| dc.contributor.advisor | Lüder, Carsten Prof. Dr. | de |
| dc.contributor.author | Graumann, Kristin | de |
| dc.date.accessioned | 2013-01-14T15:07:25Z | de |
| dc.date.available | 2013-01-30T23:50:52Z | de |
| dc.date.issued | 2012-11-22 | de |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-000D-EF86-2 | de |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-1476 | |
| dc.format.mimetype | application/pdf | de |
| dc.language.iso | ger | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/3.0/ | de |
| dc.title | Hemmung der Cytochrom c-induzierten Caspase-Aktivierung durch Toxoplasma gondii in vitro und in vivo:molekulare Mechanismen und parasitäre Effektormoleküle | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Groß, Uwe Prof. Dr. | de |
| dc.date.examination | 2012-04-16 | de |
| dc.subject.dnb | 000 Allgemeines, Wissenschaft | de |
| dc.subject.gok | WU 000 | de |
| dc.description.abstracteng | Toxoplasma gondii is an obligate
intracellular parasite and is able to infect almost all nucleated
cells of humans and warm-blooded animals. Infection of
immunocompetent individuals is mostly asymptomatic and leads to a
lifelong persistence of the parasite particularly in the central
nervous system and muscle cells of the host. T. gondii has evolved
different strategies to evade the immune system of the host and to
facilitate its own intracellular survival. Apoptosis is a common
form of programmed cell death which eliminates damaged or
unnecessary cells in a multicellular organism. Apoptosis also
represents an important defense mechanism of both innate and
adaptive immune responses to combat intracellular pathogens. Some
pathogens including T. gondii are able to inhibit apoptosis of its
host cell and thereby facilitate its own survival and development
in the host. T. gondii inhibits cytochrome c-induced activation of
the caspase 9-caspase 3/7-cascade in a cell-free system in a
dose-dependent manner. In the present study it could be shown for
the first time that inhibition of cytochrome c-induced caspase
3/7-activity by the parasite also plays an important role in
infected cells. Studies in caspase 9-deficient Jurkat cells and
caspase 9-retransfected cells showed a 30% stronger inhibition of
apoptosis in caspase 9-expressing cells than in caspase 9-deficient
cells thus indicating that this level of inhibition was due to
parasite interference with caspase 3/7-activation. After
electroporation of Jurkat cells in medium with and without
cytochrome c, it was shown that the cytochrome c-induced caspase
3/7-activity was completely inhibited by the parasite without
affecting cytosolic levels of cytochrome c in the host cell. In
addition, we observed a dose-dependent inhibition of cytochrome
c-induced caspase 3/7-activity in cytosolic lysates from cells
infected with increasing numbers of the parasite. Efforts to
unravel the molecular mechanism of the reduced activation of
cytochrome c-induced caspase-activation in the presence of T.
gondii showed that the recruitment of caspase 9 to the apoptosis
activating factor-1 (APAF-1) and therefore activation of the
initiator caspase 9 was inhibited by the parasite. In a
reconstituted test system consisting of recombinant APAF-1, caspase
9 and cytochrome c, complete lysate of T. gondii inhibited
cytochrome c-induced activation of caspase 9 dose-dependently.
Thus, in a test system with defined components, our result
displayed for the first time that T. gondii inhibits activation of
caspase 9 and even blocks this activation after the apoptosome was
already formed. The cell-free system was also used to characterize
effector proteins of the T. gondii-mediated inhibition of the
caspase cascade. In previous studies, NTPase and HSP70 of T. gondii
were identified as possible effectors for the anti-apoptotic effect
of the parasite. This study revealed that the NTPase of T. gondii
indeed represents an anti-apoptotic protein of the parasite.
Immunodepletion of NTPase from T. gondii lysate showed a
dose-dependent abrogation of the inhibitory effect of complete
parasite lysate on cytochrome c-induced caspase activity.
Investigations with a non-hydrolyzable ATP analogon, App(NH)p, as
well as use of high concentration of ATP indicated that the
hydrolase-activity of T. gondii NTPase was not required for the
inhibition of cytochrome c-induced caspase activation. Clonal
lineages of T. gondii express different isoforms of NTPase with
different hydrolase activities. However, investigations on a
possible strain specificity of the parasite-mediated inhibition of
the caspase cascade showed comparable inhibition by members of the
three different clonal lineages on cytochrome c-induced caspase
activation. This inhibition thus occurred independent of the
expression of a specific isoform of NTPase. In this study,
recombinant NTPase and HSP70 of T. gondii were expressed in E.
coli. Bacterial expressed T. gondii HSP70 had no effect on
cytochrome c-induced caspase 3/7 activity. In contrast, after
purification under denaturing conditions, recombinant T. gondii
NTPase showed an inhibition of cytochrome c-induced caspase
activity. Furthermore, a functionally active NTPase inhibited
cytochrome c-induced activation of caspase 3/7 dose-dependently and
at a concentration of 0,5 | de |
| dc.contributor.coReferee | Braus, Gerhard Prof. Dr. | de |
| dc.subject.topic | Biology (incl. Psychology) | de |
| dc.subject.ger | Toxoplasma gondii | de |
| dc.subject.ger | Apoptose | de |
| dc.subject.ger | NTPase | de |
| dc.subject.ger | Cytochrom c-induzierte Caspase-Aktivität | de |
| dc.subject.eng | Toxoplasma gondii | de |
| dc.subject.eng | apoptosis | de |
| dc.subject.eng | NTPase | de |
| dc.subject.eng | cytochrome c-induced caspase-activity | de |
| dc.subject.bk | 42.30 | de |
| dc.subject.bk | 44.43 | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-webdoc-3805-1 | de |
| dc.identifier.purl | webdoc-3805 | de |
| dc.affiliation.institute | Biologische Fakultät | de |
| dc.identifier.ppn | 737897732 | de |