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龐貝氏症患者及帶原者酵素活性差異性之原因

2021-06-13T07:48:06Z

標題: 龐貝氏症患者及帶原者酵素活性差異性之原因; Etiology of enzyme activity variability in Pompe’s disease 作者: Cheng-Ni Liu; 劉正暱摘要: 龐貝氏症(Pompe’s disease)是一種肝醣儲積症(Glycogen storage disease, GSD)，又稱為肝醣儲積症第二型(GSD II)。是由於先天缺乏溶小體酵素-酸性麥芽糖酶(Acid Maltase，又稱acid α-glucosidase, GAA)，使得進入溶小體的肝醣無法被分解而持續堆積，進而影響到細胞的功能。臨床上依發病年齡分為嬰兒型、青少年型及成人型。龐貝氏症確定診斷的方法為測定皮膚纖維芽細胞或血液中GAA酵素活性。通常患者的酵素活性很低(<5%)，而帶原者的活性會介於正常人和患者之間。臨床上發現，部分帶原者的酵素活性偏低，約只有正常人5 ~ 10%，可能會增加檢測困難度。因此，我們認為可能存在某些因數會影響酸性麥芽糖酶的表現，如基因多型性或其他機轉，影響到酸性麥芽糖酶的蛋白質穩定性。在本實驗室近年來的研究，發現熱休克蛋白Hsp27對於突變蛋白GTP cyclohydrolase I的dominant-negative effect會有影響。Hsp27是一種small heat shock蛋白，和蛋白質摺疊、細胞骨架之穩定性，以及細胞凋亡有關。突變酸性麥芽糖酶可能在某些情況下，如Hsp27存在下，其蛋白質穩定性會有所改變，也可能會受到其他因數的影響，導致細胞酵素活性降到如此的低。本研究進行的方法(1)分析所有個案的GAA酵素活性、mRNA與蛋白質表現量。(2)藉著基因序列分析找尋是否有基因多型性的存在，與突變蛋白GAA酵素活性具有關聯性。(3)以表現Hsp27的質體轉染龐貝氏症患者或是帶原者的皮膚纖維芽細胞，瞭解Hsp27對於突變蛋白GAA酵素活性是否具有調控的能力。本實驗結果證實GAA酵素活性表現與蛋白質表現量有正相關性。也證實GAA突變蛋白的存在。RNA實驗結果顯示，帶原者的GAA蛋白表現量和其mRNA間並無絕對相關性存在。以蛋白結構推測，GAA突變蛋白與正常蛋白具有差異性。雖然基因多型性可能會影響GAA突變蛋白的穩定性，在基因序列分析中，也發現許多序列多型性，並形成三個區塊，但並無特殊的基因多型性與酵素活性間有關聯性。Hsp27 -S3D實驗中發現Hsp27-S3D會降低患者與帶原者的酵素活性。影響GAA酵素活性的因數，還是有其他基因多型性，或其他分子伴隨者調控的可能。本研究主要目的希望能應用於未來的臨床診斷及治療，在臨床上提供更精確診斷，治療上能有其他新藥物。我們的研究指出蛋白質的穩定性是影響酵素活性的重要因數。可能在基因的其他位置，例如是intron仍存在著有影響的序列多型性。或是細胞內的其他熱休克蛋白會影響GAA蛋白的穩定性，這些都還待進一步之研究。; Pompe’s disease is a lysosomal storage disease involving the storage of glycogen. It is used to be called type II glycogenosis. The etiology of this disease is the deficiency of acid maltase (or acid alpha-glucosidase, GAA). The deficiency of GAA leads to a progressive storage of glycogen in the lysosome, which affects the function of the cells. According to the onset clinically, there are three subtypes – infantile, juvenile, and adult type. The diagnosis of Pompe’s disease depends on the measurement of GAA activity in either skin fibroblast or in peripheral blood mononuclear cells. Theoretically, GAA activities in obligatory carriers should be 50% of normal. However, we frequently met carriers with GAA activities as low as 5~10% of normal. The current hypothesis is the presence of some important factors, ex. polymorphism or some other mechanisms that will alter the stability of GAA. In our previous studies, Hsp27 can alter the stability of the GTP-cyclohydrolase I protein. Hsp27 is a member of the small HSP that is involved in protein folding, stability of the cytoskeleton, and cell apoptosis. It is possible that the stability of GAA will be changed under certain circumferences, and then causes the excessive low GAA activity in some individuals. In this study, we are going to perform the assays: (1) the GAA activity, mRNA, and protein expression; (2) the gene polymorphism analysis; (3) to overexpress Hsp27 in skin fibroblasts from either Pompe’s patients or the carriers. We will observe the changes of GAA protein by the western blot analysis, and also GAA activities by enzyme assays, to see if these parameters will be changed by the expression of Hsp27. Special interests will be on Pompe’s carriers with excessive low GAA activities. In the study, there is a positive correlation between the GAA activity and the protein expression. From the RNA study, there is no correlation between the expression of the GAA protein and of the mRNA. From the structure analysis, the polymorphism (V816I) may influence the stability of mutant GAA protein. We also find some polymorphisms and form three blocks in the gene polymorphism study. But, no relation between the polymorphisms and the GAA activity. The data of the Hsp27 study shows Hsp-S3D will decrease the GAA activity, either in the patients or carriers. There still exists the possibility of some other factors that will influence the GAA activity, ex. Polymorphisms, or heat shock proteins. This study will contribute significantly the knowledge of the pathogenesis of disease, the mechanism of changes in protein stability, the diagnostic technology, and also deeply to the future treatment of the diseases, including those with similar molecular mechanisms. Our data showed the stability of GAA protein is one important factor to the GAA activity. Maybe some polymorphisms exists in some position of the GAA gene, ex. Intron, that will play some roles. Or, the other Hsp in the cells may affect the stability of the GAA protein. Those all will be needed further study.

