No description available.

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has demonstrated that the regulatory domain of CaN (RD) is disordered. The binding of CaM to CaN results in RD folding. Folding of the RD in turn causes the autoinhibitory domain (AID) located C-terminal to the RD to be ejected from CaN’s active site. This binding-induced disorder-to-order transition is responsible for the activation of CaN by CaM. In this work, we explore the nature of the disorder in the RD and its transition to an ordered state, demonstrating that the RD exists in a compact disordered state that undergoes further compaction upon CaM binding. We also demonstrate that a single CaM molecule is responsible for binding to and activating CaN. Finally, we determine that the CaM binding to CaN induces an amphipathic helix (the distal helix) C-terminal to the CaM binding region. The distal helix undergoes a hairpin-like chain reversal in order to interact with the surface of CaM, resulting in the removal of the AID from CaN’s active site. We employ site-directed mutagenesis, size-exclusion chromatography, protein crystallography, circular dichroism spectroscopy, fluorescence anisotropy and correlation spectroscopy, and phosphatase activity assays to investigate the ordering of CaN’s regulatory domain, the stoichiometry of CaN:CaM binding, and the impact of the distal helix on CaM activation of CaN.

The spatio-temporal attributes of intracellular calcium (Ca2+) transients activate various biological functions. These Ca2+ signaling events are triggered extracellularly through different stimuli and controlled intracellularly by the major Ca2+ storage organelle and by numerous Ca2+ pumps, channels, and Ca2+ binding proteins. Ca2+ transients can be significantly altered as a result of defects with signal modulation, leading to different diseases. Because of the fragility and intricacy of the Ca2+ signaling system, with the endo- and sarcoplasmic reticulum at the center, genetically-encoded Ca2+ probes that have been optimized for mammalian expression and fast kinetics are needed to observe global and local Ca2+ changes in different cells. Here, we first report the crystal structure determination of our genetically-encoded Ca2+ sensor CatchER which utilizes EGFP as the scaffold protein. Crystal structures of CatchER were resolved in the Ca2+-free, Ca2+-loaded, and gadolinium-loaded forms at 1.66, 1.20, and 1.78 Å, respectively. Analysis of all three structures established conformational changes in T203 and E222 produce the varying ratios of the neutral and anionic chromophore reflected in the absorbance spectrum where Ca2+ stabilizes the anionic chromophore and enhances the optical output. Since CatchER has miniscule fluorescence when expressed at 37˚C in mammalian cells, we enhanced its brightness by improving the folding at 37˚C, facilitating better chromophore formation. The resulting mutants are the CatchER-T series of Ca2+ sensors with CatchER-T’ having the most improvement in brightness at 37˚C. We also introduced the N149E mutation in the binding site to alter the Kd along with the brightness mutations. The resulting mutants were characterized and found to have weaker Kds compared to wild-type CatchER, similar quantum yields, and altered ratios of the neutral and anionic chromophore in the apo form. Then, CatchER-T’ was applied in situ to monitor Ca2+ changes globally in the ER/SR of C2C12, HEK293, and Cos-7 cells. A new construct consisting of CatchER-T’ and JP-45 was created to monitor local Ca2+ dynamics in the SR lumen of skeletal muscle cells. The results showed a difference between global and local SR Ca2+ release. We also examined the potential and spectroscopic properties to utilize some of our sensors in T cells to monitor the magnesium (Mg2+) flux in immune cells with faulty MagT1 receptors to understand the role of Mg2+ in the immune response.

No description available.

