Modelar a dinâmica de neurônios é relevante em estudos de neurociências. Neste trabalho, propõe-se um modelo computacional de neurônio baseado no comportamento dos canais iônicos presentes na sua membrana. O modelo combina elementos microscópicos, como o comportamento dos canais individuais, com elementos macroscópicos, como a tensão ao longo de um trecho de membrana. Simulações foram realizadas com o objetivo de reproduzir dados biológicos e resultados obtidos de modelos teóricos clássicos da área. Foi possível reproduzir com boa concordância o potencial de ação, o fenômeno da adaptação, a curva da corrente de entrada versus a frequência de disparos e o potencial excitatório pós-sináptico. / Modelling the dynamics of neurons is relevant in studies on neurosciences. In this work, a computational model of neuron based on the behavior of the ionic channels found in its membrane is proposed. The model comprises microscopic elements, as the behavior of the individual channels, and macroscopic elements, as the tension along a membrane patch. Simulations were performed with the aim of reproducing biological data and results derived from classical theoretical models of the field. It was possible to reproduce with good agreement the action potential, the phenomenon of adaptation, the curve of the input current versus the spike frequency, and the excitatory postsynaptic potential.

Fosfolipases A2 miotóxicas (Lys49, enzimaticamente inativa, e Asp49, com atividade) isoladas do veneno de Bothrops asper, induzem hipernocicepção. Assim, avaliamos os mecanismos estruturais, moleculares e mediadores centrais envolvidos neste efeito. A injeção intraplantar das FLA2s acarretou hiperalgesia, enquanto que apenas a FLA2-Asp49 induziu alodinia. A região C-terminal é a responsável pelo efeito da FLA2-Lys49, enquanto que a atividade catalítica da FLA2-Asp49 parece ser responsável pela indução de hipernocicepção. Canais de Ca2+ e Na+ participam deste efeito. Na medula espinhal, receptores NK1 e para CGRP, receptores ionotrópicos para glutamato, NO, IL-1, prostanóides e adenosina participam da hiperalgesia induzida pelas FLA2s. Adicionalmente, receptores metabotrópicos para glutamato e o TNF?, estão envolvidos na hiperalgesia induzida pela FLA2-Asp49. Receptores NK1 e NK2 e para CGRP, receptores para glutamato, TNF? e prostanóides medeiam a alodinia. A ativação de astrócitos e microglia, na medula espinhal, contribui para a gênese do efeito hipernociceptivo. / Phospholipase A2 (Lys49, catalytically-inactive and Asp49, catalytically active), isolated from Bothrops asper snake venom, induce pain. The present studies examined the molecular, structural and central mechanisms involved in hypernociception induced by both PLA2s. These PLA2s induced mechanical hyperalgesia, whereas only PLA2-Lys49 evoked allodynia. The C-terminal region of the PLA2-Lys49 seems to be responsible for hyperalgesia, whereas the enzymatic activity of PLA2-Asp49 contributes to such an effect. Calcium and sodium channels are involved in PLA2s-induced hyperalgesia. In the spinal cord, NK1 and CGRP receptors, glutamate ionotropic receptors, NO, IL-1, prostanoids and adenosine contribute to hyperalgesia caused by PLA2s. Additionally, metabotropic glutamate receptors and TNF are involved in hyperalgesia induced by PLA2-Asp49. NK1, NK2 and CGRP receptors, glutamate receptors, TNF and prostanoids mediate allodynia. Activation of spinal astrocytes and microglia contribute to the generation of hyperalgesia and allodynia induced by both toxins.

