@phdthesis{Clemen2010,
  author    = {Clemen, Holger},
  title     = {Analyse der Expression und m{\"o}glicher signalinduzierender Eigenschaften des CD1d-Molek{\"u}ls der Ratte},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-65242},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Wie MHC Klasse I und II-Molek{\"u}le pr{\"a}sentieren CD1d-Molek{\"u}le dem TCR Antigene, allerdings Lipide und Glykolipide und nicht Proteinfragmente. Die Entdeckung der massiven TH1- und TH2-Zytokinproduktion von Typ I-NKT-Zellen nach CD1d-vermittelter Erkennung von α-Galactosylceramid, einem aus dem Meeresschwamm gewonnenen Glykosphingolipid, weckte großes Interesse an ihrem immunregulatorischen Potential und ihrem m{\"o}glichen Nutzen f{\"u}r neue Immun- und Tumortherapien. Um die Funktion und die Bedeutung von CD1d besser zu verstehen, wurden in dieser Arbeit die Expressionslevel der lymphatischen Gewebe der Ratte und der Maus untersucht. Hierf{\"u}r wurden die neu generierten monoklonalen Antik{\"o}rper 232 und 58/4 verwendet, die die CD1d-Molek{\"u}le von Ratte und Maus binden und so den direkten Vergleich beider Spezies erm{\"o}glichen. Sowohl die isolierten Zellen des Thymus und der Milz als auch des Lymphknotens waren in der LEW- und F344-Ratte sowie in der BALB/c-Maus schwach bis stark CD1d positiv. In der LEW-Ratte und in der F344-Ratte wiesen jeweils ca. 18\% der Milzzellen eine vergleichsweise erh{\"o}hte CD1d-Expression auf. Dabei handelte es sich in erster Linie um Marginalzonen-B-Zellen. Bestimmte Subpopulationen der Dendritischen Zellen und vermutlich Makrophagen stellten die restlichen CD1d stark positiven Populationen dar. Nur ca. 2\% der isolierten Zellen der Lymphknoten der LEW-Ratte waren stark CD1d positiv, wohingegen der LEW-Thymus gem{\"a}ß dem noch geringeren Anteil an APC kaum Zellen mit erh{\"o}hter CD1d-Expression enthielt. In der BALB/c-Maus war der Anteil CD1d stark positiver Milzzellen mit 4\% deutlich geringer als in der LEW- oder F344-Milz. Abgesehen von MZ-B-Zellen konnten in der Maus kaum Populationen mit starker CD1d-Expression in den verschiedenen F{\"a}rbungen festgestellt werden. Demnach stellt CD1d sowohl in der Ratte als auch in der Maus einen guten Marker f{\"u}r MZ-B-Zellen dar. Demgegen{\"u}ber zeigten vereinzelt kleine Populationen der Milz, des Lymphknotens und des Thymus beider Spezies eine verminderte oder gar keine CD1d-Expression. Zur Analyse m{\"o}glicher signalinduzierender Eigenschaften der verschiedenen Anti- CD1d-Antik{\"o}rper wurden ihre Effekte auf rCD1d+ Transduktanten und prim{\"a}re Zellen untersucht. 58/4 konnte im Gegensatz zu 232 spezifisch {\"u}ber Bindung an Ratten- CD1d Zelltod und Aggregatbildung in Tumor-B-Zellen des Menschen und der Maus, aber nicht in Tumor-T-Zellen, induzieren. Der zytoplasmatische Schwanz der CD1d-Molek{\"u}le scheint an der Aggregatbildung beteiligt zu sein. Die Bindung von 58/4 oder 232 f{\"u}hrte in {\"u}berlebenden rCD1d+ Raji-Zellen zu einer {\"a}hnlich starken Internalisierung der CD1d-Molek{\"u}le. W{\"a}hrend nach 5-st{\"u}ndiger Inkubation mit 232 und erneuter CD1d-F{\"a}rbung wieder die vorherige CD1d- Expression festgestellt wurde, konnte nach Inkubation mit 58/4 eine bleibende Herunterregulierung beobachtet werden. Folglich bewirkte 58/4 ein anderes bzw. st{\"a}rkeres Signal in den Zellen als 232. Diese Beobachtungen st{\"u}tzen die Signaltransduktion als m{\"o}gliche weitere Funktion der CD1d-Molek{\"u}le neben der Antigenpr{\"a}sentation und definieren die monoklonalen Antik{\"o}rper 232 und 58/4 als n{\"u}tzliche Werkzeuge f{\"u}r weitere Studien zur Analyse der molekularen Mechanismen der CD1d-vermittelten Signaltransduktion. Das Verst{\"a}ndnis solcher Mechanismen bildet wiederum die Grundlage f{\"u}r die Entwicklung neuer Therapien z. B. zur Eliminierung CD1d exprimierender Tumore.},
  subject      = {Zelltod},
  language  = {de}
}
@phdthesis{Hondke2014,
  author    = {Hondke, Sylvia},
  title     = {Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109641},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {WISP3 is a member of the CCN family which comprises six members found in the 1990's: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes. WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis. Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.},
  subject      = {Knorpelzelle},
  language  = {en}
}
@phdthesis{Martens2011,
  author    = {Martens, Theresa Maria Christina},
  title     = {Lobaplatin als Agens zur Induktion von Zelltod in triple-negativen Brustkrebszelllinien},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71974},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2011},
