Thesis (Ph. D.)--University of Wisconsin, 1951. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [82]-86).

La protéine MLN51, surexprimée dans environ 30% des cancers du sein, est un facteur clé du métabolisme des ARNm, en tant que membre du complexe de jonction des exons (EJC). Ce graffiti moléculaire est un régulateur essentiel de l’expression génique de par son implication dans l’épissage, l’export, la traduction, la stabilité et la dégradation des ARNm. A l’échelle structurale, l’EJC est organisé autour d’un coeur protéique avec l’hélicase eIF4A3, MLN51 et l’hétérodimère Magoh/Y14. Ce tétramère sert de plateforme d’ancrage à d’autres facteurs périphériques. Les mécanismes de recrutement du coeur EJC sur l’ARNm ont été élucidés par des approches biochimiques. Dans ce contexte, nous avons initié un travail original destiné à mettre en évidence la localisation cellulaire de l’assemblage du coeur EJC in vivo. L’utilisation de techniques d’imagerie photonique et électronique a permis d’établir un lien véritable entre la localisation du coeur EJC et l’architecture nucléaire. Nous avons montré que la plupart des facteurs EJC sont localisés et interagissent à la périphérie des speckles nucléaires, lieux de stockage des facteurs d’épissage. Ces régions discrètes nucléaires ont été appelées «perispeckles» et sont des entités distinctes des speckles. De manière intéressante, la localisation des protéines coeur coïncide avec celle des ARNm dans les perispeckles et est spatialement reliée aux sites transcriptionnels. Ces données démontrent que l’assemblage du coeur EJC a lieu dans le compartiment nucléaire et définissent le perispeckle comme un territoire intermédiaire entre les speckles nucléaires et sites de transcription où s’opèrent des évènements post-transcriptionnels fondamentaux. / The MLN51 protein, overexpressed in around 30% of breast cancers, is a key factor for mRNA metabolism, as a member of the Exon Junction Complex (EJC). The EJC marks the splicing history of an mRNA and influences many stages of its subsequent metabolism: splicing, dynamic cytoplasmic export, efficient and localized translation, quality-control and stability. Structurally, the EJC is organized around a core complex that is formed by four proteins (eIF4A3, MLN51, Magoh, Y14). The core complex serves as a binding platform for more than a dozen peripheral factors. The EJC is not a pre-assembled complex; however, its assembly mode is well described in vitro using recombinant proteins and splicing extracts. Nevertheless, where this complex assembles in vivo was a matter of debate. By using light and electron microscopy approaches, we established an original link between the cellular distribution of the EJC core factors and the nuclear architecture. The core and most of the peripheral EJC factors are colocalized and interact together in discrete regions of the nucleus, located at the periphery of nuclear speckles. This doughnut-shaped region appears to be a novel nuclear territory that we termed “the perispeckle”. This territory is distinct from nuclear speckles; it contains nascent mRNAs and it is close to active transcription sites. Overall, this study supports a model in which the deposition of the EJC core takes place in the nucleus, and that assembled EJC core factors concentrate in discrete subnuclear territories termed perispeckles. These regions contribute to the compartmentalization of the nucleus as an active domain implicated in mRNP packaging.

Digital averaging and analysis of miniature endplate currents (MEPCs) from mouse diaphragm was used to characterize the normal MEPC and its modification by a variety of drugs. Under normal conditions the decay of MEPCs showed consistent deviations from a simple exponential consisting in a progressive increase of rate constant, followed by a slow tail. Receptor blockade by d-tubocurarine (dTC), a-bungarotoxin, and other agents thought to occupy ACh-binding sites reduced MEPC amplitude, accelerated MEPC decay by about 30% (making it about equal to decay rate of channels opened by exogenous acetylcholine), and eliminated the early deviations from an exponential decay; dTC also abolished the late tail. Examination of the interaction of acetylcholinesterase (AChE) poisoning and receptor blockade on MEPC height and time course indicated that normally most quantal ACh is captured by receptors and, as predicted by theoretical consideration, a rather large degree of receptor blockade is necessary to reduce MEPC height. MEPC tails were exaggerated by AChE poisoning and exogenous ACh or carba-chol. The latter agents reduced MEPC height in a fashion inconsistent with blockade of ACh binding and concurrent modulation of the tail suggested an important role of desensitized receptors in tail generation. A number of other drug actions are also described quantitatively: (a) channel prolongation, typical of alcohols but also found with ketones and some amines; (b) 'channel plugging', typical of local anaesthetics but also found with many other agents, including long chain alcohols, and (c) an action to reduce MEPC size without reducing net response to exogenous agonist typical of volatile anaesthetics, associated with increase rather than decrease of ACh binding to receptor. Criteria for distinguishing different modes of modification of receptor function are discussed. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23091号 / 医博第4718号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 増永 慎一郎, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Spinal Muscular Atrophy (SMA) is a childhood form of motor neuron disease that causes a progressive paralysis that, in its most severe form, results in death before two years of age. There is currently no cure or treatment for SMA. SMA is caused by a reduction in levels of Survival Motor Neuron (SMN) protein, which results in the degeneration of lower motor neurons. This degeneration is first observed at the neuromuscular junction (NMJ), where pre-synaptic nerve terminals belonging to the motor neuron become dysfunctional and degenerate during the early stages of disease. Several previous studies have shown that the some populations of motor neurons appear to have a resistance to SMA pathology, while other neighbouring populations are vulnerable. In this study, we attempted to elucidate the cause of this vulnerability spectrum. Initially, we characterised the relative vulnerability of ten different motor unit pools in an established mouse model of severe SMA and attempted to correlate these vulnerabilities with quantified aspects of motor unit morphology. From this study, no significant correlation could be found with any aspect of motor unit morphology examined, suggesting that morphological parameters of motor neurons do no influence their relative susceptibility. We then attempted to identify changes in basal gene expression between protected and vulnerable pools of motor units using microarray analysis. Motor unit pools were labelled using a retrograde tracer injected into muscles that had previously been identified as having highly vulnerable or resistant motor units. Labelled motor neuron cell bodies were then isolated from the spinal cord using laser capture micro-dissection and RNA was extracted for microarray analysis. From this study, we identified several molecular pathways and individual genes whose expression levels compared the gene expression profiles of vulnerable and resistant motor units. Thus, molecular differences between motor neuron pools likely underlie their relative vulnerability to degeneration in SMA. Lastly, we attempted to identify a novel peptide that could be used to label synapses, including neuromuscular junctions, in vivo. This would allow us to non-invasively visualise degenerating NMJs and other synapses in human patients without the need for a biopsy. Such a tool would be extremely valuable in assessing the effectiveness of drug trials, both in human patients and animal models, and may also contribute to earlier diagnosis of motor neuron disorders. To identify a potentially suitable peptide, we used a phage display library and panned for peptides that specifically bound to the outer surface of synapses using synaptosome preparations. From this panning we successfully enriched two peptides, the sequences of which were used to manufacture fluorescently tagged peptides.