活性氧在內皮細胞生理發展比如細胞生長增殖和病理中起到非常重要的作用。在病理條件下，活性氧在血管功能失調和重構起到關鍵作用。氧化應激現在被認為存在於多種形式的心血管疾病中。諸多證據表明著活性氧誘導的心血管系統中很多功能異常之前會伴隨有細胞內鈣離子濃度的上升。 / 在本論文的第一個部分，我比較了活性氧在大血管（主動脈）和小血管（腸系膜動脈）的內皮細胞裡引起的鈣應激的相似和差異之處。在這兩種細胞中，活性氧均可引起細胞內鈣離子濃度的上升。這種鈣離子濃度增加可被磷酸酯酶C (PLC) 的抑製劑U73122或者磷酸肌醇受體 (IP₃R) 抑製劑 （Xestospongin C， XeC）大幅度的減弱。此外，用過氧化氫預處理後的細胞會降低細胞對ATP的鈣應激反應。這種鈣應激反應的抑制可能是由於過氧化氫引發的鈣庫流失。令人關注的是，腸系膜動脈的內皮細胞對過氧化氫的作用更為敏感。次黃嘌呤 (hypoxanthine; HX) 加上黃嘌呤(xanthine; XO) 也能引起這兩種內皮細胞鈣離子濃度的上升，而這種鈣離子的增加源於超氧陰離子而不是氫氧離子。在腸系膜動脈的內皮細胞中，過氧化氫在此事件中起到的作用明顯比在主動脈細胞大。總之，過氧化氫可以引起大血管和小血管的內皮細胞裡磷酸酯酶C-磷酸肌醇受體依賴的鈣應激反應。而這種鈣應激後的鈣庫耗竭會對ATP引起的鈣應激起作用。綜上所述，小血管的內皮細胞的鈣應激比大血管的內皮細胞對過氧化氫更為敏感。 / 基於以上的結果，在第二部分的內容中，我們以培植的微血管內皮細胞系（H5V）為小血管內皮細胞的模型，研究了TRPM2通道在過氧化氫誘導的的鈣應激和凋亡中的作用。TRPM2是表達在動物是血管內皮組織中的氧化敏感的和陽離子無選擇性通道。我們開發了TRPM2通道的抑制性抗體 (TM2E3)，這種抗體可以結合到TRPM2通道的離子孔道的E3區域。對H5V細胞進行TM2E3的預處理後，可以降低細胞對過氧化氫刺激下的鈣離子的增加。用TRPM2特異的短發卡核糖核酸 （shRNA）也有同樣的抑制反應。我們用了3種方法來檢測過氧化氫誘導的細胞凋亡：四甲基偶氮唑盐（MTT）檢測，脫氧核糖核酸凋亡片段的檢測和4,6-联脒-2-苯基吲哚(DAPI) 核染色。基於以上的試驗結果，TM2E3 和TRPM2特異的shRNA都表現出了對過氧化氫引起的細胞凋亡的保護作用。相反，在細胞中過表達TRPM2會導致過氧化氫引起的鈣離子濃度上升的增加和細胞凋亡程度的加重。 這些發現強有力的證明了TRPM2 介導了過氧化氫引起的鈣離子濃度的上升和細胞凋亡。此外，我們還研究了TRPM2激活後的下游事件：半胱氨酸蛋白酶-3，-8和9是否參與到這個過程。我的數據表明過氧化氫誘導細胞凋亡是通過內源和外源通路導致半胱氨酸酶-3激活，而TRPM2在這個過程中起到了重要的決定作用。總括而言，TRPM2 介導了過氧化氫誘導的內皮細胞凋亡，下調內源性的TRPM2的表達會保護血管內皮細胞。 / Reactive Oxygen Species (ROS) play a key role in normal physiological processes such as cell proliferation and growth, as well as in pathological processes. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. Oxidative stress is now thought to underlie many cardiovascular diseases. Accumulating evidence also demonstrate that many ROS-induced functional abnormalities in the cardiovascular system are preceded by an elevation of intracellular Ca²⁺. / In the first part, I compared the Ca²⁺ responses to ROS between mouse endothelial cells derived from large-sized artey aortas (aortic ECs), and small-sized mesenteric arteries (MAECs). Application of hydrogen peroxide (H₂O₂) caused an increase in cytosolic Ca²⁺ levels ([Ca²⁺]i) in both cell types. The [Ca²⁺]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP₃) receptors. In addition, treatment of endothelial cells with H₂O₂ reduced the Ca²⁺ responses to subsequent challenge of ATP. The decreased Ca²⁺ responses to ATP were resulted from a pre-depletion of intracellular Ca²⁺ stores by H₂O₂. Interestingly, we also found that Ca²⁺ store depletion was more sensitive to H₂O₂ treatment in endothelial cells derived from mesenteric arteries than those of derived from aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca²⁺]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H₂O₂ but not by hydroxyl radicals. H₂O₂ made a greater contribution to HX-XO-induced [Ca²⁺]i rises in endothelial cells from mesenteric arteries than those from aortas. In summary, H₂O₂ could induce store Ca²⁺ release via phospholipase C-IP₃ pathway in endothelial cells. Emptying of intracellular Ca²⁺ stores contributed to the reduced Ca²⁺ responses to subsequent ATP challenge. Furthermore, the Ca²⁺ responses in endothelial cells of small-sized arteries were more sensitive to H₂O₂ than those of large-sized arteries. / In the second part, I used murine heart microvessel endothelial cell line H5V as a model of endothelial cells from small-sized arteries to investigate the role of Melastatin-like transient receptor potential channel 2 (TRPM2) channels in H₂O₂-induced Ca²⁺ responses and apoptosis. TRPM2 is an oxidant-sensitive cationic non-selective channel that is expressed in mammalian vascular endothelium. A TRPM2 blocking antibody channel (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the Ca²⁺ responses to H₂O₂. