@phdthesis{Aurbach2021,
  author    = {Aurbach, Katja},
  title     = {Studies on the role of the cytoskeleton in platelet production},
  doi       = {10.25972/OPUS-23466},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-234669},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Platelets are small anucleated cell fragments that originate from megakaryocytes (MKs), which are large cells located in the bone marrow (BM). MKs extend long cytoplasmic protrusions, a process which is called proplatelet formation, into the lumen of the sinusoidal vessels where platelets are sized by the bloodstream. During the process of platelet biogenesis, segments of the MK penetrate the endothelium and, through cytoskeletal remodeling inside the MK, proplatelet fragments are released. Rho GTPases, such as RhoA and RhoB, are critically involved in cytoskeletal rearrangements of both the actin and the tubulin cytoskeleton. The first part of this thesis concentrated on the protein RhoB and its involvement in cytoskeletal organization in MKs and platelets. Single knockout (KO) mice lacking RhoB had a minor microthrombocytopenia, which means a smaller platelet size and reduced platelet number, accompanied by defects in the microtubule cytoskeleton in both MKs and platelets. In particular, tubulin organization and stability, which is regulated by posttranslational modifications of α-tubulin, were disturbed in RhoB-/- platelets. In contrast, RhoB-/- MKs produced abnormally shaped proplatelets but had unaltered posttranslational modifications of α-tubulin. The second part focused on the influence of RhoA and RhoB on MK localization and platelet biogenesis in murine BM. Many intact RhoA-/- MKs are able to transmigrate through the endothelial layer and stay attached to the vessel wall, whereas only 1\% of wildtype (wt) MKs are detectable in the intrasinusoidal space. Concomitant deficiency of RhoA and RhoB reverts this transmigration and results in macrothrombocytopenia, MK clusters around the vessel in the BM and defective MK development. The underlying mechanism that governs MKs to distinct localizations in the BM is poorly understood, thus this thesis suggests that this process may be dependent on RhoB protein levels, as RhoA deficiency is coincided with increased RhoB levels in MKs and platelets. The third part of this thesis targeted the protein PDK1, a downstream effector of Rho GTPases, in regard to MK maturation and polarization throughout thrombopoiesis. MK- and platelet-specific KO in mice led to a significant macrothrombocytopenia, impaired actin cytoskeletal reorganization during MK spreading and proplatelet formation, with defective MK maturation. This was associated with decreased PAK activity and, subsequently, phosphorylation of its substrates LIMK and Cofilin. Together, the observations of this thesis highlight the importance of Rho GTPases and their downstream effectors on the regulation of the MK and platelet cytoskeleton.},
  subject      = {Megakaryozyt},
  language  = {en}
}
@phdthesis{Nehring2021,
  author    = {Nehring, Helene},
  title     = {Role of cholesterol intermediates in supporting cell survival},
  doi       = {10.25972/OPUS-21763},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217631},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Cell death is an essential aspect of life that plays an important role for successful development and tissue remodeling as well as for diseases. There are several different types of cell death that differ from each other in morphological, functional and biochemical ways. Regulated cell death that occurs in physiological processes is generally equated with programmed cell death (PCD), whereby apoptosis is the most studied form of PCD. Ferroptosis is a form of regulated cell death and unique in its requirements for iron and lipid peroxidation. It is linked to numerous biological processes, such as amino acid metabolism, phospholipid metabolism and sterol synthesis. Cholesterol biosynthesis is a complex pathway with a large number of enzymes and substrates that are potential target points for cellular dysfunctions. Motivated by the results from a CRISPR-based genetic screening in this thesis, we focused on 7-dehydrocholesterol reductase (DHCR7), the enzyme responsible for conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. In this work we focused on the ferroptosis sensitive cell line HT1080 and generated a series of models to address the importance of DHCR7 in ferroptosis. Using CRISPR/Cas9, HT1080 DHCR7_KO and DHCR7/SC5D_KO cell lines were generated and used to validate their sensitivity against ferroptosis inducers and sterol consumption. We could show that 7-DHC is a strong antiferroptotic agent that could prevent cell death in genetic models as well as when supplemented directly to cells. Importantly, all the results obtained were subsequently confirmed in isogenic reconstituted pairs from the HT1080 DHCR7/SC5D_KO. Moreover, we demonstrate that this protective effect is not due to an inherent and unspecific resistance as the sensitivity to non-ferroptotic stimuli was equally effective in killing the HT1080 DHCR7_KO and DHCR7/SC5D_KO cell lines. We could also show that selenium present in the media has a strong impact on the activity of 7-DHC and this is because in its absence the effective concentration is rapidly decreased. Surprisingly we also demonstrate that removing sterol from cell culture triggers ferroptosis in cells unable to synthesize 7-DHC, suggestive that this could be used as a novel mechanism to trigger ferroptosis. Ultimately, in the present work we could show that unlike previously reported, 7-DHC is not only a toxic intermediate of the cholesterol biosynthesis pathway but under specific circumstances it has a strong pro-survival effect.},
  subject      = {Zelltod},
  language  = {en}
}
@phdthesis{Stetter2021,
  author    = {Stetter, Maurice},
  title     = {LC3-associated phagocytosis seals the fate of the second polar body in \(Caenorhabditis\) \(elegans\)},
  doi       = {10.25972/OPUS-23198},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-231981},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {This work investigates the death and degradation of the second polar body of the nematode C. elegans in order to improve our understanding how pluripotent undifferentiated cells deal with dying cells. With the use of fluorescence microscopy this work demonstrates that both polar bodies loose membrane integrity early. The second polar body has contact to embryonic cells and gets internalized, dependent on the Rac1-ortholog CED-10. The polar body gets degraded via LC3-associated phagocytosis. While lysosome recruitment depends on RAB-7, LC3 does not improve lysosome recruitment but still accelerates polar body degradation. This work establishes the second polar body as a genetic model to study cell death and LC3-associated phagocytosis and has revealed further aspects of phagosome maturation and degradation.},
  subject      = {Polk{\"o}rper},
  language  = {en}
}