by Wai-on Fu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 88-95). / Chapter Chapter I --- Introduction --- p.1 / Chapter I .1 --- Structure and functions of the epididymis --- p.1 / Chapter I.2 --- Cellular mechanisms for electrolyte secretion --- p.3 / Chapter I.3 --- Second-messenger modulation of chloride secretion in epididymis --- p.5 / Chapter I.3.1 --- Cyclic AMP pathway --- p.5 / Chapter I.3.2 --- Ca2+ as second messenger --- p.6 / Chapter I.4 --- Pathophysiology of electrolyte transport in the epididymis --- p.6 / Chapter I.5 --- Objectives of the study --- p.9 / Chapter Chapter II. --- Materials and Methods --- p.10 / Chapter II. 1 --- Materials --- p.10 / Chapter II. 1.1 --- Culture media and enzyme --- p.10 / Chapter II. 1.2. --- Drugs --- p.10 / Chapter II. 1.3 --- Chemicals --- p.11 / Chapter II. 1.4. --- Animals and human tissue --- p.11 / Chapter II.2 --- Preparation of solutions --- p.12 / Chapter II. 3. --- Preparation of cultured cells --- p.12 / Chapter II.3 .1 --- Culture of rat epididymal epithelial cells --- p.12 / Chapter II.3.2 --- Cultured of human epididymal epithelial cells --- p.16 / Chapter II.4. --- Patch-Clamp technique --- p.21 / Chapter II.4.1 --- Electrode --- p.23 / Chapter II.4.2 --- Pulling of electrode --- p.23 / Chapter II.4.3 --- Coating of electrode --- p.23 / Chapter II.4.4 --- Polishing of the electrode --- p.24 / Chapter II.4.5 --- Filling of the electrodes --- p.26 / Chapter II.4.6 --- Mounting of Electrode to the Headstage Pipette Holder --- p.26 / Chapter II.4.7 --- Electrical Isolation --- p.28 / Chapter II.4.8 --- Vibration Isolation --- p.28 / Chapter II.4.9 --- "Formation of ""Giga-seal"" and Whole-cell configuration" --- p.28 / Chapter II.4.10 --- Correction for liquid junction potential --- p.30 / Chapter II.4.11 --- "Data Acquisition and Analyses," --- p.32 / Chapter II.4.12 --- Statistics --- p.34 / Chapter Chapter III. --- Results --- p.35 / Anion Secretion in human epididymal cells --- p.35 / Chapter III. 1 --- Whole-cell current in human epididymal cells --- p.35 / Chapter III. 2 --- Effect of adrenergic receptor blockers on whole-cell current --- p.38 / Chapter III.3 --- Effect of inhibitors of the cyclic AMP pathway --- p.42 / Chapter III.4 --- Effect of altering intracellular calcium concentration --- p.42 / Anion secretion in rat epididymal cells --- p.46 / Chapter III. 5 --- Effect of cAMP on whole cell C1- current in rat epididymis --- p.46 / Chapter III.6 --- Effect of ionomycin on whole cell C1- current --- p.53 / Chapter III.7 --- Differences between cAMP- and ionophore- dependent C1- current --- p.57 / Chapter III. 8 --- The swelling-induced whole-cell currents --- p.63 / Chapter III.9 --- The swelling-induced current was mainly C1- selective --- p.65 / Chapter III. 10 --- Anion selectivity of the swelling-induced C1- current --- p.68 / Chapter III. 11 --- Inhibition of the swelling-induced C1- conductance by anion channel blockers --- p.68 / Chapter III. 12 --- The role of Ca2+ and cAMP in swelling-induced C1- conductance --- p.74 / Chapter Chapter IV. --- Discussion --- p.79 / Signal transduction mechanism of adrenaline stimulated C1- current in human epididymal cell --- p.79 / Anion secretion in rat epididymal cell The role of Ca2+ and cAMP --- p.83 / Chapter Chapter V. --- Reference --- p.88