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect in H₂O₂-induced Ca²⁺ responses. H₂O₂-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect on H₂O₂-induced apoptotic cell death. In contrast, overexpression of TRPM2 in H5V cells increased the Ca²⁺ responses to H₂O₂ and aggravated the apoptotic cell death in response to H₂O₂. These findings strongly suggest that the TRPM2 channel mediates Ca²⁺ overload in response to H₂O₂ and contributes to oxidant-induced apoptotic cell death in vascular endothelial cells. I also examined the downstream cascades of TRPM2 activation and explored whether caspase-3, -8 and -9 were involved in this process. My data indicates that H₂O₂-induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways, leading to activation of caspases-3. Furthermore, TRPM2 played an essential role in the process. Together, my data suggest that TRPM2 mediates H₂O₂-induces endothelial cell death and that down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 101-114). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.I / Abstract --- p.II / 論文摘要 --- p.IV / Acknowledgments --- p.VI / Abbreviations and Units --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and Reactive nitrogen species --- p.1 / Chapter 1.1.1 --- What is oxidative stress? --- p.1 / Chapter 1.1.2 --- Types of ROS --- p.2 / Chapter 1.1.2.1 --- Hydroxyl radical (*OH) --- p.2 / Chapter 1.1.2.2 --- Hydrogen peroxide (H₂O₂) --- p.3 / Chapter 1.1.2.3 --- Superoxide (O₂*⁻) --- p.4 / Chapter 1.1.2.4 --- Nitric oxide (NO) --- p.5 / Chapter 1.1.3 --- ROS-producing systems --- p.6 / Chapter 1.1.3.1 --- NAD(P)H oxidase --- p.6 / Chapter 1.1.3.2 --- Xanthine oxidase (XO) --- p.7 / Chapter 1.1.3.3 --- The mitochondrial respiratory chain --- p.8 / Chapter 1.1.3.4 --- Uncoupled endothelial NO synthase --- p.8 / Chapter 1.1.4 --- Antioxidant defense mechanisms in the cardiovascular systems --- p.9 / Chapter 1.1.4.1 --- SOD --- p.9 / Chapter 1.1.4.2 --- Catalase --- p.10 / Chapter 1.1.4.3 --- Glutathione peroxidase (GPx) --- p.10 / Chapter 1.1.4.4 --- Small molecules --- p.11 / Chapter 1.1.5 --- Role of oxidative stress in human diseases --- p.12 / Chapter 1.1.6 --- Endothelium dysfunction in oxidative stress-relating human diseases --- p.12 / Chapter 1.1.7 --- Role of Ca²⁺ in oxidative stress-relating human diseases --- p.14 / Chapter 1.1.8 --- Differential effects of ROS on endothelial calcium signaling --- p.15 / Chapter 1.1.8.1 --- Multiple Oxidative Stress-induced Ca²⁺ signaling pathway --- p.16 / Chapter 1.1.9 --- Effects of ROS on Agonist-induced endothelial calcium signaling --- p.19 / Chapter 1.1.10 --- Role of H₂O₂ as EDHF --- p.20 / Chapter 1.1.11 --- Differential effect of ROS on cells derived from large-sized and small-sized artries --- p.21 / Chapter 1.2 --- The transient receptor potential (TRP) Channels --- p.21 / Chapter 1.2.1 --- TRP Channel structure --- p.22 / Chapter 1.2.2 --- TRP Channel function --- p.23 / Chapter 1.2.3 --- TRPM subfamily --- p.23 / Chapter 1.2.3.1 --- TRPM2 Property and Structure --- p.24 / Chapter 1.2.3.2 --- TRPM2 Expression --- p.25 / Chapter 1.2.3.3 --- TRPM2 Activator --- p.25 / Chapter 1.2.3.4 --- TRPM2 Physiological and pathophysiological function --- p.28 / Chapter Chapter 2 --- Objectives of the Present Study --- p.35 / Chapter Chapter 3 --- Materials and methods --- p.37 / Chapter 3.1 --- Ethics statement --- p.37 / Chapter 3.2 --- Materials --- p.37 / Chapter 3.3 --- Methods --- p.38 / Chapter 3.3.1 --- Cell culture --- p.38 / Chapter 3.3.1.1 --- Primary Cell Culture --- p.38 / Chapter 3.3.1.2 --- H5V endothelial cell line --- p.39 / Chapter 3.3.1.3 --- Human embryonic kidney 293 (HEK293) cells --- p.39 / Chapter 3.3.4. --- TRPM2-specific shRNA, TRPM2 and transfection --- p.39 / Chapter 3.3.5 --- Western blotting --- p.40 / Chapter 3.3.6 --- [Ca²⁺]i Studies --- p.43 / Chapter 3.3.6.1 --- Fluo-4/AM- Measuring intracellular [Ca²⁺]i --- p.43 / Chapter 3.3.6.2 --- Fura-2/AM-Measuring intracellular [Ca²⁺]i --- p.44 / Chapter 3.3.6.3 --- Mag-fluo-4-Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.45 / Chapter 3.3.7 --- IP₃ measurement --- p.45 / Chapter 3.3.8 --- Electrophysiology --- p.46 / Chapter 3.3.9 --- TRPM2 blocking antibody (TM2E3) and Pre-immune