Molecular insights into the mechanisms of transport and energy coupling in membrane transport proteins

Membrane transport proteins are the main gatekeepers controlling the traffic of molecules in and out the cell. The mechanism by which they mediate selective and regulated transport across the membrane is of broad physiological and biophysical relevance. In this dissertation, several critical aspects of the transport process have been studied through molecular dynamics (MD) simulations, including ion binding and its coupling to chemical processes such as H+ transport, translocation of the transported substrate and cotransported ions, dynamics of the catalytic site, coordinated motions of the remote regions, as well as other molecular events facilitating the transport of the cargo. The first part of the dissertation covers topics on a Cl-/H+ transporter from the CLC superfamily, which catalyzes stoichiometrically coupled exchange of Cl- and H+ across biological membranes. CLC transporters exchange H+ for halides and certain polyatomic anions, but exclude cations, F-, and larger physiological anions, such as PO4^3- and SO4^2-. Despite comparable transport rates of different anions, the H+ coupling in CLC transporters varies significantly depending on the chemical nature of the transported anion. Although the molecular mechanism of exchange remains unknown, studies on bacterial ClC-ec1 transporter have revealed that Cl- binding to the central anion-binding site is crucial for the anion-coupled H+ transport. This study shows that Cl-, F-, NO3-, and SCN- display distinct binding coordinations at the central site and are hydrated in different manners. Consistent with the observation of differential bindings, ClC-ec1 exhibits markedly variable ability to support the formation of the transient water wires, which are necessary to support the connection of the two H+ transfer sites (Gluin and Gluex), in the presence of different anions. These findings provide structural details of anion binding in ClC-ec1 and reveal a putative atomic-level mechanism for the decoupling of H+ transport to the transport of anions other than Cl-. Another important question concerning the functional mechanism of CLC transporters is that no large conformational change have been detected crystallographically, even though transporters usually undergo global conformational change to alternately expose substrate-binding sites to opposite sides of the membrane. The collaborative work here demonstrates the formation of a previously uncharacterized `outward-facing open' state enrich by high H+ concentration, which involves global structural changes ~20 A away from the outer gate. This long distance conformational change highlights the coupled motions as well as the relevance of global structural changes in CLC transport cycle. The second part of the dissertation focus on a phospholipid scramblase which mediates rapid transbilayer redistribution (scrambling) of phospholipids at plasma membrane. This process dissipates lipid asymmetry in response to signals for critical cellular events like apoptosis that elevate cytoplasmic Ca^2+ concentration. The work here shows that the hydrophilic aqueduct on the surface of the fungal scramblase nhTMEM16 serves as the path for lipid translocation, and that Ca$^{2+}$ binding plays a key role in determining an open conformation of the path for lipid diffusion. The fully occupied lipid track connects the inner and outer leaflets and forms a “proteolipidic” pore, which allows ion conduction through the aqueous pathway formed between the protein and lipid headgroups under transmembrane electric potentials. Supporting this mechanism, site-specific mutagenesis experiments show that nhTMEM16 ionic currents are synergistically linked to phospholipid scrambling. To further validate the idea that ions permeate through TMEM16s via the same structural pathway taken by phospholipids, two specific residues in the pore region were pinpointed, which are able to convert TMEM16A Ca^2+-activated Cl- channel (CaCC) into robust scramblase upon point mutations. This novel view of flexible pore structure explains a number of unusual features of the TMEM16 ionic currents, especially the highly variable ionic selectivity and the ability to permeate large ions, which also provides crucial information on the functional dichotomy in TMEM16s.Ope

Investigation Into The Development And Function Of The Gastrointestinal Tract Of Zebrafish

This study aimed to further elucidate how the gastrointestinal tract of zebrafish functions to better utilize the zebrafish model for studying intestinal motility. Zebrafish share a high percentage of genes with humans and have similar gastrointestinal morphology. An assay that allows for the comparison of healthy motility and altered motility was developed. Using the intestinal transit assay that I developed with other lab members, I found that it takes approximately four hours for nine days post fertilization (dpf) larval zebrafish to empty their intestinal bulb when kept under normal conditions. Using trans-cinnamaldehyde as a trpa1a agonist, I was able to determine that activation of trpa1a increased the intestinal transit rate in 9 dpf zebrafish. In the absence of other nutrients and using Nile red as a tracer, trans-cinnamaldehyde was sufficient to increase the intestinal transit rate. In future studies, the Kinkel lab aims to spatiotemporally map kita, ano1, trpa1a, and trpa1b to gain a better understanding of the ion channels and receptors that regulate proper intestinal motility. The zebrafish model may provide potential targets for therapeutics for various intestinal motility disorders

