利用生物體外的工具來操控生物體內的各式活性，一直都是人們夢寐以求的技術。而在基礎研究中，數個能夠遠程控制細胞活性的技術已被建立且被廣泛的使用。這些技術主要是藉由藥物、光子及磁場，三種物質作為刺激源，雖然它們都有個別的優勢，但在技術的劣勢層面卻也難以平衡。綜合來說，其劣勢包含:刺激的時間點以及位置難以精準掌控；刺激源在生物組織之穿透力低弱，需輔以侵入性手段植入裝置；無法準確聚焦於目標區域且不影響刺激路徑上的組織。與此同時，另一個長期與人類生活有所連結的─超音波，具有以下優點:穿透力強，可將能量傳遞至深層生物組織達到非侵入性的刺激；普遍使用的醫療超音波，能聚焦在毫米等級的範圍內，而不影響傳遞路徑之組織活性。上述優勢都表明了其作為刺激源的安全性。而此篇論文主要是利用了超音波作為外部刺激源，來尋找改善這些劣勢的可能性，並開發出新的超音波遠端遙控生物活性之技術。
第一個開發的技術為結合了聲孔作用以及化學性誘導蛋白聚合系統。首先對小分子藥物進行改造，使其無法穿透細胞膜後，利用聲孔作用，在超音波的刺激下，瞬間引入此改造藥物至目標細胞內，引起蛋白質聚合，控制我們感興趣的生理活性。實驗結果證明了此技術能夠快速且有效的調控細胞膜上特定磷脂質的代謝。第二個技術則是超音波遺傳工具。在一連串的資料蒐集後，我們鎖定了一個可能的超音波感應蛋白，源自哺乳類動物聽覺系統。並進一步比對了不同超音波物種間的遺傳訊息，依此對此蛋白進行改造，替換了特定兩處的氨基酸序列。實驗結果顯示，將此遺傳改造的感應蛋白表現於哺乳類細胞內後，成功的使其對超音波的刺激產生反應，引發鈣離子流入，並能活化大腦深處的神經。另一方面，以上兩種技術所使用的音波刺激皆為低頻率及低聲壓，實驗結果也證明不會影響細胞的生存率。
此論文所開發的兩個技術，成功改善傳統技術的缺點，拓展了超音波技術領域的應用性，達到非侵入性、高精準性的遠程調控深層組織活性之目的，並期望在未來能提供臨床醫療上的應用以及加速基礎研究之發展。

Several external stimulus have been utilized to remotely control a range of cellular events in genetically-modified target cells. However, the conventional methods using stimuli including chemicals, photons, and magnetic fields are limited by different drawbacks: slow pharmacokinetic, invasiveness, or poor spatiotemporal resolution. While the intrinsic properties of stimuli hinder their feasibility in therapeutic applications, ultrasound, a well-developed tool with good penetrability and spatiotemporal resolution, provide a potential solution to address this issue. Therefore, we focused on developing new approaches to precisely manipulate cellular activities in target cells by ultrasound stimulation. The first developed approach is a hybrid system composed of the US-driven sonoporation and a chemically-inducible dimerization tool. Excitation of ultrasound on microbubble-attached cells transiently increase the membrane permeability, resulting in the influx of synthetic dimerizer and manipulate specific cellular activities via CID system. With this approach, we successfully manipulated membrane phospholipid composition in target cells by ultrasound excitation. The second approach we established is a new sonogenetic tool which is achieved by an engineered auditory-sensing protein, prestin. Heterogeneous expression of mouse prestin carrying with two parallel amino acid substitutions, N7T and N308S that frequently appear in echolocating mammals, sensitizes genetically-modified mammalian cells to ultrasound stimulation at 0.5 MHz, leading to the influx of calcium from extracellular space and activate neurons in deep regions of mouse brain. Most importantly, low-frequency, low-pressure, and focused ultrasound are utilized in both approaches without inducing apparent adverse effects to cells. These two ultrasound-based approaches allow us to spatiotemporally manipulate the cellular activities and offer potential strategies for non-invasive therapy in deep tissue.