IgG Generation --- p.46 / Chapter 3.3.10 --- DNA fragmentation assay --- p.47 / Chapter 3.3.11 --- DAPI Staining --- p.48 / Chapter 3.3.12 --- MTT assay --- p.48 / Chapter 3.3.13 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca²⁺: Comparison of Endothelial Cells from Large-sized and Small-sized Arteries --- p.50 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Primary Cell Culture --- p.52 / Chapter 4.2.2 --- [Ca²⁺]i Measurement --- p.52 / Chapter 4.2.3 --- Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.52 / Chapter 4.2.4 --- IP₃ measurement --- p.53 / Chapter 4.2.5 --- Data Analysis --- p.53 / Chapter 4.3 --- Results --- p.53 / Chapter 4.3.1 --- Both Ca²⁺ entry and store Ca²⁺ release contributed to H₂O₂-induced [Ca²⁺]i rises.. --- p.53 / Chapter 4.3.2 --- H₂O₂ enhanced IP₃ production and store Ca²⁺ release --- p.54 / Chapter 4.3.3 --- H₂O₂ reduced the Ca²⁺ responses to ATP in a H₂O₂ concentration and incubation time dependent manner --- p.54 / Chapter 4.3.4 --- H₂O₂ induced Ca²⁺ store depletion --- p.55 / Chapter 4.3.5 --- Ca²⁺ responses to ATP in the absence of H₂O₂ --- p.56 / Chapter 4.3.6 --- Non-involvement of hydroxyl radical --- p.56 / Chapter 4.3.7 --- HX-XO-induced [Ca²⁺]i rises were caused by superoxide anion and hydrogen peroxide --- p.56 / Chapter 4.4 --- Discussion --- p.68 / Chapter Chapter 5 --- Role of TRPM2 in H₂O₂-induced cell apoptosis in endothelial cells --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.73 / Chapter 5.2.1 --- Cell Culture --- p.74 / Chapter 5.2.2 --- [Ca²⁺]i measurement --- p.74 / Chapter 5.2.3 --- DNA fragmentation assay --- p.74 / Chapter 5.2.4 --- MTT assay --- p.74 / Chapter 5.2.5 --- TRPM2-specific shRNA, TRPM2 and transfection --- p.75 / Chapter 5.2.6 --- Electrophysiology --- p.75 / Chapter 5.2.7 --- Western blotting --- p.75 / Chapter 5.2.8 --- DAPI Staining --- p.76 / Chapter 5.2.9 --- Data analysis --- p.76 / Chapter 5.3 --- Results --- p.76 / Chapter 5.3.1 --- Involvement of TRPM2 channels in H₂O₂-induced Ca²⁺ influx in H5V cells --- p.76 / Chapter 5.3.2 --- Involvement of TRPM2 channels in H₂O₂-elicited whole-cell current change in H5V cells --- p.77 / Chapter 5.3.3 --- Role of TRPM2 channels in H₂O₂-induced apoptotic cell death in H5V cells --- p.78 / Chapter 5.3.4 --- Involvement of caspases in H₂O₂-induced apoptotic cell death --- p.79 / Chapter 5.3.5 --- Involvement of TRPM2 in TNF-α-induced cell death in H5V cells --- p.79 / Chapter 5.3 --- Discussion --- p.90 / Chapter Chapter 6 --- General Conclusions, Disscussion and Future work --- p.94 / Chapter 6.1 --- General Conclusions --- p.94 / Chapter 6.2 --- Discussion --- p.95 / Chapter 6.2.1. --- Comparative study --- p.95 / Chapter 6.2.2. --- IP₃ receptor (IP₃R) --- p.95 / Chapter 6.2.3. --- TM2E3-Specific blocking antibody of TRPM2 --- p.95 / Chapter 6.2.4. --- Pathological effect of H₂O₂ at high concentration --- p.96 / Chapter 6.2.5 --- Non-change on Basal [Ca²⁺]i --- p.97 / Chapter 6.3. --- Future work --- p.98 / References --- p.101

Cheng Chin Hung. / Thesis submitted in: December 2000. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 138-145). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.i / ACKNOWLEDGMENT --- p.iv / ABBREVIATIONS --- p.v / ABSTRACT --- p.vi / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1 --- Endogenous Antisense RNAs --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Class --- p.2 / Chapter 1.3 --- Natural Antisense RNAs in Prokaryotes and Viruses --- p.3 / Chapter 1.4 --- Endogenous Antisense RNAs in Eukaryotes --- p.8 / Chapter 1.4.1 --- Distribution --- p.8 / Chapter 1.4.2 --- Conserved Pattern of Antisense Transcription --- p.10 / Chapter 1.5 --- Potential Functions of Antisense RNAs --- p.10 / Chapter 1.5.1 --- Template for Translation --- p.11 / Chapter 1.5.2 --- Regulation of Sense Gene Expression --- p.12 / Chapter 1.5.2.1 --- Nucleus --- p.13 / Chapter 1.5.2.1.1 --- Transcriptional