Functional characterization of four CDK-like kinases and one Calmodulin-dependent kinase of the human malaria parasite Plasmodium falciparum [Elektronische Ressource] / Shruti Agarwal

Shruti Pandalai

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 40
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Functional characterization of four CDK-like kinases
and one Calmodulin-dependent kinase of the
human malaria parasite Plasmodium falciparum





Dissertation for the completion of doctorate degree in Natural
Sciences at the Julius-Maximilians-Universität Würzburg








M.Sc.
Shruti Agarwal
New Delhi, India

February 2010











Eingereicht am:
.....................................................................................................................

Mitglieder der Promotionskommission:
Vorsitzender:
1. Gutachter: PD Dr. Gabriele Pradel
2. Gutachter: Prof. Dr. Thomas Dandekar
3. Gutachter: Prof. Dr. Christian Doerig

Tag des Promotionskolloquiums:
.........................................................................................

Doktorurkunde ausgehändigt am:
........................................................................................


Erklärung
Gemäß § 4 Abs. 3 S. 3, 5 und 8 der Promotionsordnung der Fakultät für Biologie der
Bayerischen Julius-Maximilians-Universität Würzburg.







Hiermit erkläre ich, dass ich die Dissertation

„ Functional characterization of four CDK-like kinases and one
Calmodulin-dependent kinase of the human malaria parasite, Plasmodium
falciparum”

selbstständig angefertigt und keine anderen als die von mir angegebenen Quellen und
Hilfsmittel benutzt habe.

Ich erkläre außerdem, dass die Dissertation bisher weder in gleicher noch in ähnlicher
Form in einem anderen Prüfungsverfahren vorgelegen hat.

Ich habe bisher außer den mit dem Zulassungsgesuch urkundlich vorgelegten Graden,
keine weiteren akademischen Grade erworben oder zu erwerben versucht.

Würzburg, Februar 2010




Shruti Agarwal

Acknowledgements
The present study has been conducted in the research group of PD Dr.
Gabriele Pradel at the Research Center for Infectious Diseases, University of
Würzburg from April 2006 to February 2010.
First and foremost, I would like to thank my supervisor, PD Dr. Gabriele Pradel
for giving me an opportunity to pursue my doctoral studies in a beautiful foreign
land. She introduced me to the complex yet exciting field of the malaria
parasite, P. falciparum. Her constant guidance and encouragement have
helped me in paving my way towards successful completion of this project. I am
indebted to her for fruitful discussions that have helped me in defining targets,
planning the experiments and drawing solid conclusions. I thank Gabi for being
not only a good boss but also for all her care and support in every possible
way.
I thank Prof. Dr. Dr. h.c. mult. J. Hacker and Prof. Dr. Matthias Frosch (former
chairs, IMIB/ZINF) for providing me the possibility to work in this well equipped
institute.
With a deep sense of gratitude, I would like to thank my co-supervisor, Prof.
Christian Doerig (EPFL, Lausanne) for introducing me to the field of
P. falciparum kinases. His continuous guidance has been very helpful for the
progress of this project. I am thankful to him for providing me the possibility to
perform reverse genetics experiments in his laboratory, then at the University of
Glasgow, U.K. I would like to extend my thanks to his group members
especially to Jean Halbert for acquainting me with the new techniques.
I am grateful to my second co-supervisor Prof. Thomas Dandekar for giving me
an overview of the basic bioinformatic analysis. I am thankful for his scientific
suggestions for the betterment of this project and for a careful review of my
progress reports. In addition to his scientific input in the project, he introduced
me to the scholarship from the BioMedTec International Graduate School of
Science (BIGSS), “Lead structures of cell function” of the Elite Network
Bavaria.
Further, I am indebted to Prof. Paul Rӧsch and Dr. Stephan Schwarzinger
(Univesrity of Bayreuth) for providing me an opportunity to become an
associated member of this graduate school. Apart from gaining a scope to interact with graduate students of Würzburg, Erlangen and Bayreuth, the
school financed my four months stay in Glasgow and the participation in a
distinguished Molecular Parasitology meeting at Woods Hole, USA in the year,
2008. These scientific gains would not have been possible without the
scholarship from the BIGSS.
I am also thankful to Prof. G. Krohne and his team for help with the immuno
electron microscopy. Thanks to Dr. Jude Przyborski and Dr. Stefan Baumeister
for their collaboration on Mass spectrometry analysis.
I am grateful to all past and present members of our “TEAM” to provide a very
friendly and helpful work atmosphere. I highly appreciate Ludmilla Sologub and
Nina Simon for their time and effort in the immunization of mice. Thanks to
Selina Kern for her input in diagnostic PCRs on PfCLK-3-tag and PfCLK-4-tag
parasite cultures. I am also thankful to Rebecca Schillig and Dr. Matthias
Scheuermeyer for their contribution in experiments related with the PfPKRP
kinase. Special thanks to Sabrina for her scientific discussions and friendship,
thanks to Andrea and Matthias for correcting my thesis and providing their
valuable inputs. I further thank Andrea for German translation of the summary
section. I thank all members of AG Pradel for a wonderful time throughout my
stay.
I appreciate Hilde Merkert for the technical help, Barbara Plaschke for the
assistance with sequencing and all members of ZINF and IMIB for their
co-operation.
Words fail to express my indebtedness to my Indian friends who have been
always there for me to share my sorrows and happiness. Thanks for giving me
the warmth of home, being miles away.