中文摘要-------i
Abstract-------ii
致謝-------iii
Table of Contents-------v
Abbreviation-------ix
Ch1. Research Synopsis-------1
1.1 The background and aims of this thesis study-------1
1.2 Ultrasound, an ideal stimulus for non-invasive stimulation-------1
1.2.1 Introduction-------1
1.2.2 Biological effects-------2
1.2.3 Overview of biological effect studies-------4
1.3 Basic concept of experimental strategy-------4
Ch2. Ultrasound-Chemical Hybrid Tool-------6
2.1 Introduction-------6
2.1.1 Chemically inducible dimerization system-------6
2.1.2 An extended approach of ultrasound: Sonoporation-------7
2.1.3 The combination of CID system and sonoporation-------8
2.2 Materials and methods-------9
2.2.1 Cell Culture-------9
2.2.2 Plasmid construction-------9
2.2.3 In vitro transfection-------9
2.2.4 Preparation of Folate-conjugated, fluorescent-labeling microbubbles (FMBs)-------10
2.2.5 Setup of FUS sonication-------12
2.2.6 Live cell imaging-------12
2.2.7 Cytotoxicity measurements-------14
2.3 Results-------15
2.3.1 Synthesis of membrane-impermeable dimerizers based on rapamycin for sonoporation-inducible dimerization system-------15
2.3.2 Rapa conjugated with biotin prevent the membrane permeation and remains functional in inducing dimerization-------16
2.3.3 Pulsed FUS at low-frequency efficiently increase membrane permeability by disrupting membrane-bound MBs-------17
2.3.4 Sonoporation rapidly triggers the influx of RPB and subsequently induces dimerization for modulation of phospholipid metabolism-------18
2.3.5 Sonoporation trigger the GA3-dependent dimerization system-------19
2.3.6 SonoCID enables simultaneously triggering of multiple CID systems in various cell-types-------20
Ch3. Identification of Genetically Encoded Ultrasound-Responsive Proteins (URPs) for Non-Invasive Neuromodulation-------22
3.1 Introduction-------22
3.1.1 The challenges of applying ultrasound to non-invasively control neural activity-------22
3.1.2 Overview of chemogenetics, optogenetics and magnetogenetics-------23
3.1.3 Recent development of sonogenetics-------25
3.1.4 Prestin, a candidate of URP-------27
3.2 Materials and methods-------29
3.2.1 Cell Culture-------29
3.2.2 Plasmid construction-------29
3.2.3 In vitro transfection-------30
3.2.4 Preparation of MBs-------31
3.2.5 Preparation of Adeno-associated virus (AAV) and Lentivirus-------32
3.2.6 Acoustic peak negative pressure and US focal zone measurement-------33
3.2.7 In vitro FUS stimulation-------34
3.2.8 Live-cell imaging and data analysis-------35
3.2.9 Immunofluorescence staining-------36
3.2.10 Primary neuronal culture and lentivirus transduction-------37
3.2.11 In vivo gene delivery-------38
3.2.12 In vivo FUS stimulation-------39
3.2.13 Immunohistochemistry staining (IHC) of mouse brain slices and quantification of c-Fos expression-------40
3.2.14 Cell viability test-------41
3.2.15 Measurement of the thermal effect upon ultrasound stimulation-------42
3.3 Results-------43
3.3.1 The setup of FUS stimulation and live-cell imaging for investigating the ultrasound-sensitivity of various Prestin mutants-------43
3.3.2 mPrestin(N7T, N308S) enables HEK 293T cells to sense the ultrasound stimulation-------44
3.3.3 mPrestin(N7T, N308S) is responsive specifically to 0.5 MHz ultrasound-------45
3.3.4 Plasma membrane targeting of prestin is important for its ultrasound sensitivity-------46
3.3.5 Self-association of mPrestin(N7T, N308S) forms puncta and responds to FUS stimulation in an oscillation manner-------47
3.3.6 mPrestin(N7T, N308S) triggers the calcium influx from extracellular space upon FUS stimulation-------49
3.3.7 mPrestin(N7T, N308S) enables selectively non-invasive activation of VTA neurons via FUS stimulation-------51
3.3.8 FUS with low-PRF exclusively activate mPrestin(N7T, N308S)-expressed neurons in target region without taking effect via activation of auditory neurons-------52
Ch4. Discussion-------54
4.1 Summary of this thesis study-------54
4.2 Discussion of the established approaches and findings in this thesis study-------54
Tables, Figures and Movies-------59
Tables-------59
Figures-------62
Movie list-------109
References-------110

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