Investigating the role of anoctamin 1 in the calcium dependent depolarisation of the myometrium

The myometrial smooth muscle of the uterus is responsible for force generation during labour, leading to successful delivery and expulsion of the foetus. This contraction occurs as a result of electrical signalling, and the firing of the myometrial action potential. Development of a more detailed understanding the mechanisms that regulate electrical excitability in the myometrium may allow for the identification of new strategies to manage preterm birth and prolonged labour clinically. The calcium activated chloride channel (CaCC) anoctamin 1 (ANO1) has been suggested to play an important role in promoting myometrial contractility, though its potential mechanism is unclear. In this work, a novel model of uterine excitability has been developed to allow investigation of these processes in silico. Simulations run using this model suggest that ANO1 may play a role in initiating the myometrial action potential and coupling the electrical signalling to chemical signalling via oxytocin. ANO1 may also play a role in stabilising and prolonging the action potential. These hypotheses are investigated experimentally using multiple model systems. Cultured immortalised uterine myocytes and intact human tissue were investigated using specific pharmacological inhibition of ANO1 with the novel, potent pharmacological agent Ani9. Experiments using the Cre-Lox system to achieve inducible smooth muscle specific deletion of ANO1 were also carried out. These experiments observed no evidence of ANO1 promoting uterine contractility, suggesting that previously reported findings were the result of non-specific actions of inhibitors. Transcriptional analysis of cultured myocytes and myometrial tissue suggests ANO6 as a potential alternative candidate for forming a CaCC in the myometrium. Simulations also suggest potential roles for the canonical transient receptor protein channel (TRPC6) in initiating the action potential in response to diacyl glycerol generation, and potassium channels such as TWIK-related Potassium Channel 1 (TREK1) in mechano-sensing and regulation of the duration of the myometrial action potential. Plasticity of the myometrial conductome is also considered as a mechanism that underlies the heterogeneity in electrical behaviours between cells, and may dynamically modulate the electrical properties of the cell over physiologically relevant timescales to regulate the action potential. These potential electrophysiological mechanisms are suggested as potential avenues for further investigatio

Emerging Diversity in Lipid-Protein Interactions

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions

Amino acid residues constituting the agonist binding site of the human P2X3 receptor and subunit stoichiometry of heteromeric P2X2/3 and P2X2/6 receptors

Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.:Index of contents Introductory remarks „Wissenschaftlicher Anteil des Promovenden an der Publikation“ „Bibliographische Beschreibung“ I. Introduction Pain as a sensory quality Neuronal circuitry for pain processing and sensation in the PNS and CNS Transformation of thermal, mechanical and chemical stimuli into electrical activity by nociceptors; nociceptor-targeted therapeutic approaches Release mechanisms for nucleotides and their fate in the extracellular space Nucleotide receptor-types ATP-sensitive P2 receptors and pain-sensation References II. Scientific background and aims of my thesis ATP binding-sites of P2X3 receptors; subunit composition of P2X2/3 and P2X2/6 heteromeric receptors The aims of the present work III. Publications IV. Summary and conclusions Amino acid residues constituting the agonist binding site of the human P2X3 receptor ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors „Eigenständigkeitserklärung“ Curriculum vitae Acknowledgement

An integrative and systems biology approach to Drosophila melanogaster transcriptomes