Above all, I would like to thank the almighty for giving me the strength to fulfill
my goals and my family, the treasure of my life! It is their love, immense care,
strength and support that have shaped me into an individual, I am today.
Special thanks to my husband Vaibhav, for being with me always and
especially during the tough times.

To close with, I thank one and all who contributed directly or indirectly for the
success of this project.
Contents


1. Introduction………………………………………………………………… 1

1.1 The tropical disease, Malaria……………………………………… 1
1.2 Malaria control strategies………………………………………...... 3
1.2.1 Vaccine research......................................................... 5
1.2.2 Malaria chemotherapy................................................. 7
1.3 Evaluation of kinases as drug targets…………………………….. 9
1.4 The Plasmodium falciparum kinome…………………………....... 12
1.5 The CMGC group…………………………………………………… 13
1.6 CDK-like kinases in the malaria pathogen, P. falciparum………. 16
1.7 The cluster of Calmodulin-dependent protein kinases in 19
P. falciparum……………
1.8 Aim of the present study…………………………………………. 21

2. Materials and Methods…………………………………………………… 23

2.1 Materials…………………………………………………………….. 23

2.1.1 Computer programmes for in silico analysis………….. 23
2.1.2 Instruments, chemicals and disposables used……….. 23
2.1.3 Purification kits…………………………………………… 26
2.1.4 Enzymes and DNA/Protein Ladders…………………… 26
2.1.5 Medium and buffers for cell culture…………………….. 27
2.1.6 Solutions for nucleic acid isolation and Southern blot.. 29
2.1.7 Reagents and solution for Protein purification, SDS- 30
PAGE and Western blot………………………………….
2.1.8 Medium and agar plates for bacterial cultivation……... 33
2.1.9 Cell lines and bacteria…………………………………… 34
2.1.10 Plasmids…………………………………………………... 35
2.1.11 Antibodies…………… 36
2.1.12 Primers for Reverse Transcriptase PCR for PfCLK 37
kinases…......................................................................
2.1.13 Recombinant protein primers…………………………… 38 2.1.14 Primers for gene disruption using the pCAM-BSD 39
vector………………………………………………………
2.1.15 Primers for gene tagging using the pCAM-BSD 39
vector
2.1.16 Primers for genotype characterization ………………… 40
2.1.17 Primers for amplification of probes for Southern blot 41
analysis…………………………………………………….
2.1.18 Gene IDs...................................................................... 41

2.2 Methods……………………………………………………………... 42

2.2.1 Cell biology methods……….…………….................... 42
2.2.1.1 Cultivation and storage of Plasmodium 42
falciparum……………………………………..
2.2.1.2 Transfection………………………………...... 45
2.2.1.3 Clonal dilution and Malstat assay………….. 46
2.2.1.4 Parasite culture and membrane feeds……. 47
2.2.1.5 Indirect immunofluorescence assay………. 48
2.2.1.6 Immunoelectron microscopy……………….. 48
2.2.2 Molecular biology methods…………………………... 49
2.2.2.1 Genomic DNA isolation……………………... 49
2.2.2.2 Polymerase chain reaction …………........... 50
2.2.2.3 Spin pur