  abstract  = {Das triple-negative Mammakarzinom ist Hormonrezeptor- und HER2 negativ und mit einer ung{\"u}nstigen Prognose verbunden. Triple-negative Patientinnen sind unempfindlich gegen die endokrine Therapie und den HER-2/neu Antik{\"o}rper Trastuzumab und deshalb auf die zytostatische Chemotherapie angewiesen. Lobaplatin ist ein Platinderivat mit wenigen Nebenwirkungen und guter Antitumorwirkung in vitro und deshalb m{\"o}glicherweise auch f{\"u}r die Behandlung triple-negativer Tumoren geeignet. F{\"u}r die vorliegende Arbeit haben wir zwei triple-negative und eine klassische Brustkrebszelllinie verwendet. Die beiden triple-negativen Zelllinien wurden anhand einer vorbeschriebenen Mutation identifiziert, um eine Verwechslung auszuschließen. Die Aktivit{\"a}t des p53 Gens wurde in allen drei Zelllinien {\"u}berpr{\"u}ft, ein aktives p53 Gen jedoch nur in der hormonrezeptor-positiven MCF7 Zelllinie nachgewiesen. Ein Zusammenhang zwischen p53 Status und Ansprechen der Zellen auf Lobaplatin oder Cisplatin konnte nicht belegt werden. Wir haben die zytotoxische Wirkung von Lobaplatin und Cisplatin verglichen. Lobaplatin war in unseren Untersuchungen {\"a}hnlich wirksam wie Cisplatin und bei der Behandlung der MCF7 Zelllinie und einer Cisplatin-resistenten Ovarialkarzinomzelllinie sogar dem Cisplatin {\"u}berlegen. Lobaplatin wies in diesen Zellen keine komplette Kreuzresistenz zu Cisplatin auf. In einem n{\"a}chsten Schritt wurden unterschiedliche Zytostatika mit Lobaplatin kombiniert. Additive Synergien zwischen den einzelnen Substanzen konnten nicht bewiesen werden, jedoch war ein gutes Zusammenwirken von Lobaplatin und dem in der klinischen Erprobung befindlichen Todesliganden TRAIL zu erkennen. Mittels Nachweis von Spaltprodukten haben wir gezeigt, dass Lobaplatin und Cisplatin eine Aktivierung von Caspasen bewirken. Die aktivierte Caspase 3 konnte allerdings nicht dargestellt werden. Die Begriffe Apoptose und Nekroptose wurden diskutiert und eine Beteiligung beider Prozesse an der Wirkung von Lobaplatin und Cisplatin nachgewiesen. Ver{\"a}nderungen des Zellzyklus wurden untersucht. Die beiden Platinderivate f{\"u}hrten zu einem Arrest in der G2-Phase und in h{\"o}heren Konzentrationen teilweise zu einem G1-Arrest. Insgesamt sind an der zytotoxischen Wirkung von Lobaplatin und Cisplatin und der Entscheidung {\"u}ber Leben oder Tod viele wichtige Mechanismen in wechselnder Rangfolge beteiligt. In unseren Versuchen an zwei triple-negativen und einer klassischen Brustkrebszelllinie erweist sich Lobaplatin als ebenb{\"u}rtiger Nachfahre des Cisplatin mit einer vergleichbaren Zytotoxizit{\"a}t, weniger schwerwiegenden Nebenwirkungen und der F{\"a}higkeit, vorhandene Cisplatinresistenzen teilweise zu umgehen. Lobaplatin verspricht dar{\"u}ber hinaus in Kombination mit anderen Zytostatika interessante therapeutische Perspektiven, die bisher nur unzureichend erprobt sind.},
  subject      = {Brustkrebs},
  language  = {de}
}
@phdthesis{Niederlechner2013,
  author    = {Niederlechner, Stefanie},
  title     = {Assessment of the basic molecular mechanisms underlying L-glutamine's cytoprotective effects after intestinal injury},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77399},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Critical illness like sepsis, shock, and intestinal bowel disease are one of the leading causes of morbidity and mortality in the US and around the world. At present, studies to define new therapeutic interventions that can protect tissues and cells against injury and attenuate inflammation are fields of intense investigation. While research over the past decade has clearly identified GLN as a vital stress substrate facilitating cellular survival following injury, the initiation steps in GLN's cytoprotective molecular mechanism still remain elusive. Previously published work suggested that stabilization of ECM proteins and activation of ECM receptor osmosignaling may play a central role in the orchestration of many cellular pathways following stress. Thus, I hypothesized that preservation of ECM protein and EGFR levels as well as ECM receptor signaling play key roles in the molecular mechanisms underlying GLN's protection against thermal injury in the intestine. I was able to confirm via Western blotting and by using silencing RNA against FN, Ntn-1, EGFR, and their negative controls, that GLN-mediated preservation of FN, Ntn-1, and EGFR levels is critical in GLN's protection against hyperthermia in IEC-6 cells. By using a selective FN-Integrin interaction inhibitor GRGDSP, its negative control peptide GRGESP, and Src-kinase inhibitor PP2, I showed that FN-Integrin signaling and Src-kinase activation are essential in GLN-mediated protection