The availability of fully sequenced genomes of the model organisms including Drosophila, and their subsequent annotation has afforded seamless opportunities for reverse genetics in a complex model organism. With the advent of DNA microarrays to assay the levels of tens of thousands of genes in a single sample, functional genomics has been significantly aided to understand the functions in systems context. These microarrays have been employed predominantly on the RNA samples that are extracted from the whole animals for example at different developmental stages or in response to external stimuli. However, these approaches relied on the expression patterns that represent the sum of transcription coming from all the organs, which do not estimate the tissue-specificity of transcription. The purpose of this thesis is to provide tissue-specific transcriptomes of Drosophila melanogaster that were generated as part of the large FlyAtlas project using Affymetrix Drosophila GeneChips® (or microarrays). These chips, one at a time interrogate the levels of 18,500 transcripts (that represent all known genes) using 18,880 distinct probe sets in a single, total RNA sample. For each tissue, four biological replicates were analysed using the chips and the normalised signal intensities were obtained that represent the relative levels of mRNA expression. Using the transcriptomes, a general analysis was performed for potential novel insights into tissue-specific functions (Chintapalli et al., 2007) (Chapter 3). Then, a comparative analysis of epithelial tissues was performed to understand how the epithelia are organised in terms of their transcriptomes (Chapter 4). The Malpighian tubules are the Drosophila epithelial counterparts of the human kidney. They show asymmetric organisation in the body cavity. FlyAtlas segment-specific tubule transcriptomes allowed the comparison of their potential functional similarities and differences, thus to understand the asymmetry in function (Chapter 5)(Chintapalli, 2012). This identified a human Best vitelliform macular dystrophy (BVMD) disease homolog, Best2 in only the anterior pair of tubules that have the morphologically and functionally distinct enlarged initial (or distal) segment, a storage organ for Ca2+. Bestrophins were accordingly selected as candidate genes to analyse organismal functions, and thus to validate previous two theories that implicated bestrophins as Ca2+-activated Clˉ channels and/or Ca2+ channel regulators (Chapter 6). The confocal microscopy analysis of bestrophin YFP fusion proteins revealed interesting and novel localisations of bestrophins, in that Best1 was found in the apical plasma membranes, Best2 localised to peroxisomes, Best3 and Best4 were found intracellular. The salt survival analysis showed that Best1 is essential in regulating extra salt levels in the body. Furthermore, the fluid secretion analysis showed Best1’s potential role in Ca2+-dependent Clˉ function. Interestingly, the flies with reduced levels of Best2 expression showed increased ability to survive on extra salt food; the basis for this was investigated further in Chapter 7. Best2 was also found abundant in the eyes than anywhere else in the head. A comparative analysis of anterior tubule- and eye-specific transcriptomes revealed a potential overlap of Ca2+ signaling components, in that the PLCβ signaling was one. A neuropeptide Ca2+ agonist, capa1 evoked secondary cytosolic Ca2+ responses were found high in Best2 knockdowns. A quantitative PCR (qPCR) analysis of candidate Ca2+ signaling and homeostasis genes in Best2 mutants revealed their gene expression upregulation, under control-fed and salt-fed conditions than their wildtype controls, fed on similar diet regimes. The norpA that encodes PLCβ was found significantly enriched in the mutants. Cyp6a23 is another gene that was highly upregulated in Best2 mutants; it is a Drosophila homologue of human Cyp11b, a Ca2+-responsive gene implicated in renal salt wasting. Upon the downregulation of Cyp6a23, flies became sensitive to salt diet feeding. Other genes investigated and found to be upregulated in the mutants include transient-receptor-potential (trp) Ca2+ channel and retinal degeneration C (rdgC). Together, these results strongly suggest Best2 as a potential Ca2+ channel regulator, and provide fascinating insight into bestrophin function. Peroxisomal localisation of Best2 in line with the implication that peroxisomes act as dynamic regulators of cell Ca2+ homeostasis led to another aspect of the project (Chapter 8). This study identified two peroxins that are most abundant in the tubules and play essential roles in the novel cyclic nucleotide-regulated peroxisomal Ca2+ sequestration and transport pathway and that are detrimental for peroxisome biogenesis and proliferation

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Hiroshima University Research and Technology Guide 2012 Version : Life Science

