Targeting flatworm signaling cascades for the development of novel anthelminthic drugs [Elektronische Ressource] / submitted by Verena Magdalena Gelmedin

Verena Mair-Briggs

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 51
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

TARGETING FLATWORM SIGNALING CASCADES FOR THE DEVELOPMENT OF NOVEL
ANTHELMINTHIC DRUGS

SIGNALKASKADEN VON PLATTWÜRMERN ALS ANGRIFFSPUNKTE ZUR ENTWICKLUNG NEUER
ANTIHELMINTHIKA


Doctoral thesis for a doctoral degree
at the Graduate School of Life Sciences,
Julius-Maximilians-Universität Würzburg,
Section Infection and Immunity

submitted by

VERENA MAGDALENA GELMEDIN
from

SAARBRÜCKEN

Würzburg 2008
















Submitted on: …………………………………………………………..……..
Office stamp

Members of the Promotionskomitee:

Chairperson: Prof. Dr. Manfred Gessler

Primary Supervisor: Prof. Dr. Klaus Brehm

Supervisor (Second): Prof. Dr. Roland Benz

Supervisor (Third): Prof. Dr. Joachim Morschhäuser


Date of Public Defence: …………………………………………….…………

Date of receipt of Certificates: ……………………………………………….


AFFIDAVIT


I hereby declare that my thesis entitled TARGETING FLATWORM SIGNALING CASCADES FOR THE
DEVELOPMENT OF NOVEL ANTHELMINTHIC DRUGS is the result of my own work. I did not receive
any help or support from third parties, i.e. commercial consultants or others. All sources and/
or material are listed and specified in the thesis.

Furthermore, I verify that this thesis, neither in identical nor similar form, has not yet been
submitted as part of another examination process.

I confirm that the information which I have given in this application is complete and true.




Würzburg,














DANKSAGUNG


An dieser Stelle möchte ich die Gelegenheit nutzen, mich bei denjenigen zu bedanken, die
zum Gelingen dieser Arbeit beigetragen haben – in wahlloser Reihenfolge:


Prof. Dr. Klaus Brehm für die sehr gute Betreuung und permanente Unterstützung dieser
Arbeit, die anschaulichen Diskussionen und die enthusiastischen und lebendigen Ausflüge in
die Parasitenkunde, aber auch in die Geschichte

Prof. Dr. Matthias Frosch

Prof. Dr. Roland Benz und Prof. Dr. Morschhäuser für die Übernahme der Gutachten und die
Teilnahme an der Prüfungskommission

Meiner Trainingsgruppe des internationalen Graduiertenkollegs Würzburg-Nice GCWN 1141

Der Echinokokkenarbeitsgruppe Kerstin Epping, Sabine Lorenz, Dirk Radloff, Sophia Müller,
Sarah Hemer, Ferenc Kiss, Markus Spiliotis und denjenigen, die nicht mehr dabei sind, für
die angenehme Arbeitsatmosphäre und das Ausstatten meines Arbeitsplatzes, vor allem
auch Christian Konrad, Monika Bergmann für die Unterstützung und die fränkischen
Weisheiten, Rainer Brand für die tierisch gute Zusammenarbeit, Michael Ullrich für die
Hilfsbereitschaft und das stets schnelle Beheben von technischen Defekten, den restlichen
Mitarbeitern des Instituts für Hygiene und Mikrobiologie, die mich meine Promotionsstätte in
guter Erinnerung behalten lassen

Xenia Schmidt, Valeska Guth und Carmen Ziegler für die langjährige Freundschaft

Meiner Familie












Contents

1. Summary - 1 -

2. Introduction - 4 -
2.1 Phylogeny and distribution of the genus Echinococcus - 4 -
2.2 Life cycle of E. multilocularis - 6 -
2.3 Alveolar echinococcosis - 8 -
2.3.1 Alveolar echinococcosis and transmission risk - 8 -
2.3.2 Alveolar echinosis and its manifestation - 8 -
2.3.3 Treatment options and chemotherapy - 9 -
2.4 Signaling in developmental and proliferation processes - 10 -
2.4.1 Peptide growth factors and their receptors - 12 -
2.4.2 The Erk1/2 MAPK module - 13 -
2.4.3 The p38 MAPK module - 14 -
2.4.4 The atypical MAPKs - 15 -
2.4.5 Alternative pathways
2.5 Host-parasite interplay during alveolar echinococcosis - 16 -
2.5.1 Early molecular and biochemical approaches - 16 -
2.5.2 In vitro cultivation of E. multilocularis - 17 -
2.5.3 Genomic sequencing project
2.5.4 Evolutionary conserved signaling in E. multilocularis - 18 -
2.6 Aim of the project - 20 -

