ORIGINAL PUBLICATIONS ........................................... 6
ABBREVIATIONS ................................................... 7
ABSTRACT ........................................................ 9
1 INTRODUCTION ................................................ 10
2 REVIEW OF THE LITERATURE .................................... 11
2.1 Programmed cell death determines tissue homeostasis .... 11
2.2 Caspases as regulators of death signaling .............. 13
2.2.1 Initiation of death signaling by caspases ....... 15
2.2.2 Caspases as executioners of cell death .......... 17
2.2.3 The mechanism of caspase activation ............. 18
2.2.4 Detection of caspase activation and execution
of apoptosis .................................... 20
2.3 Mitochondrial apoptotic signaling ...................... 21
2.3.1 The mitochondrion as a determinant of cell
death ........................................... 21
The Bcl-2 family of pro- and anti-apoptotic
proteins ........................................ 21
Other proteins involved in mitochondrial death
signaling ....................................... 23
The apoptosome - a platform for caspase
activation ...................................... 24
2.3.2 Stress-induced apoptosis ........................ 24
2.3.3 Alternative death pathways ...................... 26
2.4 Death receptor-mediated apoptosis ...................... 26
2.4.1 Death receptors and ligands ..................... 26
CD9 5-mediated apoptosis ........................ 28
TRAIL - the tumor-specific death ligand ......... 29
TNF-R1 signaling involves two signaling
complexes ....................................... 30
2.4.2 The death-inducing signaling complex -
a platform for regulation of caspase
activation ...................................... 31
Death domain and death effector domain
interactions as the backbone of the DISC ........ 31
Regulation of DISC signaling .................... 32
2.4.3 Modulation of death receptor responses by
c-FLIP .......................................... 33
Viral and cellular forms of FLIP ................ 33
c-FLIP as a modulator of caspase activation ..... 35
The expression levels of c-FLIP affect
apoptosis sensitivity ........................... 37
Regulation of the c-FLIP expression at the
level of protein synthesis ...................... 38
Regulation of c-FLIP stability by post-
translational modifications ..................... 40
2.4.4 Alternative pathways regulated via death
receptors ....................................... 41
2.4.5 DISC proteins in the regulation of lymphocyte
proliferation and cell death .................... 44
Regulation of T-lymphocyte homeostasis .......... 44
c-FLIP in T-lymphocyte survival and
proliferation ................................... 46
2.4.6 Diseases involving abnormal death receptor
responses - the role of c-FLIP .................. 48
High c-FLIP levels are associated with cancer ... 48
c-FLIP as a determinant of lymphocyte survival
in autoimmune diseases .......................... 50
2.5 Effects of hyperthermia on cell survival ............... 51
2.5.1 The heat shock response ......................... 51
2.5.2 The role of fever and inflammation in cell
survival ........................................ 51
2.5.3 The effects of hyperthermia and the heat shock
response on apoptotic signaling ................. 53
Thermotolerant cells are protected from
apoptotic signaling ............................. 53
The effect of Hsps upstream of the
mitochondria .................................... 54
The effect of Hsps downstream of the
mitochondria .................................... 55
High expression levels of Hsps protect cancer
cells from cell death ........................... 56
2.5.4 Hyperthermia and disease ........................ 57
Hyperthermia in cancer therapy .................. 57
The effect of fever and infections on allergy
and autoimmunity ................................ 58
3 OUTLINE OF THE STUDY ........................................ 59
4 MATERIALS AND METHODS ....................................... 60
4.1 Cell culture ........................................... 60
4.2 Plasmids and constructed cell lines .................... 60
4.3 Reagents and treatments ................................ 61
4.4 Detection of apoptosis ................................. 62
4.5 Western blotting ....................................... 63
4.6 Mitochondrial fractionation ............................ 63
4.7 Electrophoretic mobility shift assay ................... 63
4.8 Kinase assays .......................................... 64
4.9 Microscopy ............................................. 64
4.10 Detection of activated Bax ............................. 65
4.11 Surface expression analysis of CD95 .................... 65
4.12 DISC analysis .......................................... 65
4.13 RNase protection assay ................................. 66
4.14 Ubiquitination assays .................................. 66
4.15 Detection of caspase-3 activation by FRET
microfluorimetry ....................................... 66
4.16 Image analysis and statistics .......................... 66
5 RESULTS AND DISCUSSION ...................................... 68
5.1 Development of tools for in vivo detection of
apoptosis (I) .......................................... 68
5.2 c-FLIPs is absent in rodent genomes .................... 70
5.3 The effects of hyperthermia on CD95-mediated
apoptosis (II, III) .................................... 74
5.3.1 Hyperthermia sensitizes T-lymphocytes to CD95-
mediated apoptosis (II, III) .................... 74
5.3.2 Hyperthermia enhances CD95-mediated caspase
activation (II) ................................. 76
5.3.3 Hyperthermia does not affect the expression of
either CD95 ligand or receptor (II, III) ........ 77
5.3.4 The hyperthermia-induced sensitization to
CD95-mediated apoptosis does not involve МАРК
signaling (II) .................................. 78
5.4 The hyperthermia-induced sensitization to CD95-
mediated apoptosis is caused by c-FLIPS
downregulation (II, III) ............................... 78
5.4.1 Hyperthermia downregulates c-FLIP and
sensitizes to CD95-mediated apoptosis (II,
111) ............................................ 78
5.4.2 Downregulation of c-FLIPs is required for the
hyperthermia-induced sensitization to CD95-
mediated apoptosis (III) ........................ 80
5.4.3 The loss of c-FLIP from the DISC allows for
efficient activation of caspase-8 (III) ......... 82
5.5 Regulation of c-FLIP ubiquitination and degradation
during hyperthermia (HI) ............................... 82
5.5.1 Hyperthermia does not affect transcription or
translation of c-FLIP (HI) ...................... 82
5.5.2 The ubiquitination and degradation of c-FLIP
are increased upon hyperthermia (III) ........... 83
5.6 The hyperthermia-induced sensitization to CD95-
mediated apoptosis is independent of Hsp70 and the
heat shock response (II, HI) ........................... 84
5.7 Hyperthermia and apoptosis during fever and immune
responses (HI) ......................................... 86
6 CONCLUSIONS ................................................. 88
7 ACKNOWLEDGEMENTS ............................................ 89
PREFERENCES .................................................... 91
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