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease. A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. Spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and postsynaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here I have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/- ;SMN2 and Smn-/-;SMN2;Δ7 mouse models of SMA. Results imply that synaptic degeneration is an early and significant event in SMA, with progressive denervation and neurofilament accumulation being present at early symptomatic time points. I have identified selectively vulnerable motor units, which appear to conform to a distinct developmental subtype compared to more stable motor units. I have also identified significant postsynaptic atrophy which does no correlate with pre-synaptic denervation, suggesting that there is a requirement for Smn in both muscle and nerve and pathological events can occur in both tissues independently. Rigorous investigation of lower motor neuron development, connectivity and gene expression at pre-symptomatic time points revealed developmental abnormalities do not underlie neuromuscular vulnerability in SMA. Equivalent gene expression analysis at end-stage time points has implicated growth factor signalling and extracellular matrix integrity in SMA pathology. Using an alternative model of early onset neurodegeneration, I provide evidence that the processes regulating morphologically distinct types of synaptic degeneration are also mechanistically distinct. In summary, in this work I highlight the importance and incidence of synaptic pathology in mouse models of spinal muscular atrophy and provide mechanistic insight into the processes regulating neurodegeneration.

No description available.

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.

Gap junctions are intercellular channels that permit passage of electrical and chemical signals between neighbouring cells. Vascular endothelium typically co-expresses Cx37 and Cx40, but may downregulate its expression of Cx37 (and upregulate Cx43) in response to changes in flow. The specific regulatory roles mediated by vascular endothelial connexins, and the consequences of altered connexin expression, remain unclear. In this study, we hypothesized that Cx37 and Cx40 regulate distinct vascular responses. We further hypothesize that Cx37 is predominantly involved in vascular growth control, whereas vascular growth is not affected by ablation of Cx40 expression. We show herein that Cx37, but not Cx40 or Cx43, suppresses growth of a highly-proliferative cancer cell line by inducing G1 cell cycle accumulation. We further show that Cx37-deficient mice, lacking Cx37's putative growth inhibitory effect on the vasculature, exhibit a more extensive native and post-ischemic collateral circulation, and greater ischemia-induced microvascular density. In addition, Cx37-/- mice demonstrate a functional improvement in recovery over wild-type animals in two models of hindlimb ischemia. By contrast, Cx40-/- mice fail to recover distal limb flow following unilateral hindlimb ischemia, resulting in necrosis. Long-term angiotensin II antagonism normalized post-ischemic hindlimb bloodflow, reduced macrophage infiltration, and delayed (but did not reverse) the necrotic phenotype of these animals. In summary, we show a distinct role for each of the endothelial connexins, Cx37 and Cx40, in regulating post-ischemic vascular responses.

The LAMBDA-DIODE was invented in integrated-circuit form in 1974. There was a proposal about this device's application in memory circuits at that time. This thesis is to evaluate the circuit performance of the COMPLEMENTARY JFET LAMBDA-DIODE SRAM. It investigates the speed, power consumption and chip area of this circuit compared with the JFET CROSS COUPLED SRAM by using SPICE and breadboard simulation techniques. The results show positive signs of the Λ-DIODE's feasibility for use in VLSI static memory circuits from the chip area aspect if the parasitic capacitance of the JFET device could be minimized to reduce the power delay product.