龍伯球透鏡之波傳散射與聚焦特性研究

2021-06-13T04:14:02Z

標題: 龍伯球透鏡之波傳散射與聚焦特性研究; Wave propagation, scattering and focusing effect of Luneberg lens 作者: Hao-Hsien Lin; 林浩賢摘要: 龍伯球透鏡(Luneberg lens)為一種介電係數與半徑成一固定比例的球形透鏡，大部分是由數層不同介電係數介電質組合而成，而其主要特性能夠把入射波聚焦到球表面一點，假使在透鏡表面處鍍上一圓錐角金屬層即為龍伯球透鏡反射器(Luneberg lens reflector)，能把入射波依平面波的型態反射回原入射端，通常用於天線、雷達、衛星通訊、軍事等方面。而本論文主要在研究Luneberg lens reflector的波傳散射和聚焦的特性，首先利用球諧向量函數Mie theory在球座標下表示整個電磁場，將平面波用球諧函數方式展開以利於分析，因為在邊界上是由金屬和介電質兩種材質所組成，無法依相同的邊界條件求得，所以採用PMM數值方法去求透鏡混合邊界外場的值，其方式主要是取有限個節點滿足其所在位置邊界條件的方程式去求解，而透鏡內依照每層的邊界條件向內求出各層的電場，最後可得到平面波入射到透鏡經過散射反射之後的電磁場。在討論方面我們採用不同層數的透鏡去逼近理想值曲線，因為非理想Luneberg lens的關係所以會產生差異性以及精確度上的不同，於是我們要在不同層數與聚焦效果兩者之間取得平衡，還有在不同的波長下對於透鏡聚焦的效果的影響，透鏡的散射圖騰在鍍上金屬之後的差異性，以及整個波傳場圖在不同的波長下聚焦效果是否明顯，聚焦點是否確實處於球表面處和反射之後是否為完整的平面波型態，最後畫出球表面上的電場向量以及poynting vector以了解能量的流向是否有漸漸往球表面一點匯集的效果。; Luneberg lens is a spherical lens which its permittivity varies with normalized radial coordinates. It is fabricated by using different homogeneous dielectric centrically layered media. The lens can focus a parallel beam of rays from any direction exactly at a point on the surface. If it is made by adding a reflecting surface “metallic cap”, it is called Luneberg lens reflector. It can reflect incident wave into plane wave in incoming direction. It is usually applied in antenna, radar reflector, satellite-based, and mobile-communucation. The main points of this thesis are about wave propagation, scattering and focusing effect of Luneberg lens. First we use spherical harmonics, the Mie theory, to express the electric and magnetic field in spherical polar coordinate system. We expand a plane wave in vector spherical harmonics. This problem can not be solved by the same boundary condition, because there are two different materials, dielectric and metallic, on the surface. So we use point-matching method (PMM) to solve this mixed boundary value problem. The method consists of satisfying the boundary conditions in the contact plane only at a finite point. Then we use the boundary condition of each layer, we can find the coefficient of each layer. Finally we can find the all field of the incident wave into a Lungberg lens. In the discussion, we use different layers of lens to approach the ideal Lungberg lens. We take the balance in different layers and the focusing effect. What is the influence of the different wave length and what is the difference between Lungberg lens and Lungberg lens reflector in scattering patterns. We also provide a picture of wave propagation through Lungberg lens to see if the points are exactly on the surface, the incoming wave reflected by the metallic cap is a perfect plane wave or not. Finally, we provide the poynting vector on the special surface. The purpose is to see if the energy focuses to a point on the surface.