Leung, Pan Cheung Catherine. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 105-120). / Abstracts in English and Chinese. / DECLARATION --- p.II / ACKNOWLEDGEMENTS --- p.III / ENGLISH ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VII / Chapter MODULE 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Vascular Endothelium --- p.1 / Chapter 1.1.1. --- Vascular Endothelial Functions --- p.1 / Chapter 1.1.2. --- Calcium Signaling in Vascular Endothelial Cells --- p.2 / Chapter 1.2. --- The Founding Member of TRP Family: Drosophila TRP --- p.3 / Chapter 1.2.1. --- Discovery of Drosophila TRP and TRP-related Proteins --- p.3 / Chapter 1.2.2. --- Drosophila TRPs: Ca2+-permeable Channels? --- p.3 / Chapter 1.3. --- Mammalian TRP Superfamily --- p.5 / Chapter 1.3.1. --- The TRP Subfamily: TRPV --- p.9 / Chapter 1.3.2. --- The TRP Subfamily: TRPM --- p.9 / Chapter 1.3.3. --- The TRP Subfamily: TRPC --- p.11 / Chapter 1.4. --- Functional and Physiological Roles of Mammalian TRPCs --- p.12 / Chapter 1.4.1. --- TRPC1 --- p.15 / Chapter 1.4.2. --- TRPC2 --- p.16 / Chapter 1.4.3. --- "TRPC3, TRPC6 and TRPC7" --- p.17 / Chapter 1.4.4. --- TRPC4 and TRPC5 --- p.19 / Chapter 1.4.5. --- Over-expression of TRPCs: Physiologically Relevant Channels? --- p.20 / Chapter 1.4.6. --- Alternatives to Heterologous Expression Study --- p.21 / Chapter 1.5. --- Aims of the Study --- p.23 / Chapter MODULE 2. --- MATERIALS AND METHODS --- p.24 / Chapter 2.1. --- Functional Characterization of TRPCs by Antisense Technique --- p.24 / Chapter 2.1.1. --- Restriction Enzyme Digestion --- p.26 / Chapter 2.1.2. --- Purification of Released Inserts and Cut pcDNA3 Vectors --- p.27 / Chapter 2.1.3. --- "Ligation of TRPC Genes into Mammalian Vector, pcDNA3" --- p.27 / Chapter 2.1.4. --- Transformation for the Desired Clones --- p.28 / Chapter 2.1.5. --- Plasmid DNA Preparation for Transfection --- p.28 / Chapter 2.1.6. --- Confirmation of the Clones］ --- p.29 / Chapter 2.1.6.1. --- Restriction Enzymes Strategy --- p.29 / Chapter 2.1.6.2. --- Polymerase Chain Reaction (PRC) Check --- p.30 / Chapter 2.1.6.3. --- Automated Sequencing --- p.31 / Chapter 2.2. --- Establishing Stable Cell Lines --- p.33 / Chapter 2.2.1. --- Cell Culture --- p.33 / Chapter 2.2.2. --- Transfection Conditions Optimization --- p.33 / Chapter 2.2.3. --- Geneticin Selection --- p.35 / Chapter 2.3. --- Expression Pattern Studies of TRPC Genes in Vascular Tissues --- p.38 / Chapter 2.3.1. --- Immunofluorescence Staining in Cultured CPAE Cells --- p.38 / Chapter 2.3.2. --- Immunolocalization in Human Cerebral and Coronary Arteries --- p.40 / Chapter 2.3.2.1. --- Paraffin Section Preparation --- p.40 / Chapter 2.3.2.2. --- "Immunohistochemistry for TRPC1, 3, 4 and 6 Channels" --- p.40 / Chapter 2.3.2.3. --- Subcellular Localization of hTRPC1 and hTRPC3 Channels in Endothelial Cells --- p.42 / Chapter 2.4. --- Study of Bradykinin-induced Ca2+ Entry by Calcium Imaging --- p.47 / Chapter 2.4.1. --- Primary Aortic Endothelial Cell Culture --- p.47 / Chapter 2.4.2. --- Fura-2 Measurement of [Ca2+］］］ --- p.47 / Chapter 2.5. --- Study of Functional Role of TRPC6 in Stably Transfected H5V Cells … --- p.49 / Chapter 2.5.1. --- Protein Sample Preparation --- p.49 / Chapter 2.5.2. --- Western Blot Analysis --- p.50 / Chapter 2.5.3. --- Confocal Microscopy for Bradykinin-induced Calcium Entry --- p.51 / Chapter 2.6. --- Data Analysis --- p.52 / Chapter MODULE 3. --- RESULTS --- p.53 / Chapter 3.1. --- Bradykinin-induced Calcium Entry in Vascular Endothelial Cells --- p.53 / Chapter 3.1.1. --- Bradykinin-induced Calcium Entry --- p.53 / Chapter 3.1.2. --- Effects of cGMP and PKG on Bradykinin-induced Ca2+ Entry --- p.54 / Chapter 3.1.3. --- Effects of HOEUO on Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.1.4. --- Involvement of Phospholipase C Pathway in Bradykinin-induced Store-independent Ca2+ Entry --- p.55 / Chapter 3.2. --- Expression Pattern of TRPC Channels in Vascular Systems --- p.63 / Chapter 3.2.1. --- Immunolocalization of TRPC Homologues in CPAE Cells --- p.63 / Chapter 3.2.2. --- Immunolocalization of TRPC Homologues in Human Cerebral and Coronary Arteries --- p.66 / Chapter 3.2.3. --- Subcellular Localization of TRPC1 and TRPC3 Fused to Enhanced Green Fluorescence Protein (EGFP) --- p.77 / Chapter 3.3. --- Functional Role of TRPC6 Channels in Bradykinin-induced Calcium Entry --- p.81 / Chapter 3.3.1. --- Effect of Antisense TRPC6 Construct on Protein Expression --- p.81 / Chapter 3.3.2. --- Effect of Antisense TRPC6 on Bradykinin-induced Ca2+ Entry --- p.81 / Chapter 3.3.3. --- Effect of Antisense TRPC6 on Thapsigargin-depleted Ca2+ Stores --- p.82 / Chapter MODULE 4. --- DISCUSSION --- p.89 / Chapter 4.1. --- Characterization of Bradykinin-induced Ca2+ Entry in Endothelial Cells --- p.89 / Chapter 4.2. --- The Expression Pattern of TRPC Isoforms in Vascular Tissues --- p.93 / Chapter 4.3. --- Functional Characterization of TRPC6 Homologues in Bradykinin-induced Ca2+ Entry --- p.98 / Chapter 4.4. --- Perspectives --- p.103 / Chapter 4.5. --- Conclusion --- p.104 / Chapter MODULE 5. --- REFERENCES --- p.105

Kwok, Po Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-130). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Abstract --- p.ii / 中文摘要 --- p.v / Acknowledgements --- p.vii / List of Figures --- p.viii / List of Tables --- p.x / Table of Abbreviations --- p.xii / Contents --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- The Biology of Human Umbilical Cord Perivascular Cells (HUCPVs) and Their Potential Applications in Tissue Regeneration / Chapter 2.1 --- INTRODUCTION --- p.5 / Chapter 2.1.1 --- Stem cells --- p.5 / Chapter 2.1.2.1 --- Embryonic stem cells --- p.6 / Chapter 2.1.2.2 --- iPS cells --- p.7 / Chapter 2.1.2.3 --- Somatic stem cells --- p.8 / Chapter 2.1.3 --- Mesenchymal stem cells --- p.9 / Chapter 2.1.4 --- Pericytes --- p.11 / Chapter 2.1.5 --- CD146 positive MSCs --- p.12 / Chapter 2.1.6 --- Human umbilical cord perivascular cells (HUCPVs) --- p.13 / Chapter 2.1.7 --- The biology of stem cell microenvironment (niche) --- p.14 / Chapter 2.1.8 --- Current applications of HUCPVs --- p.17 / Chapter 2.1.9 --- Regenerative medicine --- p.17 / Chapter 2.1.10 --- Applications of stem cells in bone regeneration --- p.19 / Chapter 2.2 --- MATERIALS AND METHODS --- p.22 / Chapter 2.2.1 --- Cell culture --- p.22 / Chapter 2.2.2 --- Preparation of Human Umbilical Cord Perivascular (HUCPV) cells --- p.22 / Chapter 2.2.2.1 --- Isolation of Human Umbilical Cord Perivascular (HUCPV) cells from human umbilical cord --- p.22 / Chapter 2.2.2.2 --- Purification of HUCPV cells --- p.23 / Chapter 2.2.3 --- Immunocytochemsitry --- p.24 / Chapter 2.2.4 --- Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) --- p.25 / Chapter 2.2.4.1 --- Isolation of total cellular RNA --- p.25 / Chapter 2.2.4.2 --- Complementary DNA (cDNA) synthesis --- p.26 / Chapter 2.2.4.3 --- Polymerase chain reaction (PCR) --- p.26 / Chapter 2.2.5 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.30 / Chapter 2.2.6 --- In vitro differentiation assays --- p.33 / Chapter 2.2.6.1 --- Osteogenic differentiation --- p.33 / Chapter 2.2.6.2 --- Adipogenic differentiation --- p.33 / Chapter 2.2.6.3 --- Chondrogenic differentiation --- p.34 / Chapter 2.2.6.4 --- In vitro chondrogenic differentiation on gelfoam® --- p.34 / Chapter 2.2.7 --- Cytochemistry staining --- p.35 / Chapter 2.2.7.1 --- Alkaline Phosphatase staining --- p.35 / Chapter 2.2.7.2 --- Alizarin Red S staining --- p.35 / Chapter 2.2.7.3 --- Oil Red O staining --- p.36 / Chapter 2.2.7.4 --- Alcian Blue staining --- p.36 / Chapter 2.2.8 --- Scanning electron microscopy (SEM) --- p.37 / Chapter 2.2.9 --- Transmission electron microscopy (TEM) --- p.37 / Chapter 2.2.10 --- Paraffin tissue embedding --- p.38 / Chapter 2.2.10 --- Haematoxylin and Eosin staining --- p.38 / Chapter 2.3 --- RESULTS --- p.40 / Chapter 2.3.1 --- Isolation and purification of HUCPVs --- p.40 / Chapter 2.3.2 --- Osteogenic differentiation of HUCPVs under normoxia --- p.41 / Chapter 2.3.3 --- Osteogenic differentiation of HUCPVs under hypoxia --- p.42 / Chapter 2.3.4 --- Adipogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.5 --- Chondrogenic differentiation of HUCPVs --- p.43 / Chapter 2.3.6 --- Chondrogenic differentiation of HUCPVs on gelfoam® --- p.44 / Chapter 2.4 --- DISCUSSION --- p.59 / Chapter Chapter 3 --- Calcium and Calcium-sensing Receptor (CaSR) in osteogenesis / Chapter 3.1 --- INTRODUCTION --- p.62 / Chapter 3.1.1 --- Metabolism of calcium --- p.62 / Chapter 3.1.2 --- Calcium-sensing receptor --- p.64 / Chapter 3.1.2.1 --- The molecular structure of calcium-sensing Receptor (CaSR) --- p.64 / Chapter 3.1.2.2 --- The expression pattern of calciumsensing receptor (CaSR) --- p.67 / Chapter 3.1.2.3 --- The physiological function of calcium-sensing receptor in different tissues or organs --- p.68 / Chapter 3.1.2.4 --- Regulatory role of calcium-sensing receptor in calcium sensing and homeostasis --- p.71 / Chapter 3.1.2.5 --- The role of calcium-sensing receptor in diseases --- p.72 / Chapter 3.1.2.6 --- Genetic animal models targeting calciumsensing receptor --- p.73 / Chapter 3.1.2.7 --- Calcium-sensing receptor in mesenchymal lineage Differentiation --- p.76 / Chapter 3.1.2.8 --- The role of calcium-sensing receptor in the skeleton --- p.76 / Chapter 3.1.3 --- Calcium-sensing receptor related pathway --- p.78 / Chapter 3.1.3.1 --- Cyclic AMP pathway --- p.78 / Chapter 3.1.3.2 --- Cyclic AMP response element-binding protein (CREB) --- p.80 / Chapter 3.2 --- MATERIALS AND METHODS --- p.83 / Chapter 3.2.1 --- Preparation of primary mouse osteoblasts (MOB) from long bone --- p.83 / Chapter 3.2.2 --- Preparation of primary mouse osteoblasts (CMOB) from calvaria --- p.84 / Chapter 3.2.3 --- Immunocytochemistry --- p.84 / Chapter 3.2.4 --- Osteogenic differentiation --- p.85 / Chapter 3.2.3 --- Quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR) --- p.85 / Chapter 3.2.4 --- Cell proliferation measurement by BrdU ELISA (colorimetric) assay --- p.85 / Chapter 3.2.5 --- Western blotting analysis --- p.86 / Chapter 3.2.5.1 --- Preparation of the protein lysate --- p.86 / Chapter 3.2.5.2 --- Protein quantitation --- p.86 / Chapter 3.2.5.3 --- SDS-PAGE --- p.87 / Chapter 3.2.5.4 --- Protein transfer --- p.87 / Chapter 3.2.5.5 --- Immunodetection --- p.88 / Chapter 3.2.6 --- cAMP EIA assay --- p.89 / Chapter 3.3 --- RESULTS --- p.91 / Chapter 3.3.1 --- "Expression of CD 146 and CaSR in HUCPVs, primary mouse long bone osteoblasts and MC3T3-E1 cell line" --- p.91 / Chapter 3.3.2 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under normoxia --- p.91 / Chapter 3.3.3 --- The effect of calcium treatment on the osteogenic differentiation potential of MC3T3-E1 cells under hypoxia --- p.92 / Chapter 3.3.4 --- The effect of calcium treatment on cell proliferation in primary mouse long bone osteoblasts --- p.93 / Chapter 3.3.5 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse long bone osteoblasts --- p.94 / Chapter 3.3.6 --- The effect of calcium treatment on calcium-sensing receptor expression in HUCPVs --- p.95 / Chapter 3.3.7 --- The effect of calcium treatment on calcium-sensing receptor expression in primary mouse calvarian osteoblasts --- p.96 / Chapter 3.3.8 --- The effect of calcium treatment on cyclic AMP levels in primary mouse long bone osteoblasts --- p.97 / Chapter 3.4 --- DISCUSSION --- p.117 / Chapter Chapter 4 --- General Discussions --- p.121 / References --- p.124 / Appendices --- p.131

Yip Ham. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 132-141). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abbreviations --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Chapter Chapter 1: --- Introduction --- p.5 / Chapter 1.1 --- Calcium Signaling --- p.5 / Chapter 1.1.1 --- Importance of Calcium to Life Forms --- p.5 / Chapter 1.1.2 --- Calcium Channels in Excitable and Non-excitable Cells --- p.6 / Chapter 1.2 --- Vascular Endothelial Cells --- p.8 / Chapter 1.2.1 --- General Functions --- p.8 / Chapter 1.2.2 --- Calcium signaling in Endothelial Cells --- p.9 / Chapter 1.3 --- Capacitative Calcium Entry (CCE) or Store-operated Calcium Entry (SOC) --- p.10 / Chapter 1.3.1 --- Definition --- p.10 / Chapter 1.3.2 --- Endoplasmic Reticulum (ER) as the Main Intracellular Calcium Stores --- p.10 / Chapter 1.3.3 --- Types of Experiments leading to the Identification of SOCs --- p.11 / Chapter 1.3.4 --- Emptying the Internal Calcium Store --- p.11 / Chapter 1.3.4.1 --- Inhibition of Calcium ATPase --- p.11 / Chapter 1.3.4.2 --- IP3 Triggered Release of Calcium --- p.12 / Chapter 1.3.5 --- "Store-operated Calcium Current, Icrac" --- p.15 / Chapter 1.3.6 --- Different Types of SOCs in Animal Cells --- p.16 / Chapter 1.4 --- Transient Receptor Potential (Trp) Gene & Transient Receptor Potential Like (Trpl) Gene in Drosophila --- p.17 / Chapter 1.4.1 --- Discoverery of Trp and Trpl --- p.17 / Chapter 1.4.2 --- Expression Studies of Drosophila Trp and Trpl --- p.19 / Chapter 1.4.2.1 --- Trp and Trpl form Channels but only Trp is Store Operated --- p.19 / Chapter 1.4.2.2 --- Co-expression Studies of Trp and Trpl --- p.20 / Chapter 1.5 --- Molecular Cloning and Expression of Mammalian Trp Homologues --- p.21 / Chapter 1.5.1 --- Seven Human Homologus of Trp were found --- p.21 / Chapter 1.5.2 --- Expression Pattern of mammalian Trp Homologues in Different Tissues --- p.23 / Chapter 1.5.3 --- Expression Studies of Mammalian Trp Homologues Yields Contradictory Results --- p.27 / Chapter 1.5.3.1 --- Trpl --- p.27 / Chapter 1.5.3.2 --- Trp2 --- p.28 / Chapter 1.5.3.3 --- Trp3 --- p.29 / Chapter 1.5.3.4 --- Trp4 --- p.30 / Chapter 1.5.3.5 --- Trp5 --- p.31 / Chapter 1.5.3.6 --- Trp6 --- p.31 / Chapter 1.5.3.7 --- Trp7 --- p.31 / Chapter 1.5.3.8 --- "Activation of Trp3, Trp6 and Trp7 by Diacylglycerol (DAG)" --- p.32 / Chapter 1.5.3.9 --- Functional Consequence after Co-expression of Trp Homologues --- p.34 / Chapter 1.5.3.10 --- Antisense Strategy to Determine the Functional Subunits of Store-operated Channels --- p.35 / Chapter 1.5.3.11 --- Possible Reasons for the Contradictory Results of Trp Homologues When Expressed in a Heterologous System --- p.36 / Chapter 1.6 --- Aims Of Study --- p.37 / Chapter Chapter 2. --- Materials and Methods --- p.38 / Chapter 2.1 --- Cell Culture --- p.38 / Chapter 2.2 --- Total RNA extraction from HCAEC 5286 --- p.39 / Chapter 2.3 --- Reverse Transcription from Cultured Human Coronary Artery Endothelial Cell Line HCAEC 5286 --- p.40 / Chapter 2.4 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.41 / Chapter 2.5 --- Separation and Purification of PCR Products --- p.43 / Chapter 2.5.1 --- Separation --- p.43 / Chapter 2.5.2 --- Purification --- p.43 / Chapter 2.6 --- Confirmation of PCR Products --- p.44 / Chapter 2.7 --- Molecular Cloning of Trp Gene Family --- p.45 / Chapter 2.7.1 --- "Cloning of HTrpl, HTrp3, HTrp4,HTrp5,HTrp6, HTrp7" --- p.45 / Chapter 2.7.1.1 --- Polishing the Purified PCR Products --- p.47 / Chapter 2.7.1.2 --- Determination of the Amount of Polished PCR Products --- p.47 / Chapter 2.7.1.3 --- Inserting the PCR Products into the pPCR-Script Amp SK(+)Cloning Vector (Ligation) --- p.48 / Chapter 2.7.1.4 --- Transformation --- p.48 / Chapter 2.7.1.5 --- Preparing Glycerol Stocks Containing the Bacterial Clones --- p.49 / Chapter 2.7.1.6 --- Plasmid DNA Preparation --- p.49 / Chapter 2.8.1.7 --- Clones Confirmation --- p.50 / Chapter 2.8 --- In situ Hybridization --- p.54 / Chapter 2.8.1 --- Probe Preparation --- p.54 / Chapter 2.8.1.1 --- Trp1 Probe --- p.54 / Chapter 2.8.1.2 --- Trp3 Probe --- p.58 / Chapter 2.8.1.3 --- Trp4 Probe --- p.61 / Chapter 2.8.1.4 --- Trp5 Probe --- p.62 / Chapter 2.8.1.5 --- Trp6 Probe --- p.63 / Chapter 2.8.1.6 --- Trp7 Probe --- p.65 / Chapter 2.8.1.7 --- Control Probe --- p.66 / Chapter 2.8.2 --- Testing of DIG-Labeled RNA Probes --- p.66 / Chapter 2.8.3 --- Paraffin Sections Preparation --- p.67 / Chapter 2.8.4 --- In Situ Hybridization: Pretreatment --- p.67 / Chapter 2.8.5 --- "Pre-hybridization, Hybridization and Post-hybridization" --- p.68 / Chapter 2.8.5.1 --- Pre-Hybridization --- p.68 / Chapter 2.8.5.2 --- Hybridization --- p.68 / Chapter 2.8.5.3 --- Post-Hybridization --- p.69 / Chapter 2.8.6 --- Colorimetric Detection of Human Trps mRNA --- p.69 / Chapter 2.9 --- Northern Hybridization --- p.70 / Chapter 2.9.2 --- Labelling of Riboprobe with 32P --- p.70 / Chapter 2.9.3 --- Prehybridization and Hybridization with Radiolabeled RNA Probes --- p.73 / Chapter Chapter 3. --- Results --- p.74 / Chapter 3.1 --- Polymerase Chain Reaction (PCR) of Partial Trp Gene Fragments --- p.74 / Chapter 3.2.1 --- Expression of TRPs RNA in Human Coronary Artery --- p.78 / Chapter 3.2.1.