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Trehalose-Mediated Enhancement of Glycosaminoglycan Degradation in the Lysosomal Storage Disorder Mucopolysaccharidosis III

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Trehalose-Mediated Enhancement of Glycosaminoglycan Degradation in the Lysosomal Storage Disorder Mucopolysaccharidosis III Aus dem Institut für Humangenetik der Universität zu Köln Direktorin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth Trehalose-mediated enhancement of glycosaminoglycan degradation in the lysosomal storage disorder Mucopolysaccharidosis III Trehalose vermittelte Steigerung des Glykosaminoglykan-Abbaus in der lysosomalen Speichererkrankung Mukopolysaccharidose III Inaugural-Dissertation zur Erlangung der Doktorwürde der Hohen Medizinischen Fakultät der Universität zu Köln vorgelegt von Victor Mauri aus Stuttgart promoviert am 29. Januar 2014 Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln, 2014 Dekan: Universitätsprofessor Dr. med. Dr. h.c. Th. Krieg 1. Berichterstatterin: Frau Universitätsprofessor Dr. rer. nat. B. Wirth 2. Berichterstatter: Professor Dr. rer. nat. F.-G. Hanisch Erklärung Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten: Univ.-Prof. Dr. rer. nat. Brunhilde Wirth Marco Sardiello, PhD, Assistant Professor BCM Christian Schaaf, MD, PhD, Assistant Professor BCM Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines Promotionsberaters in Anspruch genommen. Dritte haben von mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertationsschrift stehen. Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Köln, den 19. August 2013 ______________________ Unterschrift Die in dieser Arbeit präsentierten Untersuchungen wurden zwischen März 2011 und Oktober 2011 im Department of Human and Molecular Genetics des Baylor College of Medicine am Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston, Texas, USA, in der Arbeitsgruppe von Assistant Professor Marco Sardiello, PhD, durchgeführt. Die Mehrzahl der dieser Arbeit zugrunde liegenden Experimente sind nach entsprechender Anleitung durch Herrn Dr. Marco Sardiello von mir selbstständig durchgeführt worden. Bei den Cloning-Experimenten habe ich Unterstützung von Herrn Dr. Alberto di Ronza bekommen. Das Experiment zur zeitlichen Linearität des NAG-Assays sowie die Experimente zur Z‘-Faktor-Berechnung wurden von Frau Dr. Parisa Lotfi durchgeführt. Bei der Messung der intestinalen Trehalase-Aktivität bin ich von der labor- technischen Assistentin Frau Sanagasetti unterstützt worden. Acknowledgment This thesis would not have been possible without the help, the guidance and the encouragement of several persons who in one way or another contributed to the completion of the studies and the preparation of this thesis. First, I wish to express my sincere gratitude to Dr. Marco Sardiello, my principal investigator and supervisor at Baylor College of Medicine, for giving me the opportunity to work in his lab on this highly interesting project, for being an excellent scientific mentor, for sharing his great knowledge, curiosity and experience, for valuable discussions, encouragement and for many good moments inside and outside the lab. I also owe my deepest gratitude to Professor Brunhilde Wirth, director of the Institute of Human Genetics and my supervisor in Köln, Germany, for her helpfulness and great support to make this German-American thesis happen, for her time, for valuable conversations and suggestions while writing the manuscript. Without her support it would not have been possible to write in Germany about the experiments performed in Houston. Many thanks go also to the other members of the Sardiello-Lab for providing me such a stimulating and friendly working environment, Dr. Alberto di Ronza and Dr. Michela Palmieri for teaching me basic and not so basic laboratory techniques and for their endless help with almost endless problems. Dr. Parisa Lotfi for performing experiments during the review process of our paper. Deepthi Sanagasetti for technical support. I thank Alberto di Ronza, not just for being a great teacher and lab mate and for his helpfulness at any time, but mostly for his friendship, for being such an invaluable friend inside and outside the lab. I would like to thank Dr. Christian Schaaf for his constant support and help during my time in the USA, for initially introducing me to Dr. Sardiello, for his invaluable feedback on the manuscript, for his great book “Mit Vollgas zum Doktor” and for his advice on scientific and personal issues. I would also like to thank the Team Sanfilippo Foundation as well as the Beyond Batten Disease Foundation for their generous funding of this and many other projects to eventually help children with Sanfilippo Syndrome, Batten Disease and other rare, but devastating diseases. I also owe my deepest gratitude to my parents and my sister, for their everlasting support and encouragement, their confidence in me and my ideas, and for everything they taught me, which seems to be an excellent foundation for life. Last, but not least, I owe my warmest thanks to Isabel, my “better half”. Thank you for your support at any time and your love! Table of contents Table of contents Table of contents .................................................................................... I Glossary ............................................................................................... VI 1 INTRODUCTION ............................................................................... 1 1.1. Lysosomes and Lysosomal Storage Disorders ..................................... 1 1.1.1. The lysosome .......................................................................................... 1 1.1.2. TFEB and the CLEAR network................................................................ 3 1.1.3. Lysosomal storage disorders .................................................................. 5 1.2. Mucopolysaccharidosis III/Sanfilippo Syndrome ................................. 11 1.2.1. The mucopolysaccharidoses ................................................................. 11 1.2.2. MPSIII: history, biochemistry and genetic aspects ................................ 13 1.2.3. MPSIII: epidemiology, clinical presentation and diagnosis .................... 15 1.2.3.1. Epidemiology ............................................................................................ 15 1.2.3.2. Clinical picture .......................................................................................... 15 1.2.3.3. Diagnosis ................................................................................................. 16 1.2.4. Therapeutic strategies for MPS III (and other LSDs) ............................. 17 1.2.4.1. Enzyme replacement therapy ................................................................... 17 1.2.4.2. Gene therapy ........................................................................................... 18 1.2.4.3. Cell-based therapy ................................................................................... 18 1.2.4.4. Substrate reduction therapy ..................................................................... 19 1.2.4.5. Pharmaceutical chaperon therapy (PCT) .................................................. 19 1.2.4.6. Stop codon read-through (SCRT) ............................................................. 20 1.3. Trehalose ............................................................................................. 20 1.3.1. Chemical properties and natural occurrence ......................................... 20 1.3.2. Metabolism ............................................................................................ 21 1.3.3. Safety and toxicity ................................................................................. 22 1.3.4. Functions of trehalose ........................................................................... 22 1.3.4.1. Protection from various stress conditions ................................................. 22 1.3.4.2. Induction of autophagy and degradation of macromolecules .................... 24 I Table of contents 2 AIMS ............................................................................................... 26 3 MATERIAL AND METHODS .......................................................... 28 3.1. Cell lines .............................................................................................. 28 3.1.1. Fibroblast cell lines derived from MPS III patients and control subjects 28 3.1.2. HEK 293T and HeLa cells ..................................................................... 29 3.2. Equipment, Chemicals, Reagents and Kits ......................................... 29 3.2.1. Equipment ............................................................................................. 29 3.2.2. Mouse dissection equipment ................................................................. 30 3.2.3. Chemicals ............................................................................................. 30 3.2.4. Reagents and other materials ............................................................... 31 3.2.5. Kits .......................................................................................................
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