Holdings: Centrosomal Sas-6 in regulation of stem cells and aging

Centrosomal Sas-6 in regulation of stem cells and aging:

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Main Author:	Gooi, Li Ming (Author)
Format:	Thesis Book
Language:	English
Published:	Köln 2017
Subjects:	Hochschulschrift
Online Access:	Inhaltsverzeichnis Inhaltsverzeichnis
Physical Description:	111 Seiten Illustrationen 21 cm

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adam_text	TABLE OF CONTENTS TABLE OF CONTENTS..........................................................................................................................- 2 - ABSTRACT......................................................................................................................................... - 5 - ZUSAMMENFASSUNG.......................................................................................................................- 6 - ABBREVIATIONS...............................................................................................................................- 7 - 1. INTRODUCTION........................................................................................................................ - T I L L CENTROSOMES..................................................................................................................-11- 1.1.1 CENTROSOME STRUCTURE..............................................................................................................-12- 1.1.1.1 CENTRIOLES.........................................................................................................................-12- 1.1.1.2 PERICENTRIOLAR MATERIAL (PCM)......................................................................................... -15- 1.1.2 CENTROSOME FUNCTION...............................................................................................................-16- 1.1.3 THE CENTROSOME CYCLE..............................................................................................................-1 7 - 1.1.4 SURVEILLANCE PATHWAYS FOR ABERRANT CENTROSOMAL ACTIVITY ................................................ -19- 1.1.5 SPINDLE ASSEMBLY ABNORMAL PROTEIN 6 (SAS-6) .................................................................... - 20 - 1.2 STEM CELLS..................................................................................................................... -23- 1.2.1 DROSOPHILA MALE GERMLINE STEM CELLS....................................................................................- 24 - 1.2.2 DEVELOPMENT OF THE DROSOPHILA MALE GONADS........................................................................- 25 - 1.2.3 AGING OF THE DROSOPHILA MALE GSC NICHE.............................................................................-27- 1.3 AGING............................................................................................................................ - 28 - 1.3.1 CENTROSOMES IN AGING...............................................................................................................-31- 2 OBJECTIVES AND AIMS.......................................................................................................... - 33 - 3 RESULTS................................................................................................................................. - 34 - 3.1 CENTROSOMES IN STEM CELL MAINTENANCE........................................................................-34- 3.1.1 LOSS O F SAS-6 LEADS TO ABERRANT CENTROSOMAL STRUCTURES....................................................- 34 - 3.1.2 M UTATING SAS-6 PERTURBS SELF-OLIGOMERIZATION AND RECRUITMENT TO CENTROSOM ES...........- 37 - 3.1.3 PERTURBING SAS-6 SELF-OLIGOMERIZATION LEADS TO ABERRANT CENTROSOMAL STRUCTURES ......... - 39 - 3.1.4 LOSS O F SAS-6 AND PERTURBATION O F ITS SELF-OLIGOMERIZATION COINCIDES WITH REDUCED MALE GERMLINE STEM CELLS (GSCS) IN ITS N ICHE.............................................................................................- 41 - 3.1.5 REDUCED MALE GSCS IS ASSOCIATED WITH CELL CYCLE CHANGES IN SAS-6NUU FLIE S ................ - 44 - 3.1.6 CELL CYCLE ARREST DOES NOT LEAD TO CELL DEATH O F MALE GSCS IN SAS-6NUU FLIES ................. - 47 - 3.1.7 INCREASED SYMMETRIC DIFFERENTIATION O F MALE GSCS IS OBSERVED IN SAS-6NUU FLIE S ....... - 48 - 3.1.8 OVER-EXPRESSING SAS-6 PARTIALLY RESCUES GSC LOSS DURING AGING .................................... - 49 - 3.2 CENTROSOMES IN THE STUDY OF AGING .............................................................................. - 51 - 3.2.1 LOSS O F CENTROSOME INTEGRITY COINCIDES WITH REDUCED MALE GSCS DURING A G IN G ...........-51- 3.2.2 CENTROSOMAL SAS-6 RECRUITM ENT DECREASES DURING AGING IN MALE GERM CELLS ................ - 52 - 3.2.3 AGGREGATION O F CENTROSOMAL PROTEINS INCREASES DURING AGING IN DROSOPHILA INTESTINAL CELLS .................................................................................................................................................... - 53 - 3.2.4 TOTAL CENTROSOMAL PROTEIN LEVELS INCREASES DURING AGING...................................................- 54 - 3.2.5 SAS-6 LEVELS AFFECT DROSOPHILA WDAH LIFESPAN IN A DOSE DEPENDENT M ANNER ..................... - 54 - 3.2.6 REDUCED SAS-6 EXPRESSION CORRELATES WITH BETTER M AINTENANCE O F MALE G S C S ............. - 57 - 4 DISCUSSION........................................................................................................................... - 59 - 4.1 CENTROSOMES IN STEM CELL MAINTENANCE........................................................................- 59 - 4.2 CENTROSOMES IN THE STUDY OF AGING...............................................................................- 66 - 5 MATERIAL AND METHODS........................................................................................... . - 70 - 5.1 MATERIALS....................................................................................................................... - 70 - 5.1.1 CHEMICALS, REAGENTS AND K ITS.................................................................................................- 70 - 5.1.2 INSTRUMENTS AND APPARATUS..................................................................................................... - 72 - 5.1.3 A NTIBODIES................................................................................................................................ - 73 - 5.1.4 P RIM ERS...................................................................................................................................... -74- 5.1.5 SOLUTIONS AND M IXTURES........................................................................................................... - 75 - 5.1.6 F LY STO CKS....................................................................................................................................