I Life Science Development of Treatment Strategy for Hepatocellular Carcinoma to Improve the Long Term Prognosis / Hiroshi AIKATA...2 Development of Revolutional Apatite-implant Complex with Simultaneous Bone Augmentation and Osseointegration / Yasumasa AKAGAWA...3 How Do Patients with Alzheimer’s Disease Experience Memory Impairments? / Sawako ARAI...4 Development of New Therapies for Chronic Viral Hepatitis Using Human Hepatocyte Chimeric Mice / Kazuaki CHAYAMA...5 Identification of High Risk Patients to Develop a Pulmonary Arterial Hypertension / Yoshihiro DOHI...6 The Evaluation of the Intractable Chronic Pain in Psychosocial Factor and Specificity Symptom / Mitsuru DOI...7 A Versatile Modification of Physical Properties of Organic Materials by Introduction of Oxaalkyl Chains / Koichi FUKUHARA...8 Activin A Induces Craniofacial Tissue from Undifferentiated Xenopus Ectoderm in vitro / Yasuto FUKUI...9 Functional Analysis of Signal Complexes Containing Insulin Receptor Substrates (IRSs) / Toshiaki FUKUSHIMA...10 Changes in Interhemispheric Inhibition from the Active to Resting Primary Motor Cortex during a Fine-motor Manipulation Task / Kozo FUNASE...11 Creation of High Performance Host Yeast for the Production of Human-type Sphingolipids / Koichi FUNATO...12 Development of New Technologies for the Medicine Using the Chicken Antibodies / Shuichi FURUSAWA...13 Life Science Participation of Plasminogen Activator/Plasmin System in Cell-cell Adhesion and Invasive Growth of Oral Squamous Cell Carcinoma Cells / Tomoaki HAMANA...14 Study on Activation of Salivary Secretory Function with Salivary Gland Massage / Kumiko HARA...15 New Defense System against Invasive Bacteria in Host Cells / Kae HARADA...16 Roles of P/Q Type Voltage-gated Calcium Channel in the Postnatal Development of Neuronal Circuits / Kouichi HASHIMOTO...17 Development of a Variety of Analysis Softwares for Magnetoencephalography / Akira HASHIZUME Part-time Lecturer 18 Genome-wide Profiles of the Core Clock Protein BMAL1 Target by Using Multiple High-throughputApproaches / Fumiyuki HATANAKA...19 Detection Method for Epilepsy Using 11C Flumazenil-PET / Toru HIGAKI...20 Development of the Washing-sterilizer by the Ordinary Pressure Superheated Steam Use / Takamune HINO...21 Functional Diagnosis of Cancers by the Surface Plasmon Resonance Biosensor / Takaaki HIRAGUN...22 Risk Management in Medical Practice / Tooru HIYAMA...23 Generation of Model Mice for Human Diseases and its Application for Development of Novel Therapies / Hiroaki HONDA...24 Cure Rate Improvement of Intractable Hematopoietic Malignancies by Transplantation / Hideo HYODO...25 An Electromagnetophysiological Study on Epileptogenic Zones and Epileptic Spikes UsingIntracranial Electroencephalography andMagnetoencephalography (MEG) in Epilepsy Surgery / Koji IIDA...26 Development of New Antiviral Therapy Using a Chimeric Mouse Human Hepatocytes Infected withHepatitis B Virus (HBV) and Hepatitis C Virus (HCV) / Michio IMAMURA...27 Functional Analysis of RSC Chromatin-Remodeling Complex in Budding Yeast / Yuko IMAMURA...28 Investigation of Interaction of Cancer Stem Cell to Mesenchymal Stem Cell in Oral Cancer / Yasutaka ISHIDA...29 A Study on the Architectural Planning on Children’s Home / Aya ISHIGAKI...30 Health Promotion and Prevention of Sports Injuries / Yoshimasa ISHII...31 Gene Expression Profiles of Idiopathic Interstitial Pneumonias: Identification of Disease-specific Diagnostic Markers and Molecular Therapeutic Targets / Nobuhisa ISHIKAWA...32 New Method for Gastric Cancer Screening by Serum Markers / Masanori ITO...33 Clarification of Signaling Pathway Controlling Breast Cancer / Takayuki KADOYA...34 Remarkable Factors Related to Preventing Relapse of Deciduous Anterior Crossbite / Yasutaka KAIHARA...35 Long-term Tooth Cryopreservation by Use of CAS Freezer / Masato KAKU...36 Characterization of Mesenchymal Stem Cell for Regenerative Therapy / Masami KANAWA...37 Effect of Cholesterol and its Biosynthetic