3. Results - 21 -
3.1 EmMPK2 – the p38 MAPK orthologue of E. multilocularis as a possible target for the
treatment of AE - 21 -
3.1.1 Identification and characterization of emmpk2 - 21 -
3.1.2 Expression analysis of emmpk2 in Echinococcus larval stages - 26 -
3.1.3 Expression and activity analyses of EmMPK2 in Echinococcus larval stages - 27 -
3.1.4 EmMPK2 activation in in vitro cultivated metacestode vesicles - 29 -
3.1.5 EmMPK2 – an enzymatically active MAP kinase - 31 -
3.1.6 Treatment of in vitro cultivated metacestode vesicles with pyridinyl imidazoles - 34 -
3.1.7 TreatmEchinococcus primary cells with pyridinyl imidazoles - 37 -
3.1.8 Treatment of in vitro cultivated protoscoleces with pyridinyl imidazoles - 40 -
3.1.9 Effects of pyridinyl imidazoles on mammalian cells - 42 -
3.2 Treatment of the cestodes E. granulosus and T. crassiceps with pyridinyl imidazoles - 43 -
3.2.1 Partial characterization of the p38 MAPK homologue of E. granulosus - 43 -
3.2.2 The p38 MAPK homologue of T. crassiceps
3.3 EmSSY- an unusual MAPK / a new type of MAPK? - 47 -
3.4 Effects of miltefosine and perifosine on in vitro cultivated parasite material - 50 -
3.4.1 In vitro treatment of E. multilocularis metacestode vesicles - 50 -
3.4.2 In vitront of E. muris primary cells - 51 -
3.5 EmMKK2 - a factor of the E. multilocularis MAP kinase cascade - 52 -
3.5.1 Classification of EmMKK2 as MAPK kinase on the basis of structural features - 52 -
3.5.2 Expression of emmkk2 in E. multilocularis larvae - 57 -
3.5.3 Interaction studies between EmMKK2 and potential members of the E. multilocularis
MAPK cascade - 58 -
3.5.4 In vitro activity of the parasite MAPK cascade - 60 -


I Contents

3.6 Analysing and targeting the EGF signaling pathway of E. multilocularis - 60 -
3.6.1 Phosphorylation of Elp in response to host EGF and the effects of inhibitors - 60 -
3.6.2 Activation of EmMPK1 in response to host EGF - 61 -
3.6.3 Enhanced proliferation of E. multilocularis in response to host EGF - 63 -
3.7 In vitro treatment of metacestodes with tyrosine kinase and MAPK cascade inhibitors - 64 -
3.8 Influence of various host growth factors on vesicle regeneration - 65 -
3.9 EmMPK3 – a serum sensitive MAPK - 70 -
3.10 Activity assay for EmER, the EGF receptor of E. multilocularis - Preliminary data - 72 -

4. Discussion - 73 -

5. Material and methods - 86 -
5.1 Material - 86 -
5.2 Oligonucleotides - 87 -
5.3 Determination of nucleic acid concentration and purity - 90 -
5.4 RNA procedures - 91 -
5.4.1 Isolation of total RNA from mammalian cell lines - 91 -
5.4.2 Isolation of total RNA from E. multilocularis larvae
5.4.3 Decontamination of isolated RNA - 91 -
5.4.4 First strand cDNA synthesis
5.4.5 Synthesis of SMART cDNA - 92 -
5.5 DNA procedures - 92 -
5.5.1 Isolation of chromosomal DNA from the metacestode larval stage with purification - 92 -
5.5.2 BrdU-Staining
5.5.3 DAPI staining of DNA blotted on nitrocellulose membrane - 93 -
5.5.4 Isolation of plasmid DNA from E. coli
5.5.5 Gel electrophoresis of DNA - 93 -
5.5.6 Purification of DNA - 94 -
5.5.7 DNA precipitation
5.5.8 Sequencing - 94 -
5.5.9 Amplification of DNA via PCR
5.5.10 Rapid amplification of cDNA ends (RACE) - 95 -
5.5.11 Semi-quantitative RT PCR
5.5.12 TA cloning - 95 -
5.5.13 Colony-PCR
5.5.14 Restriction digest of DNA - 95 -
5.5.15 Ligation of DNA fragments - 96 -
5.6 Protein procedures - 96 -
5.6.1 Determination of protein concentration - 96 -
5.6.2 In vitro activity assay with myelin basic protein as substrate
5.6.3 In vitro activity assay for the MAPK cascade - 96 -
5.6.4 Co-immunoprecipitation - 97 -
5.6.5 SDS-PAGE
5.6.6 Coomassie staining of protein gels - 98 -
5.6.7 Western blotting
5.7 Working with bacteria - 99 -
5.7.1 Bacteria strains and media - 99 -
5.7.2 Chemically competent E. coli - 100 -
5.7.3 E. coli transformation
5.7.4 Heterologous expression in E. coli and purification of the recombinant proteins - 100 -
II Contents

5.8 Working with yeast - 102 -
5.8.1 Yeast strains and media - 102 -
5.8.2 Yeast two hybrid analysis - 103 -
5.9 Working with mammalian cell lines - 104 -
5.9.1 Cell lines and media - 104 -
5.9.2 Quantitative live-dead staining o