Availability: Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1

Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1:

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Bibliographic Details
Main Author:	Monteserin Garcia, Jose Luis (Author)
Format:	Thesis/Dissertation Book
Language:	English
Published:	2013
Subjects:	Hochschulschrift
Links:	https://nbn-resolving.org/urn:nbn:de:bvb:19-171129 http://d-nb.info/1053618646/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027470810&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
Physical Description:	VII, 109 S. Ill., graph. Darst.
Format:	Langzeitarchivierung gewährleistet, LZA

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adam_text	LUDWIG MAXIMILIAN UNIVERSITY, MUNICH FACULTY OF BIOLOGY MAX PLANCK INSTITUTE OF PSYCHIATRY NEUROENDOCRINOLOGY GROUP REGULATION OF PITUITARY GROWTH HORMONE SYNTHESIS BY NAD + DEPENDENT DEACETYLASE SIRTL DISSERTATION SUBMITTED BY JOSE LUIS MONTESERIN GARCIA MUNICH, FEBRUARY 18 TH , 2013 HTTP://D-NB.INFO/1054305129 TABLE OF CONTENTS ABBREVIATIONS IV SUMMARY V ZUSAMMENFASSUNG VI 1 INTRODUCTION 1 1.1 GROWTH HORMONE (GH) 1 1.2 THE PITUITARY GLAND 3 1.3 SOMATOTROPH DEVELOPMENT 5 1.4 REGULATION OF GH SYNTHESIS 6 1.4.1 GHRH 7 1.4.2 SOMATOSTATIN (SST) 9 1.4.3 INSULIN-LIKE GROWTH FACTOR I (IGF-I) 10 1.4.4 OTHER REGULATORS: 10 1.4.5 ENERGY AVAILABILITY & GH SYNTHESIS 13 1.5 INTRA-CELLULAR ENERGY SENSORS 13 1.5.1 AMPK 14 1.5.2 SIRT1 16 1.5.2.1 PROTEIN ACETYLATION & DEACETYLATION 17 1.5.2.2 MAMMALIAN SIRTUINS 18 1.5.2.3 SIRTUIN & CALORIC RESTRICTION 21 1.5.2.4 SIRTUIN ACTIVATING COMPOUNDS 21 2 AIM OF THE STUDY 25 3 MATERIALS & METHODS 27 3 .1 R EAGENTS 27 3 .2 S OLUTIONS 29 3.3 METHODS 32 3.3.1 IMMUNOHISTOCHEMISTRY 32 3.3.2 ANIMALS 33 3.3.3 CELL CULTURE 33 3.3.3.1 CELL LINES 33 3.3.3.2 RAT PITUITARY PRIMARY CELL CULTURE 34 3.3.3.3 HUMAN GH SECRETING PITUITARY ADENOMAS 34 3.3.4 RNA EXTRACTION & REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR) 37 3.3.4.1 RNA EXTRACTION 37 3.3.4.2 RT-PCR 39 3.3.5 WESTERN BLOT ANALYSIS 41 3.3.5.1 PREPARATION OF CELL LYSATES FOR WB ANALYSIS FROM CELLS IN CULTURE 42 3.3.5.2 DETERMINATION OF THE PROTEIN CONCENTRATION 42 I 3.3.5.3 SAMPLE PREPARATION 43 3.3.5.4 PREPARATION OF 12% SDS-POLYACRYLAMIDE RESOLVING GEL 43 3.3.5.5 PROTEIN TRANSFER 43 3.3.5.6 MEMBRANE BLOCKING AND BLOTTING 43 3.3.6 COIMMUNOPRECIPITATION (CO-IP) 45 3.3.6.1 PREPARATION OF CELL LYSATES FOR CO-IP 45 3.3.6.2 PREPARING PROTEIN A/G PLUS-AGAROSE 46 3.3.6.3 PRE-CLEARING THE CELL LYSATES 46 3.3.6.4 ADDING THE ANTIBODY 46 3.3.7 CHROMATIN IMMUNOPRECIPITATION (CHIP) 48 3.3.7.1 PREPARATION OF CELLS FOR CHIP ANALYSIS OF GH PROMOTER 48 3.3.7.2 PREPARATION OF CELLS FOR CHIP ANALYSIS OF PITL PROMOTER 51 3.3.8 TRANSFECTION 51 3.3.8.1 RNA INTERFERENCE 52 3.3.8.2 PLASMIDS 52 3.3.9 CAMP INTRACELLULAR