齒輪機構之運動特徵分析與合成

2021-06-08T05:57:59Z

標題: 齒輪機構之運動特徵分析與合成; Kinematic Characteristics and Synthesis of Geared Mechanisms Using the Concept of Kinematic Fractionation 作者: Yu-Ching Yeh; 葉又菁摘要: 本論文主要利用運動分解的概念進行齒輪機構運動的特徵分析與合成，以及經由運動特徵進行齒輪機構的分類。齒輪機構可被分解為數個單自由度的運動單元，每個運動單元則被視為齒輪機構內的運動傳輸模組。運動單元間的拓樸構造連接可分為雙鏈結型與同軸三角型，內文將分析內含四個運動單元，單自由度至六桿，以及兩個自由度至七桿的齒輪機構，並且說明及表列其對應的拓樸構造；藉由選擇輸入與輸出端的位置，可得到齒輪機構內所有可能的運動傳輸路徑並將路徑以控制區塊圖表示。利用控制區塊的增益計算公式，將輸入端與輸出端的運動關係公式化為增益矩陣。接著進一步討論運動單元內部條件對整體運動增益的影響；運動單元內輸出入桿件的不同及運動單元間的共同連接部分的接頭條件會共同影響整體增益的表現形式，利用增益表現形式的差異可將內含四個運動單元，單自由度至六桿，以及兩個自由度至七桿的齒輪機構做完整分類，根據增益型式分類及運動特徵可得到功能導向之齒輪設計方法。; A methodology based on the concept of kinematic fractionation for the revelation of kinematic characteristics and classification of geared mechanisms is presented. It is shown that structurally non-fractionated geared mechanisms can be considered as the combination of kinematic units (KUs). Each KU is considered as the basic motion transmission module inside a geared mechanism. Admissible connections of KUs are identified according to the structural characteristics of one- and two-DOF geared mechanisms of up to four KUs. Such configurations are then used to construct possible propagation paths of motion via the assignments of input and output links. Since the propagation paths can be modeled by the control block diagram problems, the kinematic relations between input and output links are formulated to gain matrices. According to the types of entities in a gain matrix, various kinematic behaviors are disclosed. The complete kinematic behavior of single KU is revealed and three gain forms of KU is basic of global gain since the geared mechanism is combination of KUs. The global gain of the mechanism is determined by three factors: the configuration decides the transmission flow at KU level, the common linkage between KUs of the mechanism limit the thin edge type of local input and output in the connection of KUs, the assignment of input, ground and output decides the thin edge type of global input and output. From the factor of global gain, there are three gain type are identified and characteristics of geared mechanism are more clear. It is believed that such kinematic characteristics can be readily transformed into the functional requirements and synthesis of geared mechanisms of up to four KUs can be accomplished much easier.

