@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}
}
@phdthesis{Grimm2019,
  author    = {Grimm, Johannes},
  title     = {Autocrine and paracrine effects of BRAF inhibitor induced senescence in melanoma},
  doi       = {10.25972/OPUS-18116},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181161},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels. In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.},
  subject      = {Inhibitor},
  language  = {en}
}