Characterization of caspases from Lepidoptera and their role in apoptosis [Elektronische Ressource] / Juliette Courtiade. Gutachter: David G. Heckel ; Günter Theißen ; Rollie J. Clem

Von Courtiade

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2011
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Characterization of caspases from
Lepidoptera and their role in apoptosis.




Dissertation
zur Erlangung des akademischen Gra des
doctor rerum naturalium (Dr. rer. nat .)


Vorgelegtd em Rat der Biologisc-hPharmazeutischen Fakul tät
der Friedrich-Schiller-Universität Jen a



von Juliette Courtiade (M. Sc. )

geboren am 18. Juni 1980 in Longjum;eau Frankreich
































Gutachter:

Prof. Dr. David G. Hecke , Ml ax Planck for Chemical Ecology, Jena, German y

Prof. Dr. Günter Th eiβen, Friedrich Schiller University, Jena, German y

Prof. Dr. Rollie J. Clem, Kansas State University, Kansas, U SA




Tag des öffentlichen Verteidigun 15. Julg: y 2011


Table of Contents


Table of Contents
Acknowledgments ............................................................................................ 7
Summary ........................................................................................................... 9
Zusammenfassung .......................... 11
General Introduction ...................................................................................... 15
Apoptosis versus Necrosis ........................... 15
Apoptotic pathways ..................................... 16
Consequences of apoptosis.......................................................... 18
Apoptosis and development ....................................................... 18
Apoptosis and homeostasis ........................ 19
Apoptosis and immunity ............................ 19
Importance of apoptosis in holometabolous insects ................... 20
Aims of this work ......................................................................................................... 21
Chapter 1. Characterization of the Caspase gene family in Lepidoptera ..... 23
Introduction ................. 23
Metacaspases and Paracaspases ................................................................................ 23
The True caspases .................................... 24
Inflammatory caspases .............................................................. 25
Apoptotic caspases .................................... 26
Caspases in Lepidoptera ............................................................ 26
Methods ....................................................... 27
Preparation of cDNA libraries and EST sequencing ...................................................... 27
Database mining for putative caspase sequences ........................ 28
Caspase gene amplification and sequencing ................................ 28
Sequence alignment and phylogenetic analysis ........................... 29
BAC library screening and sequencing ........................................ 30
Intron-Exon structure ................................ 31
Results and discussion ................................. 31
Lepidopteran caspases cluster into six distinct clades .................. 31
Classification of the lepidopteran caspases .................................. 34
Alternative splicing of Caspase-3 and -5 in B. mori ...................... 36
A noctuid-specific caspase gene arose from duplication of Caspase-1 ........................... 37
Ms-Caspase-4 subfamily has evolved through duplication events .................................. 39
Future directions and conclusions ............................................... 40
Supplementary materials ............................................................. 41
Chapter 2. Functional analyses of Caspases in Helicoverpa armigera .......... 49
Introduction ................................................................................................................. 49
Materials and methods ................................ 50
Apoptosis induction ................................... 50
Feeding experiments, tissue and developmental stages expression .............................. 50

Page 3 Table of Contents

Real time quantitative RT-PCR ................................................................................... 51
Results ......................................................... 52
Caspase expression patterns in H. armigera ............................... 52
Caspase expression after immune challenge .............................................................. 53
Caspase expression upon induction of apoptosis ........................ 55
Discussion .................................................................................... 56
Caspase-1 and -2, shared functions? ......................................... 56
Caspase-5, the development manager? ..................................... 56
Caspase-3 and -6, to serve and protect? .... 57
Caspase-4, an outcast? ............................................................. 59
Conclusion and further directions ............................................... 59
Chapter 3. Comparative proteomic analysis of Helicoverpa armigera cells
undergoing apoptosis ..................................................................................... 61
Introduction................. 61
Material and Methods .................................. 62
Cell culture, apoptosis induction and extraction of protein samples. ............................. 62
Two-dimensional Differential Fluorescence Gel Electrophoresis (DIGE)......................... 63
Protein identification by mass spectrometry. .............................................................. 64
Heterologous expression of Ha-Caspase-1.................................. 65
Caspase activity assays. ............................................................ 65
RNA isolation and quantitative real-time PCR. 66
Results and Discussion ................................ 67
Comparative analysis and protein identification. ......................... 67
Functional characterization of Ha-Caspase-1. ............................. 70
Actin. ....................................................................................................................... 73
Voltage dependent anion channel. ............. 73
Glyceraldehyde 3-phosphate dehydrogenase. 74
Annexin IX. .............................................................................................................. 74
Aldo-keto reductase. . 75
Receptor for activated protein kinase C 1. .. 75
Chaperone proteins. ................................................................................................. 76
Seryl tRNA synthetase (SerRS). ................................................................................. 77
Conclusion ............................................... 78
Supplementary materials ............................ 79
General discussion .......................................................... 82
References ...................................................................... 85
Selbständigkeitserklärung ............................................. 96
Curriculum vitae ............................................................. 97


Page 4



























Acknowledgments

Acknowledgments
First of all I would like to thank all the teachers, professors, supervisors, and scientisst of all kind
who, during the past 10 years, showed me how wonderful it is to study the steries om f Nature .

I would like to thank Professor David G. Heckel for giving me the opportunity to do my PhD in
his department.
I‟m incredibly grateful to Yannick Pauchet for helping and supervising me even when it was not
his role.
Many thanks to Heiko Vogel for his advice in molecular biology and his help duringque thest
for caspase sequenc . es
I‟m also grateful to Dr. Dalial Freitak and Dr. Hanna Heidel-Fischer for being such good PhD
companion s and for teaching me some new technique s.
I also would like to thank Bianca Ulitzch for her technical assistance and for making my life in
the lab easier, Domenica Schnabelrauch hforer assistance in the sequencing facility and
HenrietteR ingys-Beckstein for technical advice .
Of course, his t whole enterprise wouldn‟t have been possible without the financial support of the
Max Planck Gese lclhaft, but also :
Antje Dudda and Olaf Kniemeye,r from the Hans Knöll Institute, who helped me with the DIGE
experiments.
The MS group who analyzed a lot of samples for mnd e aespecially Alexander Muck who spet n
quite some time explaining me the arcnaeness of mass spectrometr y.
The members of the IT department, who we re always there when computers turned rebellious
and started a lifeof their own.
Regina and her army of Hiwi s, for rearing the insects.
All my colleagues in the Entomology department, for the good atmosp ahend rethe , scientific
and non scientific discussions .
At last but n ot least, I would like to tha