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.79 / Chapter 3.2.1.2 --- Expression of Trp Transcripts in the Tunica Adventitia --- p.88 / Chapter 3.2.2 --- Expression of TRPs RNA in Human Cerebral Artery --- p.97 / Chapter 3.2.2.1 --- Expression of Trp Transcripts in Tunica Intima and Media --- p.97 / Chapter 3.3 --- Northern Blot Analysis of Human Trp5 RNA in Human Multiple Tissue Blot --- p.115 / Chapter Chapter 4: --- Discussion --- p.117 / Chapter 4.1 --- Co-expression of Trps in Vascular Tissues --- p.117 / Chapter 4.1.1 --- Expression of Trps in Endothelia --- p.117 / Chapter 4.1.2 --- In Smooth Muscle Cells --- p.118 / Chapter 4.2 --- Trp Channel and Store-operated Channel in Endothelial Cells --- p.119 / Chapter 4.3 --- Heteromultimerization of Trps Subtypes --- p.120 / Chapter 4.4 --- Northern Blot Analysis --- p.124 / Chapter 4.5 --- Potential Physiological Functions of Trps --- p.125 / Chapter 4.6 --- Trp Channels as a Therapeutic Target? --- p.128 / Chapter 4.7 --- Technical Aspects in the Present Studies --- p.129 / Chapter 4.8 --- Conclusion --- p.131 / Reference --- p.133

Epithelial to mesenchymal transition (EMT) is an important process in embryonic development, tissue repair, inflammation, and cancer. During EMT, epithelial cells disassemble cell-cell adhesions, lose apicobasal polarity, and initiate migratory and invasive processes that allow individual cells to colonize distant sites. It is the means by which non-invasive tumors progress into malignant, metastatic carcinomas. In vitro, EMT occurs in two steps. First, cells spread out, increasing in surface area and pushing the colony borders out. Then cells contract, pulling away from neighboring cells and rupturing cell-cell junctions, resulting in individual highly migratory cells. Recent discoveries indicate that calcium signaling is central in EMT. Both previous data with patch clamping and new calcium imaging data show a series of calcium influxes in cells induced to undergo EMT with hepatocyte growth factor (HGF). It has also been shown that blocking calcium signaling prevents EMT from progressing normally. However, it is not known if calcium alone is sufficient to drive EMT behaviors. By experimentally triggering calcium influxes with an optigenetic cation channel, the behaviors that calcium influxes induce can be determined noninvasively, without use of drugs that may have secondary effects. The results of using the optigenetic set up along with live cell imaging are that cells become more motile and disrupt normal epithelial cell-cell adhesions. This behavior is believed to be due to increased cell contractility downstream of calcium signaling, and is dependent on Ca2+/calmodulin-dependent protein kinase II (CaMKII). When cells are pre-treated with CaMKII inhibitor before HGF addition, they undergo the spreading step of EMT without subsequent cellular contraction and rupture of cell-cell junctions. CaMKII is a protein kinase that is activated by binding Ca2+/calmodulin, and is a known downstream component of calcium signaling. CaMKII is known to affect the actin cytoskeleton by both physically bundling actin filaments to increase their rigidity, and through signaling by activation of myosin light chain kinase (MLCK), which has a role in stress fiber formation. Immunofluorescence did not show colocalization of CaMKII with actin, ruling out regulation through actin