@phdthesis{Winkler2015,
  author    = {Winkler, Ann-Cathrin Nicole},
  title     = {Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Staphylococcus aureus is both a human commensal and a pathogen. 20\%-30\% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options. In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen. In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death. In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger. All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys. Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@article{UeceylerBikoSommer2010,
  author    = {Ueceyler, Nurcan and Biko, Lydia and Sommer, Claudia},
  title     = {MDL-28170 Has No Analgesic Effect on CCI Induced Neuropathic Pain in Mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68359},
  year      = {2010},
  abstract  = {The calpain inhibitor MDL-28710 blocks the early local pro-inflammatory cytokine gene expression in mice after chronic constriction nerve injury (CCI). Onehundred- thirteen wild type mice of C57Bl/6J background received CCI of the right sciatic nerve. Mechanical paw withdrawal thresholds and thermal withdrawal latencies were investigated at baseline and at 1, 3, and 7 days after CCI. Three application regimens were used for MDL-28170: a) single injection 40 min before CCI; b) serial injections of MDL- 28170 40 min before and up to day three after CCI; c) sustained application via intraperitoneal osmotic pumps. The control animals received the vehicle DMSO/PEG 400. The tolerable dose of MDL-28170 for mice was 30 mg/kg body weight, higher doses were lethal within the first hours after application. Mechanical withdrawal thresholds and thermal withdrawal latencies were reduced after CCI and did not normalize after single or serial injections, nor with application of MDL-28170 via osmotic pumps. Although the calpain inhibitor MDL-28170 inhibits the early local cytokine upregulation in the sciatic nerve after CCI, pain behavior is not altered. This finding implies that local cytokine upregulation after nerve injury alone is only one factor in the induction and maintenance of neuropathic pain.},
  subject      = {Medizin},
  language  = {en}
}
@phdthesis{Becher2006,
  author    = {Becher, Jan},
  title     = {Untersuchung des Calcineurin Signalweges bei der Myokardhypertrophie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-21889},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2006},
  abstract  = {Die Myokardhypertrophie ist eine Anpassungsreaktion des Herzens auf verschiedene pathologische Stimuli wie der arteriellen Hypertonie, Herzklappen-Vitien, Myokardinfarkt, endokrine Stoffwechselst{\"o}rungen sowie Mutationen von Genen kontraktiler Proteine. Die Antwort der Herzmuskelzelle auf einen hypertrophen Reiz ist gekennzeichnet durch ein verst{\"a}rktes Zellwachstum ohne Zellteilung und auf molekularer Ebene durch die Induktion fetaler kardialer Gene kontraktiler Proteine. Auch wenn {\"u}ber den klinischen Aspekt der Myokardhypertrophie bereits ein umfangreiches Wissen vorliegt, wurden Details {\"u}ber die bei der Hypertrophie ablaufenden intrazellul{\"a}ren Signalwege erst in den letzten Jahren entdeckt. Der Calcineurin/NFAT Signalweg ist bei der Entstehung der Herzhypertrophie von großer Bedeutung. Die Aktivierung der Ca²+-Calmodulin-abh{\"a}ngigen Phosphatase Calcineurin f{\"u}hrt zur einer Dephosphorylierung des Transkriptionsfaktors NFAT. Dies erlaubt die nukle{\"a}re Translokation, was zur einer Induktion von Genen f{\"u}hrt, welche an der Entstehung der Myokardhypertrophie beteiligt sind. Nach dem heutigen Verst{\"a}ndnis ben{\"o}tigt die Aktivierung des Calcineurin/NFAT-Signalweges die Bindung von Calmodulin sowie dauerhaft erh{\"o}hte Ca²+-Spiegel. Die Folge der Calcineurinaktivierung ist eine strukturelle Ver{\"a}nderung mit einem Shift der C-terminalen Autoinhibitorischen Dom{\"a}ne, so dass das aktive Phosphatase-Zentrum freigegeben wird. In dieser Arbeit untersuchen wir einen Mechanismus der gezielten proteolytischen Abspaltung der Autoinhibitorischen Dom{\"a}ne, welcher ebenfalls zu einer Enzymaktivierung f{\"u}hrt. Die Stimulation von Ratten-Kardiomyozyten mit Angiotensin II f{\"u}hrte zu einer Erh{\"o}hung der Calpain-Aktivit{\"a}t, was eine proteolytische Abspaltung der autoinhibitorischen Dom{\"a}ne von Calcineurin bedingte. Durch die Blockierung von Calpain konnte die Proteolyse verhindert werden. Die Calcineurin-Aktivit{\"a}t war nach Angiotensin II-Stimulation erh{\"o}ht (310±29\%) und bleib auch nach Wegnahme des Stimulus auf erh{\"o}htem Niveau (214±17\%). Wurde dem Medium w{\"a}hrend der Stimulation Calpain-Inhibitor hinzu gegeben, kam es nach Wegnahme des Angiotensin II-Stimulus zu einem deutlichen Absinken (110±19\%) der Calcineurin-Aktivit{\"a}t. An Hand von immunhistochemischen F{\"a}rbungen und von Transfektionsversuchen mit einem GFP-fusionierten Calcineurin konnte gezeigt werden, dass die Angiotensin II-Stimulation eine nukle{\"a}re Translokation von Calcineurin bewirkt. Die Calpain-Blockierung und somit die Unterdr{\"u}ckung des Calpain vermittelten Verdaus von Calcineurin erlaubt es diesem, den Zellkern wieder zu verlassen. Zusammenfassend zeigen die Ergebnisse dieser Arbeit, dass die Angiotensin II-Stimulation der Kardiomyozyten zu einer Erh{\"o}hung der Calpain-Aktivit{\"a}t f{\"u}hrt. Diese wiederum bedingt eine proteolytische Abspaltung der Autoinhibitorischen Dom{\"a}ne von Calcineurin. Die Folge ist eine erh{\"o}hte Calcineurin-Aktivit{\"a}t, was eine nukle{\"a}re Translokation von Calcineurin ausl{\"o}st. Nach dem Verlust der Autoinhibitorischen Dom{\"a}ne ist Calcineurin dauerhaft aktiv und nukle{\"a}r lokalisiert, sogar nach Wegnahme des Hypertrophie-ausl{\"o}senden Stimulus.},
  language  = {de}
}