Precursors on the Cause of BRONJ / Taku KANDA...38 Research on Functions of a New Protein Regulating Energy Metabolism / Takashi KANEMATSU...39 Antibody Arrays for Parallel Analysis of Surface Marker Expression / Koichi KATO...40 Novel Functions of Silk Protein, Sericin / Norihisa KATO...41 Regenerative Medicine Using Mesenchymal Stem Cells Expanded with Serum-free Media / Yukio KATO...42 Physical Properties of Food Products and Biomaterials and Their Practical Application / Kiyoshi KAWAI...43 Regulation of Circadian Clock / Takeshi KAWAMOTO...44 Cell Cryopreservation / Toshitsugu KAWATA...45 Stroma-directed Molecular Targeted Therapy in Gastrointestinal Cancer / Yasuhiko KITADAI...46 Development of a Novel Screening and Evaluation System for Useful Peptides by Changing Peptide Utilization Machinery in Yeast / Kenji KITAMURA...47 Identification of Mesenchymal Stem Cell Gene Markers for Quality Control / Kazuko KITAYAMA...(NEDO Grant Contract) 48 Search for Novel Key Role Molecule of Pain Signaling / Tomoya KITAYAMA...49 Construction of the Tongue Pressure and the Lips Pressure Counting System Using a Superthin Pressure Sensor, and its Clinical Application / Myongsun KOH...50 Long-term Effects of Poison Gas Exposure on the Incidence of Respiratory Diseases / Nobuoki KOHNO...51 Utility of KL-6/MUC1 in the Clinical Management of Interstitial Lung Diseases / Nobuoki KOHNO...52 Development of Molecular Target Therapy to VEGF and VEGFR against Oral Cancer / Koichi KOIZUMI...53 Establishment of Standards for Dental Implant Removal with Resorption of Surrounding Bone / Katsunori KORETAKE...54 Molecular Cloning and Analysis of a Biosynthetic Gene Cluster for the Anti-tubercular Agent D-cycloserine / Takanori KUMAGAI...55 Effects of Statin on Serum Fatty Acid Levels / Satoshi KURISU...56 Human Biomechanics Model and Product Usability Evaluation / Yuichi KURITA...57 Tactile and Haptic Models of Humans and Their Applications to Computer Interface / Yuichi KURITA...58 The Morphology and Function of Cells in the Body / Yoshisuke KUSUMOTO...59 Evaluation of the Usefulness of a New Electronic Device for Gingival Massage / Atsue MATSUMOTO...60 Discovery of Bioactive Compounds from Okinawa Subtropical Plants / Katsuyoshi MATSUNAMI...61 Structural and Functional Analysis of Three Enone Reductases from Nicotiana Tabacum / Akihito MATSUSHIMA...62 A Colored Frog Strain for Visual Identification of the Genetic Sex of Tadpole / Ikuo MIURA...63 Functional Analysis of the Gnathodiaphyseal Dysplasia Gene TMEM16E/GDD1 / Kuniko MIZUTA...64 Development of a Novel Kind of Radioprotectors That Inhibit Radiation-induced Cell Death / Akinori MORITA...65 Development of Chronic Disease Management Programs and the Care Delivery System / Michiko MORIYAMA...66 Application of Manufacturing Systems Technology to Dentistry / Takeshi MURAYAMA...67 Development of Protamine as a Protective Agent against Drug-induced Nephrotoxicity / Junya NAGAI...68 Modification of Plant Fundamental Metabolism by the Use of nMat1 Gene / Naoki NAKAGAWA...69 Biomechanical Function of Anterior Cruciate Ligament Remnants / Atsuo NAKAMAE...70 Effects of Knee Immobilization on Regeneration of the Semitendinosus Tendon and on the Proximal Shift of the Semitendinosus Muscle-tendon Junction after Hamstring Harvesting for Anterior Cruciate Ligament Reconstruction: Evaluation Using Three-dimensional Computed Tomography / Atsuo NAKAMAE...71 Development of a New Objective Diagnostic Method for Detecting Motor and Sensory Disturbance Using Higher Brain Function Analysis / Kazuyoshi NAKANISHI...72 Risk Stratification for Brugada Syndrome / Yukiko NAKANO...73 Evaluation of Substance P Content in Biological Samples by Using a High Sensitivity Radioimmunoassay / Yoshihiro NAKATA...74 Simple and Visible Detection Method for Drug and Poison Analysis / Akira NAMERA...75 Yogurt Fermented by Lactobacillus Rhamunosus L8020, Reduce the Risks of Dental Caries, Periodontal Disease and Oral Candidosis / Hiroki