鼠鬚刺激系統之開發與眶下神經重合之術後功能性回復評估

2021-06-15T13:29:34Z

標題: 鼠鬚刺激系統之開發與眶下神經重合之術後功能性回復評估; Stimulation system for rat whisker and functional recovery after infraorbital nerve transection 作者: Ji-Lin Chen; 陳吉麟摘要: 臨床上許多狀況會造成周邊感覺神經的截斷，如刀傷、車禍碰撞、撕裂傷、移植手術等。少數神經重建術接合斷裂神經的患者在經過長時間復原後可達到部分的感覺回復，究竟這些重拾感覺輸入的患者其大腦發生了哪些轉變是我們所好奇的課題。本研究以大鼠的觸鬚-桶狀皮質系統作為模型，截斷與重新接合支配觸鬚感受器的眶下神經模擬臨床情況，建立出刺激參數與神經反應的調性方程式以評估手術前後感覺皮質發生的變化。本研究開發了定量化且精確的觸鬚刺激系統以應用於動物實驗。兩組軟體產生的訊號經類比轉換及電壓放大後控制雙軸向壓電致動元件，利用黏附其上的夾具帶動鼠鬚產生雙自由度的撓動。藉由夾具的設計以及軟體的校正，本系統達到高精度的振幅刺激，此外，系統加入了應變量測裝置對輸出振幅作即時監測以減低實驗誤差。於感受域分析結果，超過半數的樣本在術後可以重新獲得周邊觸鬚受器與皮質感受域的對應關係。其中約三成樣本的感受域分布在手術前後具有相似性。針對感受域回復的樣本，刺激速度辨識能力在手術之後立即降低，但會隨著復原時間增長而回升。另外，皮質神經的刺激後反應時間在手術後增加，但會隨著復原之過程而逐漸減少。本研究呈現了大鼠觸鬚系統在受損的感覺神經功能性回復期間所伴隨的生理變化。; Functional loss of peripheral nerve, such as cutting injury, blunt injury, laceration injury and nerve transplantation, is a common clinical condition. For example, the sensation of the finger will take a long time to recover following a nerve reconstruction surgery for some patients with nerve injury. Therefore, the neuroplasticity mechanisms in the primary somatosensory cortex by a nerve reconstruction surgery were investigated. The whisker-to-barrel model was used to evaluate the change of neuronal tuning following neurotomy and neurorrhaphy surgery, an approach that simulates a clinical nerve reconstruction surgery. We first developed a whisker stimulator that can present precise whisker bending at various directions and speeds. The whiskers were bended by the piezoelectric actuator that has two degrees of freedom, each of which was driven by amplified voltage signals controlled by the software. We designed the whisker holder and completed the software calibration to provide whisker movement with precise amplitude and phase control. Furthermore, we developed a measuring device to monitor the amplitude movement of stimulator in real time. Multi-unit neuronal activities were recorded before and after the neurotomy and neurorrhaphy surgery, and the follow-up period reached 4-6 weeks post-surgery. The results showed that all units lost their sensory responses immediately after the surgery. Surprisingly, the sensory responses could recover as early as 7 days. In the long-term follow-up, one third of the unit regained whisker tuning that was analogous to that before the surgery. Also, their tuning strength to stimulus speed was weakened in the early recovery period and then gradually increased in the late recovery period. Similarly, the onset latency of a cortical response to whisker stimulation also prolonged in the early recovery period and gradually decreased in the late recovery period. In conclusion, the present study reveals the neuronal mechanisms that account for the functional recovery following nerve reconstruction in the whisker-to-barrel model.