多巴胺為一種單胺類神經傳導物質，負責調控動物的學習、記憶、認知、行動等功能，一旦多巴胺神經元的失調將會造成嚴重的中樞神經退化疾病，例如: 帕金森氏症。因此我們致力於發展偵測多巴胺水平的探針。先前本實驗室已成功開發偵測細胞內多巴胺水平的探針是以代謝多巴胺的人類單胺氧化酶 B (MAO B)為主體，結合綠色螢光蛋白(GFP)。氧化態MAO B的輔酶FAD能夠吸收488奈米的波段，造成GFP無法被激發產生螢光，我們稱此現象為「遮蔽效應(Shield effect)」。在本研究中我將應用之前的胞內探針之特性，進行細胞膜上的多巴胺探針開發，將MAO B及GFP透過穿膜蛋白表現在細胞表面。於免疫螢光染色結果發現完整的MAO B較難被帶到細胞膜表面，需要再透過修飾來減少MAO B穿膜部分對探針拓樸結構的影響。另外，我亦測試胞內探針在活體果蠅大腦的多巴胺即時動態，在功能性影像的實驗結果發現探針可以即時的偵測到大腦中外加的多巴胺訊號。多巴胺探針的開發可以幫助我們進行神經迴路的分析以及神經退化性疾病的研究，未來也能應用在臨床的診斷。

Dopamine is a neurotransmitter for regulating the behaviors including locomotion, memory, as well as cognition. The homeostasis of dopamine is vital for the nervous system. To explore dopamine circuit, we aim to develop a probe that could visualize the dopamine circuit in the brain. Through modifying a previous reported MMG1 probe from this lab, we are testing a series of plasma membrane probe designed for detecting dopamine release. These probes are modified from MMG1 of which the conjugation of a fluorescent protein, GFP, and an enzyme monoamine oxidase B (MAO B) that can catalyze dopamine oxidation via its co-factor FAD. The redox reaction of FAD could switch GFP fluorescence upon detecting dopamine release from dopaminergic neurons in vivo and has been coined as the shield effect. The early version of the probes was unsuccessful due to the inadequate topology. Here, I made some modifications of these probes with the goal to embed them in the plasma membrane with both MAO B and GFP facing the extracellular space. However, through the analyses of immunostaining results, I found the modified versions were unable to make MAO B facing outside. It seems that MAO B has some intrinsic limitation that blocks its targeting to the plasma membrane. Several prediction tools reveal a potent helical domain at the N terminus of MAO B that may be the sticking point. Despite these negative results in the attempt of making the probe for monitoring DA releasing, I have also validated the cytosolic probe, MMG1, in living brains using the functional imaging procedure. Real-time analyses showed the reversion of GFP signals upon an artificial manipulation of dopamine content in the live fly brains. We wish that with this molecular probe, a systematic study of how brain processes information in dopamine circuits, either in healthy or diseased conditions, could be implemented.

Abstract I
摘要 II
Acknowledgement III
Table of Contents IV
Introduction 1
Materials and Methods 7
Molecular Cloning 7
Cell culture 8
Transfection of HEK293 cell line 8
Immunostaining 9
MAO B activity assay 10
Fly strain for functional image 10
Fly brain dissection and fixation 11
Drug treatment and time series confocal scanning 11
Analysis of the functional image results 11
Results 12
The molecular probes (sp)MS70G and (sp)MS70G11 12
Make MAO B be presented on plasma membrane 14
Test the MAO B activity of spMCD4 on cell surface 15
Utilize GPI-anchored protein way to anchor MAO B on cell surface 16
Dopamine treatment in live fly brain elevate GFP signal of MMG1 17
Discussion 18
Figures 24
References 45

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