NIKAWA...76 Genetic Relationships among Breeds Using Porcine Autosomal SNP Genotypes Using DNA Markers (SNPs) / Masahide NISHIBORI...77 Genetic Variant Associated with the Development of Hepatocellular Carcinoma in Japanese Chronic Hepatitis C Patients / Hidenori OCHI...78 Articular Cartilage Repair with Magnetically Labeled Mesenchymal Stem Cells and External Magnetic Device / Mitsuo OCHI...79 CX3CL1 Expression Induced by Candida Albicans in Oral Fibroblasts / Kouji OHTA...80 Statistical Analysis of Multidimensional Data of Complex Systems / Megu OHTAKI...81 Biodiversity and Ecology of Marine Symbionts and Parasites; Fisheries of Edible Zooplankters / Susumu OHTSUKA...82 Unite for Stroke / Toshiho OHTSUKI...83 Caries Incidence Associated with Streptococcus Mutans and S. Sobrinus in Children / Mitsugi OKADA...84 Upregulation of Notch2 and Six1 is Associated with Progression of Early-stage Lung Adenocarcinoma and a More Aggressive Phenotype at Advanced Stages / Morihito OKADA...85 Identification of Responsible Genes for Patients with Chronic Mucocutaneous Candidiasis / Satoshi OKADA...86 Research Infrastructure for Conducting a Safe and Secure Cell Therapy Using Mesenchymal Stem Cells Derived from Human Bone Marrow / Kosei OKAMOTO...87 Screening of Useful Materials from Unculturable Bacterial Metagenomes / Yoshiko OKAMURA...88 Assessment of the Safety Behavior in the Workplace / Yasumasa OTSUKA...89 Probabilistic Considerations on Eclosion and Copulation of Cicadas / Yasumasa SAISHO...90 Development of Method to Screen Novel Therapeutical Drugs for Neurodegenerative Disease Using Imaging Technique / Norio SAKAI...91 Evaluation of Dural Arteriovenous Fistulas of Cavernous Sinus before and after Endovascular Treatment Using Time-resolved MR Angiography / Shigeyuki SAKAMOTO...92 Application of Thermally Stable Cytochrome c / Yoshihiro SAMBONGI...93 Prediction of Human Drug Metabolism and Pharmacokinetics Using Chimeric Mice with Humanized Liver / Seigo SANOH...94 Assessment of Trypsinogen-2 levels as an Early Diagnostic for Post-endoscopic Retrograde Cholangiopancreatography Pancreatitis / Tamito SASAKI...95 Functional Model of Temporomandibular Joint / Takahiro SATODA...96 Functional Model of Swallowing / Takahiro SATODA...97 Statistical Data Analysis / Kenichi SATOH...98 Statins Induce Apoptosis and Inhibit Proliferation in Cholangiocarcinoma Cells / Masahiro SERIKAWA...99 A Novel Pig Artificial Insemination Technique Using Frozen-thawed Sperm / Masayuki SHIMADA...100 Intracellular Behavior and Elimination of Extrachromosomal Elements / Noriaki SHIMIZU...101 Mechanism of Gene Amplification and its Application to the Protein Production / Noriaki SHIMIZU...102 Research and Development to Minimize the Impact of Electromagnetic Radiation on the Human Body for Hybrid Vehicles and Electric Vehicles Parts / Kenji SHODAI...(Special Appointment) 103 Development of Simulator for Extracorporeal Circulation / Taijiro SUEDA...104 Spinal Cord Protection during Thoracic Aortic Aneurysm / Taijiro SUEDA...105 Surgery for Atrial Fibrillation / Taijiro SUEDA...106 Neuronal Bases of Pleasure and Displeasure for Taste / Makoto SUGITA...107 See-through Frogs Created by Breeding / Masayuki SUMIDA...108 Clinical Application of an Artificial Nerve for Peripheral Nerve Injury / Osami SUZUKI...109 Treatment of Arthritis Using MicroRNA Th17 Cell Differentiation / Osami SUZUKI...110 Regulation of Gastrointestinal Function by Food Factors / Takuya SUZUKI...111 Medium-chain Acyl-CoA Dehydrogenase (MCAD) Deficiency: Functional Analysis of Mutant Enzymes Found in Japanese Patients / Go TAJIMA...112 Indigenous Symbiotic Environmental Bacteria in the Intestinal Mucosa Drive the Quiescent Mucosal Immune Responses / Ichiro TAKAHASHI...113 Study on Target Gene of miRNA miR224 Which is Overexpressed in Hepatocellular Carcinoma / Shoichi TAKAHASHI...114 Transport of Insulin and its Regulation in Alveolar Epithelial Cells / Mikihisa TAKANO...115 Establishment of Jaw Bone Reconstruction