Analyses of diurnal rhythms in human post-mortem tissues [Elektronische Ressource] / vorgelegt von Katrin Ackermann

Katrinsche

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Analyses of diurnal rhythms in human
post-mortem tissues




Dissertation

zur Erlangung des Doktorgrades
der Naturwissenschaften

vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main

von
Katrin Ackermann
aus Neunkirchen/Saar

Frankfurt 2007
(D30)
vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität als
Dissertation angenommen.










Dekan: Prof. Dr. Volker Müller

Gutachter: Prof. Dr. Jörg H. Stehle; Prof. Dr. Jürgen H. Bereiter-Hahn

Datum der Disputation:


II ‚Because the universe was full of ignorance all around and the scientist panned
through it like a prospector crouched over a mountain stream, looking for the gold of
knowledge among the gravel of unreason, the sand of uncertainty and the little
whiskery eight-legged swimming things of superstition.
Occasionally he would straighten up and say things like “Hurrah, I´ve discovered
Boyle`s Third Law.” And everyone knew where they stood.’

from Terry Pratchett´s ‚Witches abroad’


...or in other words...


‚Still confused, but on a higher level.’

O. Vintermyr

III Table of Contents
Table of Contents
Abbreviations ............................................................................................................. 1
Zusammenfassung...................................................................................................... 3
Summary..................................................................................................................... 9
1 Introduction ......................................................................................................... 12
1.1 Biological rhythms and the photoneuroendocrine system .......................... 12
1.2 The pineal gland – a ‘hand of the clock’ ....................................................... 14
1.3 The melatonin biosynthesis pathway............................................................. 15
1.3.1 Components of the melatonin synthesis pathway....................................... 15
1.3.2 Melatonin synthesis in rodents.................................................................... 16
1.3.3 Melatonin synthesis in non-rodent mammals ............................................. 17
1.3.4 Melatonin rhythm in humans ...................................................................... 19
1.4 Clock genes ...................................................................................................... 20
1.4.1 The transcriptional-translational feedback loop model............................... 21
1.4.2 Clock genes in the rodent pineal gland ....................................................... 23
1.4.3 Clock genes in the pineal gland of non-rodent mammals........................... 24
1.5 Human chronobiological dysfunctions.......................................................... 24
1.6 Subject and goals of this study....................................................................... 26
2 Methods ................................................................................................................ 28
2.1 Tissue sampling ............................................................................................... 28
2.1.1 Human tissue 28
2.1.2 Animal tissue 29
2.2 RNA isolation, reverse transcription, and polymerase chain reaction ...... 30
2.2.1 RNA isolation and transcription ................................................................. 30
2.2.2 Standard PCR and real-time PCR ............................................................... 30
2.2.2.1 Standard PCR........................................................................................ 30
2.2.2.2 Real-time PCR ...................................................................................... 31
2.3 Protein analyses............................................................................................... 32
2.3.1 Preparing pineal and pituitary homogenates............................................... 32
2.3.2 AANAT and HIOMT enzyme activity ....................................................... 33
2.3.3 Immunoblotting (Western Blot analysis).................................................... 33
2.3.4 Dephosphorylation of AANAT protein ...................................................... 34
2.3.5 Immunofluorescence................................................................................... 35
2.3.6 Immunohistochemistry................................................................................ 37
2.3.7 Co-immunoprecipitation ............................................................................. 37
2.3.8 Melatonin enzyme-linked immuno-sorbent assay (ELISA) ....................... 38
2.4 Statistical analyses........................................................................................... 38
3 Results................................................................................................................... 39
IV Table of Contents
3.1 Pineal analyses................................................................................................. 39
3.1.1 Statistical pretests........................................................................................ 39
3.1.2 Degradation experiment.............................................................................. 39
3.1.3 Melatonin biosynthesis parameters............................................................. 41
3.1.3.1 Aanat and Hiomt mRNA levels 41
3.1.3.2 Protein analyses..................................................................................... 43
3.1.3.2.1 Enzyme activity assays .................................................................. 43
3.1.3.2.2 Immunoblot analyses of AANAT protein content......................... 47
3.1.3.2.3 Intracellular localization of AANAT protein................................. 49
3.1.3.2.4 Co-localization of AANAT and 14-3-3 protein............................. 51
3.1.3.2.5 Co-localization of AANAT and HIOMT....................................... 51
3.1.3.2.6 Co-immunoprecipitation of AANAT and 14-3-3 protein .............. 52
3.1.3.2.7 Melatonin content and correlation to time of death ....................... 54
3.1.4 Clock genes and their protein products....................................................... 56
3.1.4.1 Presence of clock gene mRNA and proteins in human pineal tissue.... 56
3.1.4.2 Analysis of clock gene mRNA and a.... 58
3.1.4.3 Nucleo-cytoplasmic shuttling of clock gene proteins ........................... 59
3.2 Pituitary analyses ............................................................................................ 62
3.2.1 Statistical pretests........................................................................................ 62
3.2.2 Analysis of clock gene mRNA levels ......................................................... 62
3.2.3 Protein analyses........................................................................................... 63
3.2.3.1 Analysis of clock gene protein levels ................................................... 63
3.2.3.2 Intracellular localization of clock gene proteins ................................... 65
3.3 SCN analyses ................................................................................................... 68
3.3.1 Localization of the human SCN.................................................................. 68
3.3.2 Demonstration of clock gene proteins within the human SCN................... 69
4 Discussion............................................................................................................. 72
4.1 Analyzing human post-mortem tissue - a valid experimental approach ... 72
4.2 Melatonin synthesis in the human pineal gland ........................................... 74
4.3 Clock genes in human post-mortem tissue ................................................... 80
5 Conclusion and outlook....................................................................................... 87
5.1 The role of transcription and translation – an inter-species comparison.. 87
5.2 Perspectives...................................................................................................... 89
6 References ............................................................................................................ 91
7 Appendix 106
7.1 Specimens analyzed 106
7.2 Primer sequences........................................................................................... 108
7.3 Resources ....................................................................................................... 109
7.3.1 Chemicals.................................................................................................. 109
7.3.2 Antibodies ................................................................................................. 111
V Table of Contents
7.3.2.1 Primary antibodies .............................................................................. 111
7.3.2.2 Secondary antibodies .......................................................................... 112
7.3.3 Kits ...................