Regulation --- p.13 / Chapter 1.5.2.1.2 --- Post-transcriptional Nuclear Regulation --- p.14 / Chapter 1.5.2.2 --- Cytoplasm --- p.16 / Chapter 1.5.2.2.1 --- Messenger Stability --- p.16 / Chapter 1.5.2.2.2 --- Translation --- p.17 / Chapter 1.6 --- Possible Mechanism of Antisense-mediated Regulation --- p.18 / Chapter 1.6.1 --- Two Possible Mechanisms --- p.18 / Chapter 1.7 --- Novel Endogenous Antisense RNA Against Cation Channel --- p.23 / Chapter 2 --- CNG1 Cation Channel --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Classification and Distribution of CNG Channels --- p.25 / Chapter 2.3 --- Structure of CNG Channels Gene Gamily --- p.27 / Chapter 2.4 --- Interactions Between CNG Channels and Ca2+ --- p.29 / Chapter 2.5 --- Distribution of CNG Channels in the Central Nervous System --- p.30 / Chapter 2.6 --- CNG Channels Function in CNS --- p.31 / Chapter 3 --- Aim of Study --- p.33 / Chapter Chapter Two: --- Materials and Methods --- p.35 / Chapter 4 --- Materials --- p.35 / Chapter 4.1 --- Library --- p.35 / Chapter 4.2 --- Multiple Tissue Blots --- p.35 / Chapter 4.3 --- Paraffin Sections --- p.35 / Chapter 5 --- Library Screening of Human Brain cDNA Library --- p.37 / Chapter 5.1 --- Amplification of Human Brain cDNA Library Stock --- p.38 / Chapter 5.2 --- Primary Screening --- p.38 / Chapter 5.3 --- Hybridization --- p.39 / Chapter 5.4 --- Secondary Screening --- p.40 / Chapter 5.5 --- Tertiary Screening --- p.40 / Chapter 6 --- Clones confirmation by Manual Sequencing --- p.41 / Chapter 6.1 --- Plasmid DNA Preparation --- p.41 / Chapter 6.2 --- DNA Sequencing --- p.41 / Chapter 6.3 --- Primer Walking Strategy --- p.44 / Chapter 7 --- Probe Preparation for Northern Blot and In-Situ Hybridization --- p.45 / Chapter 7.1 --- Probe for Anti-CNGl --- p.45 / Chapter 7.1.1 --- Enzyme Digestion --- p.45 / Chapter 7.1.2 --- Self-ligation --- p.47 / Chapter 7.1.3 --- Transformation --- p.47 / Chapter 7.1.4 --- Insert Confirmation --- p.48 / Chapter 7.1.5 --- Second Round Modification of cDNA Clone --- p.48 / Chapter 7.2 --- Probe for Sense CNG1 Gene --- p.49 / Chapter 7.2.1 --- RT-PCR Amplification from Cultured human Epithelial Cell Line ECV304 --- p.49 / Chapter 7.2.2 --- Automatic Sequencing --- p.49 / Chapter 7.2.3 --- Cloning of PCR Product --- p.50 / Chapter 7.2.4 --- Transformation --- p.50 / Chapter 7.2.5 --- Clone Confirmation --- p.50 / Chapter 8 --- Northern Hybridization --- p.51 / Chapter 8.1 --- Probe Linealization --- p.51 / Chapter 8.2 --- Labeling of Riboprobe with Radioisotope 32P --- p.53 / Chapter 8.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.54 / Chapter 9 --- In Situ Hybridization --- p.56 / Chapter 9.1 --- Preparation of Anti-CNGl Probe --- p.56 / Chapter 9.2 --- Preparation of Sense CNG1 Probe --- p.59 / Chapter 9.3 --- Testing of DIG-labeled RNA Probe --- p.61 / Chapter 9.4 --- Pre treatment --- p.61 / Chapter 9.5 --- "Prehybridization, Hybridization and Posthybridization" --- p.62 / Chapter 9.6 --- Colorimetric Detection of DIG Label --- p.63 / Chapter Chapter Three: --- Results --- p.64 / Chapter 10 --- Isolation and Sequence Analysis of cDNA Clones --- p.64 / Chapter 11 --- Northern Blot Analysis of anti-CNGl RNA in Human Brain Multiple Tissues --- p.72 / Chapter 11.1 --- Human Brain Blot IV --- p.72 / Chapter 11.2 --- Human Brain Blot II --- p.75 / Chapter 11.3 --- Human Multiple Tissues Blot --- p.77 / Chapter 12 --- In Situ Hybridization Analysis of anti-CNGl RNA Expression in Human Embryonic and Adult Brain Regions --- p.80 / Chapter 12.1 --- Expression of Anti-CNGl RNA in Human Embryonic Brain Regions… --- p.80 / Chapter 12.1.1 --- Hippocampus --- p.80 / Chapter 12.1.2 --- Frontal Cortex --- p.84 / Chapter 12.1.3 --- Visual Cortex --- p.88 / Chapter 12.2 --- Expression of Anti-CNGl RNA in Human Adult Brain Regions --- p.91 / Chapter 12.2.1 --- Occipital Cortex --- p.91 / Chapter 12.2.2 --- Frontal Cortex --- p.95 / Chapter 