Using High Functional Material for Restoration of the Ideal Jaw Form and the Occlusal Function / Masaaki TAKECHI...116 Survey on Male Dental Hygienists / Toshinobu TAKEMOTO...117 Integrative Analyses of Neuropsychiatric Diseases / Toru TAKUMI...118 Molecular Biology of the Age-related Hearing Loss and Vertigo / Masaya TAKUMIDA...119 Analysis of Chromosome Engineered Mouse Model for Autism / Kota TAMADA...120 Study on Therapeutic Strategy for Colorectal Submucosal Carcinoma after Endoscopic Treatment / Shinji TANAKA...121 Exploitation of MICA Gene Polymorphism for Development of Personalized Medicine in Oral Cancer Patients / Ryouji TANI...122 A New Technology for Treatment of Dental Caries-Regeneration of Tooth Enamel with Amelogenin / Kotaro TANIMOTO...123 Direct Vasodilation by Transdermal Administration of Nitroglycerin: Nitroglycerin Skin Spray for Premedication before Artery Puncture / Keizo TANITAME...124 Development of a Functional Food with an Aim to Enhance Bone Volume / Kazuo TANNE...125 Molecular and Cellular Target Therapy for Hepatic Stellate Cell Using Rho Kinase Inhibitor / Hirotaka TASHIRO...126 Developing the Culture Technology of Pituitary Stem Cell in the Zero Gravity Environment / Atsushi TOMINAGA...127 The Effects on Gene Expression Profiles in Human Hepatocytes by HBV and HCV Infection / Masataka TSUGE...128 Estimation of Arterial Wall Impedance Using Ultra-sonographic Images and its Application to the Diagnosis of Arteriosclerosis / Toshio TSUJI, Masashi KAWAMOTO, Masao YOSHIZUMI...129 Measurement of Peripheral Artery Mechanical Characteristics and Evaluation of Autonomic Nerve Activity / Toshio TSUJI, Masashi KAWAMOTO, Masao YOSHIZUMI...130 Non-constrained and Non-invasive Measurement of Pulse Pressure Waves from Patients in a Supine Position and Development of a Health Monitoring System for Bedridden Individuals / Toshio TSUJI, Masashi KAWAMOTO, Masao YOSHIZUMI...131 Understanding the Mechanism of Chromosome End Maintenance and its Application for Cancer Therapy / Masaru UENO...132 Effect of the Water-soluble Extract from Cultured Medium of Ganoderma Lucidum (Reishi) Mycelia (MAK) on Murine Colitis / Yoshitaka UENO...133 Mechanism, Prediction and Countermeasure of Problematic Jellyfish Blooms / Shin-ichi UYE...134 Mechanism of Chronic Pain in Orofacial Region / Mineo WATANABE...135 Algal Synthesis of Hyaluronan and Chitin / Takashi YAMADA...136 Bacteriophage Biocontrol of Bacterial Wilt for Sustainable Agricultural Production / Takashi YAMADA...137 Detection and Monitoring of Phytopathogen Causing Bacterial Wilt / Takashi YAMADA...138 How Does Habitat Association of Plant Species Generate and Maintain? / Toshihiro YAMADA...139 Neural Regeneration by Application of Human Skull Mesenchymal Stem Cells / Satoshi YAMAGUCHI...140 Development of Engineered Nucleases that Enable the Manipulation of the Genome in Cell Lines and Organisms / Takashi YAMAMOTO...141 Development of Automatic Diagnostic System for Brain Tumors / Fumiyuki YAMASAKI...142 Regulation of Thyroid Hormone Sensitivity by Differential Expression of the Thyroid Hormone Receptor During Xenopus Metamorphosis / Yoshio YAOITA...143 Novel Diagnostic and Therapeutic Targets of Gastrointestinal Cancer Identified by “Omics” Study / Wataru YASUI...144 Clinical and Radiographical Results of 179 Thrust Plate Hip Prosthesis ―5-14 Years Follow-up Study― / Yuji YASUNAGA...145 Biological Psychology of Fish / Masayuki YOSHIDA...146 Quantitative Analysis and Development of a Computer-aided System for Gastrointestinal Lesions Observed on Endoscopy / Shigeto YOSHIDA...147 Assessment of Oral and Pharyngeal Functions for Decision Making of the Ideal Thickness of Liquid / Mineka YOSHIKAWA...148 Purification, Structure Characterization of Bioactive Substances from Marine Organisms and Application to Novel Anti-oral Cancer Drugs / Yukio YOSHIOKA...149 Low Temperature Tolerance of Mammalian Transporters and Development of a New Transport Inhibitor / Ryoko YUMOTO...15