Cell wounding, the loss of plasma membrane integrity, is a common event in the life of many cell types. Most cells are subjected to physiological events during normal functions that can lead to disruption of their plasma membranes, especially cells in the load bearing organs such as muscle, skin and bone. The capacity of the cell to repair day-to-day wear-and-tear injuries, as well as traumatic ones, is fundamental for maintaining tissue integrity. / In this thesis, we were trying to uncover single cell wound responses by applying the femtosecond laser (fs laser) technology. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscopic system. Combining real-time observations of the fs laser-induced wound and 3D reconstruction of the cells, post-damage cell and nucleus morphological transformation and wound dynamics were reported. The major findings of this study include: (1) Fs laser could induce a small hole on the plasma membrane of the targeted cell. With the same laser irradiation time, the initial hole size were positively correlated with the laser power. (2) Four typical hole evolution scenarios were reported. Hole resealing was a fast process mostly within 100 seconds in normal condition. Whether a cell could reseal the hole is dependent on the initial hole size. Cells had difficulty to cope with the bigger holes. Three ranges of hole size were given in the thesis to predict the hole resealing result. (3) After fs laser damage, the whole cell underwent a contraction. The post-damage nucleus area, footprint, and each section layer of the cell all shrank, only the thickness remained the same. The nucleus retreated a bit from the damage site after damage. (4) Oxidative stress altered some of the cellular responses to the laser damage. The fs laser- induced holes in oxidative groups were bigger than the normal condition. The cells underwent an overall swelling after fs laser damage instead of the contraction in the normal group. Section layer areas and the thickness of the cell increased after damage. But similar to the normal condition, nucleus shrinkage and retreat from the damage site were also found in the oxidative stress groups. (5) Although both acute and chronic oxidative stresses compromised the integrity of the plasma membrane, chronic oxidative stress compromised more severely with several critical post-damage cell transformations and low resealing ratio. Acute oxidative stress on the other hand may somehow promote the resealing ability of the cells. (6) The section layers closer to the bottom of the cells transformed less than the layers further away from the bottom. This probably suggested that the cell basal attachment provided a constraint force to the plasma membrane for morphological changes. / 細胞創傷，即細胞膜的完整性受損，是多種細胞生命週期中一種常見的現象。細胞在執行正常功能時可能遭遇不同程度的生理性損傷，其中大部分會導致細胞膜的破壞。這一現象對存在於承受壓力器官中的細胞更為頻繁，例如肌肉，皮膚和骨骼。細胞對於日常磨損性傷害以及意外創傷的修復能力，是維持組織完整性的基石。 / 在本論文中，我們通過使用飛秒鐳射技術模擬單細胞創傷，觀察並試圖揭示單細胞對於創傷的反應過程。在實驗中，參數可調的飛秒鐳射器與共軛聚焦顯微鏡整合為一個系統，用於在單細胞膜上進行定點損傷。我們結合了對損傷的即時觀測，細胞的三維結構重建技術，完整記錄了損傷前後的損傷部位，細胞整體以及細胞核的形態變化。以下是本研究的主要發現：（1）飛秒鐳射能夠在目標細胞的細胞膜上進行局部穿孔。在鐳射照射時間相同的情況下，鐳射穿孔的大小與鐳射的平均功率呈正相關。（2）我們發現了鐳射穿孔後穿孔部位有四種不同的變化情況。穿孔後細胞封孔是一相當快的過程，在細胞成功封孔的情況下，大部分細胞將在100秒以內將穿孔部位重新填滿。細胞是否可以將穿孔封住取決於鐳射照射後初始穿孔的大小。細胞很難修復較大的孔。我們將細胞初始穿孔大小分為三個範圍，根據這三個範圍可以利用初始孔的尺寸大致預測穿孔後細胞的封孔情況。（3）飛秒鐳射損傷細胞後，細胞將會收縮，並且細胞核的平面面積，細胞的平面面積（或稱細胞足跡），以及細胞各分層面積都有不同程度的縮小。僅細胞厚度未發生顯著變化。同時，細胞核的位置相對於損傷部位有所後退。（4）細胞在氧化應激過後，對於飛秒鐳射造成的損傷反應有所變化。具體表現為：鐳射穿孔的尺寸比正常情況下更大；穿孔後細胞將會整體腫脹而非收縮。各分層面積和細胞厚度都有不同程度的增大。但是細胞核的反應與正常情況類似，即細胞核將會收縮，並且後退以遠離鐳射損傷部位。（5）儘管急性氧化應激和慢性氧化應激都一定程度上損傷了細胞膜的完整性，但是從細胞對於鐳射創傷的反應觀察，長期慢性氧化應激對於細胞膜的損害更為嚴重，具體表現為鐳射損傷後細胞的嚴重形變和細胞膜修復比例的降低。而另一方面，急性氧化應激在某種程度上可以增強細胞對於鐳射穿孔的修復能力。（6）細胞膜穿孔後，細胞各層的面積變化不一，位置越靠近底層的分層面積變化越小。這可能表明細胞貼壁行為形成了一個對細胞形變的約束力。 / Duan, Xinxing. / Thesis M.Phil. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 73-85). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.