bundling. However, CaMKII does appear to have a role in stress fiber formation. EMT induced with HGF treatment results in increased numbers of stress fibers as well as trans-cellular actin network formation, both actin structures decorated with non-muscle myosin II (NMII). CaMKII inhibition not only blocks these actin formations, but it also decreases stress fiber levels below basal unstimulated levels in cells that have not been treated with HGF. This suggests that CaMKII has a role in regulating contractility through cellular actin networks, indicating a mechanism for calcium's role in cellular contractility in EMT.

Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope. Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP. It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins. Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

GTPases are integral components of virtually every known signal transduction pathway, and mutations in GTPases frequently cause disease. A genomic analysis identified and annotated 174 GTPases in the sea urchin genome (with 90% expressed in the embryo), covering five classes of GTP-binding proteins: the Ras superfamily, the heterotrimeric G proteins, the dynamin superfamily, the SRP/SR GTPases, and the translational GTPases. The sea urchin genome was found to contain large lineage-specific expansions within the Ras superfamily. For the Rho, Rab, Arf and Ras subfamilies, the number of sea urchin genes relative to vertebrate orthologs suggests reduced genomic complexity in the sea urchin. However, gene duplications in the sea urchin increased overall numbers, such that total sea urchin gene numbers of these GTPase families approximate vertebrate gene numbers. This suggests lineage-specific expansions as an important component of genomic evolution in signal transduction. A focused analysis on RhoA, a monomeric GTPase, shows it contributes to multiple signal transduction pathways during sea urchin development. The data reveal that RhoA inhibition in the sea urchin results in a failure to invaginate during gastrulation. Conversely, activated RhoA induces precocious archenteron invagination, complete with the associated actin rearrangements and extracellular matrix secretion. Although RhoA regulates convergent extension movements in vertebrates, our experiments show RhoA activity does not regulate convergent extension in the sea urchin. Instead, the results suggest RhoA serves as a trigger to initiate invagination, and once initiation occurs RhoA activity is no longer involved in subsequent gastrulation movements. RhoA signaling was also observed during endomesodermal specification in the sea urchin. Data show that LvRhoA activity is required, downstream of a partially characterized Early Signal, for SoxB1 clearance from endomesodermal nuclei (and subsequent expression of GataE and Endo16 genes). Investigations also suggest that within the endomesoderm, RhoA clears SoxB1 as part of Wnt8 signaling, as activated RhoA is sufficient to rescue Wnt8-inhibited embryos. These data provide evidence of the first molecular components involved in SoxB1 clearance, as well as highlight a previously unrecognized role for RhoA during endomesodermal specification. These analyses suggest RhoA signaling is integral to the proper specification and morphogenesis of the sea urchin endomesoderm. / dissertation