RADIOIMMUNOASSAY 53 3.3.10 MEASUREMENT OF CELL PROLIFERATION 54 3.3.11 GH DETERMINATION 55 3.3.12 IMMUNOFLUORESCENCE ASSAY 56 3.3.13 PROTEIN PHOSPHATASE 1 (PP1) ACTIVITY 56 3.3.14 SITE-DIRECTED MUTAGENESIS 57 3.3.14.1 SYNTHESIS OFGSK3\|3 K205R PLASMID 58 3.3.15 STATISTICAL ANALYSIS 59 RESULTS 61 4.1 AMPK ON GH SYNTHESIS 61 4.1.1 EFFECT OF AMPK ACTIVATION ON GH PROMOTER ACTIVITY 61 4.1.2 EFFECT OF AMPK ACTIVATION ON GH SECRETION IN VIVO 61 4.2 SLRTL ON GH SYNTHESIS 63 4.2.1 THE SIRT 1 ACTIVATOR RESVERATROL REDUCES GH LEVELS IN VIVO 63 4.2.2 RESVERATROL REDUCES GH SECRETION FROM PITUITARY SOMATOTROPHS IN VITRO 64 4.2.3 SIRTL IS EXPRESSED IN THE ANTERIOR PITUITARY 64 4.2.4 SIRTL ACTIVATION REDUCES GH SECRETION FROM PITUITARY SOMATOTROPHS IN VITRO 65 4.2.5 SIRTL ACTIVATION REDUCES GH SECRETION FROM HUMAN SOMATOTROPHINOMAS IN VITRO 66 4.2.6 SIRTL REDUCES GH PROMOTER ACTIVITY 68 4.2.7 SIRTL ACTIVATION REDUCES PITL BINDING TO GH PROMOTER BY SUPPRESSING PITL TRANSCRIPTION 69 4.2.8 SIRTL SUPPRESSES CREB TRANSCRIPTIONAL ACTIVITY 70 4.2.9 SIRTL SUPPRESSES CREB PHOSPHORYLATION 71 4.2.10 SIRTL ACTS THROUGH THE PROTEIN PHOSPHATASE 1 (PP1) 73 4.2.11 SIRTL ACTS THROUGH GSK3P 74 4.2.12 SIRTL AFFECTS GSK3P PHOSPHORYLATION AND ACTIVATES GSK3\|3 75 4.2.13 THE DEACETYLASE ACTIVITY IS ESSENTIAL FOR SIRTL S ACTION 76 4.2.14 SIRTL DEACETYLATES CREB 77 4.2.15 SIRTL AFFECTS GSK3P ACETYLATION AND ACTIVITY 78 5 DISCUSSION 83 6 REFERENCES 87 ACKNOWLEDGEMENTS 103 CURRICULUM VITAE 105 III
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discipline	Pädagogik Biologie
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spellingShingle	Monteserin Garcia, Jose Luis Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1
subject_GND	(DE-588)4113937-9
title	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1
title_auth	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1
title_exact_search	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1
title_full	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1 Jose Luis Monteserin Garcia
title_fullStr	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1 Jose Luis Monteserin Garcia
title_full_unstemmed	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1 Jose Luis Monteserin Garcia
title_short	Regulation of pituitary growth hormone synthesis by NAD+ dependent deacetylase Sirt1
title_sort	regulation of pituitary growth hormone synthesis by nad dependent deacetylase sirt1
topic_facet	Hochschulschrift
url	https://nbn-resolving.org/urn:nbn:de:bvb:19-171129 http://d-nb.info/1053618646/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027470810&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT monteseringarciajoseluis regulationofpituitarygrowthhormonesynthesisbynaddependentdeacetylasesirt1

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