計劃一
神經系統之正常運作對於人體機能的維持至關重要，可惜找尋有效且非侵入式的神經細胞刺激方式，進而治療不同神經疾病卻是人們長久努力但仍未能達成的目標。在本實驗室曩昔之研究中，已發現對Human 293T cells 給予特定超音波刺激後，可促進細胞產生細胞質鈣離子濃度瞬間提升的鈣離子反應。故於本實驗中，我們改為針對藉RA 分化而成的神經細胞進行研究，在給予同樣的超音波刺激後，藉由calcium sensor (鈣傳感器)的指示，我們發現確實可誘使某些細胞產生鈣離子反應；此外，我們也藉由fluorescent voltage sensor (螢光蛋白電壓傳感器)的指示，觀察對細胞施予超音波刺激後，能否直接促使其細胞膜電位發生改變。待未來進行更大量實驗後，應能更加確認可否憑藉非侵入式之超音波刺激來增進神經細胞的神經活性，最終達成操縱神經細胞之目的。

計劃二
微管蛋白所形成的微管結構參與了眾多細胞之生理作用，而多數微管皆具有不同的轉譯後修飾，這些微管蛋白轉譯後修飾之組成變化及分佈時常對於細胞其生理機制的運作扮演重要角色，細胞進行有絲分裂時，一部份的紡錘體微管會成綑聚攏，並於有絲分裂末期形成midbody 的結構。然，此由許多具轉譯後修飾之微管所形成的結構對於細胞有絲分裂運作所扮演之角色仍有眾多未知之處。為此，我們藉由添加 RO-3306 獲得眾多具有 midbody 的細胞，並期望透過Chemically inducible dimerization 的技術，誘導一個改造過的去谷氨酰化酵素 CCP5 快速地移動到 midbody 之處，使其消除構成midbody 微管其上的谷氨酰化轉譯後修飾，以了解微管蛋白的谷氨酰化修飾對於細胞有絲分裂運作的影響。

Project 1
Working normally of neural system is really important for maintain human physiological function. Unfortunately, it’s still don’t have non-invasive and effective methods to stimulate neuron, making the development of various neurological diseases’ become still unable to reach. In previous studies of other members in our laboratory, they could induce transient calcium response which elevate cytosolic Ca2+ concentration with ultrasound stimulation. In this study, we give RA-differentiated SH-SY5Y cells ultrasound stimulation. With instructions of calcium sensor, we find ultrasound stimulation can indeed induce parts of cells to create calcium response. Besides, we also observe whether could directly change neurons’ cell membrane potential with instructions of fluorescent voltage sensor. When carry out more experiments in the future, we could possibly know more about whether could induce neural activity with non-invasive ultrasound stimulation. And even more, reach the target of successfully manipulate neuronal activity.

Project 2
Microtubules (MTs) are composed of tubulin, and participate lots of cellular physiological effect. Many MTs have various posttranslational modifications (PTMs), those PTMs’ composition and distributions always be the crucial role for regulation of cellular physiological function. During cell mitosis, parts of spindle MTs bundle gradually, and form PTMs-rich structure, called “midbody” at mitosis late telophase. It’s still have many mystery for the role of midbody among cell mitosis. To explore this unknown territory, we try to get more midbody-expressed cells by adding additional reagent RO-3306. Here we hope to use a method termed STRIP (SpatioTemporally Rewriting Intraciliary PTMs) to induce modified carboxypeptidase (CCP) rapidly recruited at midbody, and remove midbody-MTs’ polyglutamylation. Finally, letting us know more about the effect of MTs’ polyglutamylation during cell mitosis.

致謝………………………………………………………………….................................…….I
CONTENTS……………………………………………………….................................…III

Project 1 Establishing a platform for monitoring membrane potential………………………………….....................................………….1
Abstract…………………………………………………………..............................……….2
中文摘要…………………………………………………………................................………3
Chapter 1 Introduction………………………………………….......................………4
1.1 Manipulating neurons with ultrasound stimulation……………....…….4
1.2 Inducing calcium response with ultrasound stimulation………....5
1.3 Sensor of action potentials………………………................……………………6
Chapter 2 Material and Methods………………………………..................……..9
2.1 Cell culture and transfection……………………….................……………9
2.2 Live cell-imaging……………………………………………….....................……..9
2.3 Immunofluorescence staining…………………………...….............………..10
2.4 List of chemical reagents………………………………................…………….11
Chapter 3 Results………………………………………..….……......................…….12
3.1 Retinoic acid-differentiated SH-SY5Y cells…………........……………12
3.2 Ionomycin can elevate the membrane potential of RA-differentiated SH-SY5Y cells which detect by ASAP2f………………..…………14
Chapter 4 Summary and Conclusion…………………………..................…….16

Project 2 Establishing a platform for recruiting proteins onto intercellular bridges…………………….......................……..……18
Abstract………………………………………….……………..............................…..19
中文摘要……………………………………………………….…...............................….20
Chapter 1 Introduction……………………………………………....................…...21
1.1 Overview of mitotic midbody……………………….................………………21
1.2 Overview of microtubular posttranslational modifications…….23
1.3 The role of midbody polyglutamylation in cytokinesis………...…24
Chapter 2 Material and Methods……………………………..…................…….27
2.1 Cell culture and transfection………………………………..............…………27
2.2 Generation of NIH3T3 cells stably expressing Cerulean3-FRB-MAP4m, CCP5CD-NF/CeR-MAP4m, or CCP5CDDM-NF/CeR-MAP4m……………………...28
2.3 Live cell-imaging……………………………………………......................………28
2.4 Immunofluorescence staining…………………………................…………….29
2.5 List of chemical reagents…………………………………………….................30
Chapter 3 Results………………………………………………….........................……31
3.1 Preparation of midbodys’ experiment system………………..…......……31
3.2 RO-3306 significantly increases the number of midbodys………..33
3.3 Both CCP5CD-NF and CeR-MAP4m have great expression in the stable cell line………………………………………………........................………..34
Chapter 4 Summary and Conclusion……………………………….................….37

REFERENCE…………………………………………………...…............................…..39

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