疫螢光染色法(Immunofluorescence) 20
七、細胞大小與凋亡細胞分析 21
八、Senescence associated β-galactosidase (SA-β-gal)之活性測試 23
九、酵母菌雙雜交分析(Yeast two hybrid assay) 23
十、報告基因分析(reporter assay) 27
第三章 實驗結果 28
第一節 確認抑制MSP58的干擾RNA 28
第二節 抑制MSP58 蛋白質表現促使HT-1080細胞凋亡 28
第三節 在HT-1080細胞中抑制MSP58 蛋白質表現呈現細胞分裂不正常 29
第四節 確認新穎蛋白質與MSP58有蛋白質交互作用 29
第五節 MSP58誘導細胞老化 30
第六節 MSP58細胞週期分析 31
第七節 MSP58下游基因的轉錄調控 32
第八節 抑制p21的表現可以回復MSP58所誘導的細胞老化 32
第九節 MSP58調控端粒酶活性 33
第四章 討論 35
第五章 參考文獻 39
自述 59
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