- 77 - 5.2 METHODS...................................................................................................................... -78- 5.2.1 GENERAL CLONING TECHNIQUES.................................................................................................... - 78 - 5.2.1.1 POLYMERASE CHAIN REACTION (PCR).................................................................................... - 78 - 5.2.1.2 GEL ELECTROPHORESIS...........................................................................................................- 79 - 5.2.1.3 GEL EXTRACTION....................................................................................................................-79- 5.2.1.4 RESTRICTION DIGEST.............................................................................................................. - 79 - 5.2.1.5 LIGATION.............................................................................................................................- 79 - 5.2.1.6 TRANSFORMATION OF PLASMIDS INTO CHEMICALLY COMPETENT E.COLI ........................................ - 79 - 5.2.1.7 PLASMID DNA PURIFICATION................................................................................................ - 80 - 5.2.1.8 GENOMIC DNA ISOLATION FROM DROSOPHILA MELANOGASTER ............................................... - 80 - 5.2.2 CELL CULTURE TECHNIQUES.......................................................................................................... - 80 - 5.2.2.1 CULTURING DROSOPHILA CELLS............................................................................................... - 80 - 5.2.2.2 PLASMID TRANSFECTION INTO DROSOPHILA CELLS.......................................................................-81- 5.2.2.3 GENERATING STABLY TRANSFECTED CELL LINES.............................................................................- 81 - 5.2.3 FLY CULTURE TECHNIQUES AND M ETHODOLOGIES.........................................................................- 81 - 5.2.3.1 GENERATION OF TRANSGENIC FLIES........................................................................................... - 81 - 5.2.3.2 MAPPING TRANSGENE INSERTIONS............................................................................................ - 82 - 5.2.3.3 LACKCROSSING....................................................................................................................- 82 - 5.2.3.4 LIFESPAN ASSAY..................................................................................................................- 82 - 5.2.3.5 MALE FERTILITY ASSAY...........................................................................................................- 82 - 5.2.4 BIOCHEMICAL TECHNIQUES........................................................................................................ - 83 - 5.2.4.1 SAMPLE PREPARATION................................................................................................................. - 83 5.2.4.1.1 DROSOPHILA 82 CELL EXTRACTS.......................................................................................... - 83 5.2.4.1.2 PREPRARATION OF DROSOPHILA SAMPLES FOR WESTERN BLOTTING............................................- 83 5.2.4.2 BRADFORD ASSAY........................................................................................................................ - 83 5.2.4.3 FLAG-IMMUNOPRECIPITATION (FLAG-IP) ........................ - 84 5.2.4.4 SUCROSE GRADIENT VELOCITY SEDIMENTATION...............................................................................- 84 5.2.4.5 WESTERN BLOTTING.................................................................................................................... - 84 5.2.5 EX VIVO EDU INCORPORATION AND DETECTION.................................................................................- 84 5.2.6 EMBRYO AND 1ST INSTAR LARVAE STAINING........................................................................................ - 85 5.2.7 TUNEL STAINING O F DROSOPHILA TE S TE S...................................................................................... - 85 5.2.8 IMMUNOFLUORESCENCE AND ELECTRON M ICROSCOPY........................................................................- 85 SUPPLEMENTARY DATA.........................................................................................................................- 87 INDEX OF FIGURES......................................................................................................................... - 91 REFERENCES.........................................................................................................................................- 93 PUBLICATION LIST......................................................................................................................... - 108 ACKNOWLEDGEMENTS................................................................................................................. -110 ERKLAERUNG U ............................................................................................................................ -ILL
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spelling	Gooi, Li Ming Verfasser (DE-588)1162959908 aut Centrosomal Sas-6 in regulation of stem cells and aging vorgelegt von Li Ming Gooi aus Kuala Lumpur, Malaysia Köln 2017 111 Seiten Illustrationen 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1162363282/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030809405&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Gooi, Li Ming Centrosomal Sas-6 in regulation of stem cells and aging
subject_GND	(DE-588)4113937-9
title	Centrosomal Sas-6 in regulation of stem cells and aging
title_auth	Centrosomal Sas-6 in regulation of stem cells and aging
title_exact_search	Centrosomal Sas-6 in regulation of stem cells and aging
title_full	Centrosomal Sas-6 in regulation of stem cells and aging vorgelegt von Li Ming Gooi aus Kuala Lumpur, Malaysia
title_fullStr	Centrosomal Sas-6 in regulation of stem cells and aging vorgelegt von Li Ming Gooi aus Kuala Lumpur, Malaysia
title_full_unstemmed	Centrosomal Sas-6 in regulation of stem cells and aging vorgelegt von Li Ming Gooi aus Kuala Lumpur, Malaysia
title_short	Centrosomal Sas-6 in regulation of stem cells and aging
title_sort	centrosomal sas 6 in regulation of stem cells and aging
topic_facet	Hochschulschrift
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