12.2.3 --- Hippocampus --- p.99 / Chapter 13 --- Expression of Sense CNG1 mRNA in Human Embryonic and Adult Brain Regions --- p.102 / Chapter 13.1 --- Expression of Sense CNG1 mRNA in Human Embryonic Brain Regions --- p.102 / Chapter 13.1.1 --- Frontal Cortex --- p.102 / Chapter 13.1.2 --- Visual Cortex --- p.107 / Chapter 13.1.3 --- Parahippocampus --- p.111 / Chapter 13.2 --- Expression of CNG1 mRNA in Human Adult Brain Region --- p.113 / Chapter 13.2.1 --- Frontal Cortex --- p.113 / Chapter Chapter Four: --- Discussion --- p.117 / Chapter 14.1 --- Cloning of Endogenous Anti-CNGl Transcript --- p.117 / Chapter 14.2 --- Neuron-specific Coexpression of Anti-CNGl and CNG1 Transcriptsin Central Nervous System --- p.124 / Chapter 15 --- Implications --- p.128 / Chapter 15.1 --- Endogenous Anti-CNGl Down-regulate Expression of CNG1 Channel --- p.128 / Chapter 15.2 --- Coordinated Co-expression of Sense and Antisense CNG1 Transcripts --- p.129 / Chapter 15.3 --- CNG1 Channel Functions in Human Nervous System --- p.130 / Chapter 15.3.1 --- CNG1 Channel Provides a Novel Ca2+ Entry Mode --- p.130 / Chapter 15.3.2 --- Activation of CNG1 Channel Through G-protein-linked Receptors --- p.130 / Chapter 15.3.3 --- Activation of CNG1 Channel Through Nitric Oxide --- p.131 / Chapter 15.3.4 --- Synaptic Plasticity and CNG1 channel --- p.131 / Chapter 15.3.5 --- A Role of CNG1 Channel in Development --- p.135 / Chapter 16 --- Conclusion --- p.136 / Chapter 17 --- Future Studies --- p.137 / References --- p.138

Tsang Lai-Ling Angel. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 136-145). / Abstracts in English and Chinese. / Abstract --- p.i / 論文撮要 --- p.iv / Acknowledgment --- p.vi / Table of Content --- p.vii / List of Publications --- p.xii / List of Figures --- p.xiv / List of Tables --- p.xvii / Abbreviations --- p.xviii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- The Human Uterus Vs Rat Uterus --- p.1 / Chapter 1.1.1 --- Myometrium --- p.1 / Chapter 1.1.2 --- Endometrium --- p.1 / Chapter 1.2 --- The Human Endometrium Vs Rat Endometrium --- p.2 / Chapter 1.2.1 --- The structure of Human Endometrium --- p.2 / Chapter 1.2.2 --- Cyclic Changes in the Endometrium --- p.4 / Chapter 1.2.3 --- Physiological Roles of the Endometrium --- p.7 / Chapter 1.2.4 --- Uterine Fluid Volume and its Composition --- p.7 / Chapter 1.2.4.1 --- Regulation of Uterine Fluid Volume and Composition --- p.7 / Chapter 1.2.4.2 --- Role of Endometrial Epithelium in the Regulation of Uterine Fluid Volume --- p.9 / Chapter 1.3 --- Epithelial Ion Channels --- p.9 / Chapter 1.3.1 --- Epithelial CI- Channels in Secretory Epithelia --- p.11 / Chapter 1.3.1.1 --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.13 / Chapter 1.3.2 --- Epithelial Na+ Channel (ENaC) in Absorbing Epithelia --- p.18 / Chapter 1.3.3 --- ENaC and CFTR in Endometrial Epithelia --- p.26 / Chapter 1.4 --- Hormonal Regulation of the Endometrial Epithelium --- p.29 / Chapter 1.4.1 --- Estrogen and Progesterone --- p.29 / Chapter 1.4.2 --- Aldosterone --- p.32 / Chapter 1.5 --- Aim of Study --- p.35 / Chapter Chapter2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Culture Medium and Enzymes --- p.38 / Chapter 2.1.2 --- Drugs --- p.38 / Chapter 2.1.3 --- Molecular Biology --- p.39 / Chapter 2.1.4 --- Experimental Tissues and Animals --- p.39 / Chapter 2.2 --- Preparations --- p.39 / Chapter 2.2.1 --- Pervious Support for Cell Growth --- p.39 / Chapter 2.2.2 --- Growth Medium --- p.40 / Chapter 2.2.3 --- Culture of Mouse Endometrium Epithelial Cells --- p.43 / Chapter 2.2.4 --- Solutions for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.5 --- Electrodes for the Short-Circuit Current Measurement --- p.44 / Chapter 2.2.6 --- Solutions for Molecular Biology Experiment --- p.44 / Chapter 2.2.6.1 --- Diethyl Pyrocarbonate (DEPC)-treated Water --- p.44 / Chapter 2.2.6.2 --- lx TAE (DNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.3 --- 5x MOPS (RNA gel electrophoresis and its running buffer) --- p.45 / Chapter 2.2.6.4 --- Formaldehyde Gel-loading Buffer --- p.45 / Chapter 2.3 --- Protocols --- p.46 / Chapter 2.3.1 --- Effect of Ovarian Hormones and Aldosterone on CFTR and ENaC Expression --- p.45 / Chapter 2.3.2 --- Possible Interaction between CFTR and ENaC upon Hormones Stimulation --- p.47 / Chapter 2.4 --- Methods of Measurement --- p.48 / Chapter 2.4.1 --- The Short-Circuit Current Technique --- p.48 / Chapter 2.4.1.1 --- The Short-Circuit Current Setup --- p.48 / Chapter 2.4.1.2 --- Experimental Procedures --- p.52 / Chapter 2.4.1.3 --- Data Analysis --- p.55 / Chapter 2.4.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.55 / Chapter 2.4.2.1 --- RNA Isolation --- p.55 / Chapter 2.4.2.2 --- RNA Gel Electrophoresis --- p.56 / Chapter 2.4.2.3 --- Reverse Transcription (RT) --- p.57 / Chapter 2.4.2.4 --- Primer used for the Polymerase Chain Reaction (PCR) --- p.58 / Chapter 2.4.2.5 --- General Procedure of PCR and Competitive RT-PCR --- p.59 / Chapter 2.4.2.6 --- DNA Gel Electrophoresis --- p.61 / Chapter 2.4.3 --- Capillary Electrophoresis - Laser Induced Fluorescence (CE-LIF) --- p.62 / Chapter 2.4.3.1 --- Capillary Tube --- p.54 / Chapter 2.4.3.2 --- Detection System --- p.65 / Chapter 2.4.3.3 --- Experimental Procedures --- p.65 / Chapter 2.4.3.4 --- Data Analysis --- p.66 / Chapter 2.4.4 --- Statistical Analysis / Chapter Chapter3 --- Results --- p.68 / Chapter 3.1 --- Influence of Ovarian Hormones on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Epithelial Na+ Channel (ENaC) Expression in Mouse Endometrial Epithelium --- p.68 / Chapter 3.2 --- Culture Condition on Expression and Function of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Mouse Endometrial Epithelial Cells --- p.92 / Chapter 3.3 --- Expression Regulation of Endometrial Epithelial Na+ Channel (ENaC) Subunits and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Na+ Diet During the Estrus Cycle in Mice --- p.98 / Chapter 3.4 --- Enhanced Epithelial Na+ Channel (ENaC) Activity in Mouse Endometrial Epithelium by Upregulation of γ-ENaC Subunit --- p.114 / Chapter Chapter4 --- General Discussion --- p.127 / Appendix --- p.132 / Chapter A. --- RNA Isolation --- p.132 / Chapter B. --- Reverse Transcription (RT) --- p.133 / Chapter C. --- Polymerase Chain Reaction (PCR) --- p.134 / Chapter D. --- Sequences and Conditions of All Primers --- p.135 / References --- p.136

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

Modelar a dinâmica de neurônios é relevante em estudos de neurociências. Neste trabalho, propõe-se um modelo computacional de neurônio baseado no comportamento dos canais iônicos presentes na sua membrana. O modelo combina elementos microscópicos, como o comportamento dos canais individuais, com elementos macroscópicos, como a tensão ao longo de um trecho de membrana. Simulações foram realizadas com o objetivo de reproduzir dados biológicos e resultados obtidos de modelos teóricos clássicos da área. Foi possível reproduzir com boa concordância o potencial de ação, o fenômeno da adaptação, a curva da corrente de entrada versus a frequência de disparos e o potencial excitatório pós-sináptico. / Modelling the dynamics of neurons is relevant in studies on neurosciences. In this work, a computational model of neuron based on the behavior of the ionic channels found in its membrane is proposed. The model comprises microscopic elements, as the behavior of the individual channels, and macroscopic elements, as the tension along a membrane patch. Simulations were performed with the aim of reproducing biological data and results derived from classical theoretical models of the field. It was possible to reproduce with good agreement the action potential, the phenomenon of adaptation, the curve of the input current versus the spike frequency, and the excitatory postsynaptic potential.

Cardiomyopathies and arrhythmias are major causes of death in untreated hereditary haemochromatosis, acute iron poisoning and during secondary iron overload resulting from repeated blood transfusions in β-thalassaemia. Iron overload cardiomyopathies are associated with systolic and diastolic dysfunction, suggesting that Ca2+ homeostasis is impaired. However, the cellular mechanisms of these dysfunctions are unknown. The data presented in this thesis establishes for the first time iron effects on cardiomyocyte Ca2+ handling, as well as the potential cellular substrates responsible for this impairment during iron overload. Exposure of isolated rat ventricular cardiomyocytes to 200μM iron led to biphasic changes in systolic Ca2+ release. Phase 1: an initial reduction of systolic Ca2+ release followed by; Phase 2: increased Ca2+ release with arrhythmogenic spontaneous Ca2+ release, cell contracture and cell death. There is evidence that Fe2+ enters cardiomyocytes via L-type Ca2+ channels (LTCC) and reduces the Ca2+ trigger. The close apposition of LTCCs to cardiac ryanodine receptors (RyR2) suggests RyR2 may be a first target. Indeed RyR2 activity was drastically reduced on exposure to nanomolar [Fe2+] in single channel studies. Together with evidence that Fe2+ may reduce the Ca2+ trigger from LTCC, this is consistent with iron reducing sarcoplasmic reticulum (SR) Ca2+ release during Phase 1. In Phase 2, the presence of spontaneous Ca2+ release events is consistent with SR Ca2+ overload. Indeed, in single rat ventricular cardiomyocytes SR Ca2+ content was found to be increased by 27% during Phase 2. The cellular substrates responsible for this increased SR Ca2+ content were 2-fold: 1) through reduced extrusion via both the Na+ Ca2+ Exchanger (NCX) and Plasmalemmal Ca2+ ATPase (PMCA) and 2) through increased resequestration via the SR Ca2+ ATPase. Iron catalyses the production of reactive oxygen species (ROS) during the Fenton reaction. To investigate whether iron effects might be due to ROS, I used the cell permeant ROS scavenger Tempol. Tempol attenuated Phase 2 effects but Phase 1 effects were not affected. This is consistent with the hypothesis that Phase 1 effects were due to direct effects of Fe2+ affecting LTCC trigger and RyR2 function. The attenuation of Phase 2 effects suggests that ROS damage to key Ca2+ handling mechanisms, such as NCX and PMCA might account for a reduced Ca2+ extrusion and subsequent SR Ca2+ overload.

abstract: The physiological phenomenon of sensing temperature is detected by transient receptor (TRP) ion channels, which are pore forming proteins that reside in the membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8 respectively, can be modulated by diverse stimuli and are finely tuned by proteins and lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold. In this dissertation, the first direct measurements between PIRT and TRPM8 are quantified with nuclear magnetic resonance and microscale thermophoresis. Using Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel small molecular binders for PIRT as well a protein named calmodulin. The ligand screening results implicate PIRT in diverse physiological functions. Additionally, sparse NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT structure predictions. Finally, the mechanism of thermosensing from the evolutionarily conserved sensing domain of TRPV1 was investigated using NMR. The body of work presented herein advances the understanding of thermosensing and TRP channel function with TRP channel regulatory implications for PIRT. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

abstract: All organisms need to be able to sense and respond to their environment. Much of this process takes place via proteins embedded in the cell membrane, the border between a living thing and the external world. Transient receptor potential (TRP) ion channels are a superfamily of membrane proteins that play diverse roles in physiology. Among the 27 TRP channels found in humans and other animals, TRP melastatin 8 (TRPM8) and TRP vanilloid 1 (TRPV1) are the primary sensors of cold and hot temperatures, respectively. They underlie the molecular basis of somatic temperature sensation, but beyond this are also known to be involved in body temperature and weight regulation, inflammation, migraine, nociception, and some types of cancer. Because of their broad physiological roles, these channels are an attractive target for potential therapeutic interventions. This dissertation presents experimental studies to elucidate the mechanisms underlying TRPM8 and TRPV1 function and regulation. Electrophysiology experiments show that modulation of TRPM8 activity by phosphoinositide interacting regulator of TRP (PIRT), a small membrane protein, is species dependent; human PIRT attenuates TRPM8 activity, whereas mouse PIRT potentiates the channel. Direct binding experiments and chimeric mouse-human TRPM8 channels reveal that this regulation takes place via the transmembrane domain of the channel. Ligand activation of TRPM8 is also investigated. A mutation in the linker between the S4 and S5 helices is found to generally decrease TRPM8 currents, and to specifically abrogate functional response to the potent agonist icilin without affecting icilin binding. The heat activation thermodynamics of TRPV1 are also probed using temperature-controlled electrophysiology. The magnitude of the gating enthalpy of human TRPV1 is found to be similar to other species reported in the literature. Human TRPV1 also features an apparent heat inactivation process that results in reduced heat sensitivity after exposure to elevated temperatures. The work presented in this dissertation sheds light on the varied mechanisms of thermosensitive TRP channel function and regulation. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019