in the intestine. This applied to EGFR signaling as demonstrated using the EGFR tyrosine kinase inhibitor AG1478. In addition to GRGDSP and AG1478, ERK1/2 inhibitors PD98059 and UO126 as well as the p38MAPK inhibitor SB203580 revealed that GLN is protective by activating ERK1/2 and dephosphorylating p38MAPK via FN-Integrin and EGFR signaling. However, GLN-mediated PI3-K/Akt/Hsp70 activation seems to occur independently of FN-Integrin and EGFR signaling as indicated by Western blots as well as experiments using the PI3-K inhibitor LY294002, GRGDSP, and AG1478. The results showed that GLN activates cell survival signaling pathways via integrins as well as EGFRs after hyperthermia. Moreover, I found that GLN-mediated preservation of FN expression after HS is regulated via PI3-K signaling. Whether GLN-mediated PI3-K signaling happens simultaneously to FN-Integrin and EGFR signaling or whether PI3-K signaling coordinates FN-Integrin and EGFR signaling needs to be investigated in future studies. Further, experiments with PD98059 and GRGDSP revealed that ERK1/2 assists in mediating transactivation of HSF-1 following HS. This leads to increases in Hsp70 expression via FN-Integrin signaling, which is known to attenuate apoptosis after thermal injury. Fluorescence microscopy results indicated that HS and GLN regulate cell are size changes and the morphology of F-actin via FN-Integrin signaling. Experiments using GRGDSP and GRGESP showed that GLN enhances cellular survival via FN-Integrin signaling in a manner that does not require increased intracellular GLN concentrations (as quantified using LC-MS/MS). In summary, my thesis work gives new and potentially clinically relevant mechanistic insights into GLN-mediated molecular cell survival pathways. These results warrant clinical translation to assess if clinical outcome of critically ill patients suffering from gastrointestinal diseases can be improved by GLN treatment and/or by targeting the molecular pathways found in my studies.},
  subject      = {Glutamin},
  language  = {en}
}
@phdthesis{Stelzner2020,
  author    = {Stelzner, Kathrin},
  title     = {Identification of factors involved in Staphylococcus aureus- induced host cell death},
  doi       = {10.25972/OPUS-18899},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-188991},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Staphylococcus aureus is a Gram-positive commensal bacterium, that asymptomatically colonizes human skin and mucosal surfaces. Upon opportune conditions, such as immunodeficiency or breached barriers of the host, it can cause a plethora of infections ranging from local, superficial infections to life-threatening diseases. Despite being regarded as an extracellular pathogen, S. aureus can invade and survive within non-phagocytic and phagocytic cells. Eventually, the pathogen escapes from the host cell resulting in killing of the host cell, which is associated with tissue destruction and spread of infection. However, the exact molecular mechanisms underlying S. aureus-induced host cell death remain to be elucidated. In the present work, a genome-wide haploid genetic screen was performed to identify host cell genes crucial for S. aureus intracellular cytotoxicity. A mutant library of the haploid cell line HAP1 was infected with the pathogen and cells surviving the infection were selected. Twelve genes were identified, which were significantly enriched when compared to an infection with a non-cytotoxic S. aureus strain. Additionally, characteristics of regulated cell death pathways and the role of Ca2+ signaling in S. aureus-infected cells were investigated. Live cell imaging of Ca2+ reporter cell lines was used to analyze single cells. S. aureus-induced host cell death exhibited morphological features of apoptosis and activation of caspases was detected. Cellular H2O2 levels were elevated during S. aureus intracellular infection. Further, intracellular S. aureus provoked cytosolic Ca2+ overload in epithelial cells. This resulted from Ca2+ release from endoplasmic reticulum and Ca2+ influx via the plasma membrane and led to mitochondrial Ca2+ overload. The final step of S. aureus-induced cell death was plasma membrane permeabilization, a typical feature of necrotic cell death. In order to identify bacterial virulence factors implicated in S. aureus-induced host cell killing, the cytotoxicity of selected mutants was investigated. Intracellular S. aureus employs the bacterial cysteine protease staphopain A to activate an apoptosis-like cell death characterized by cell contraction and membrane bleb formation. Phagosomal escape represents a prerequisite staphopain A-induced cell death, whereas bacterial intracellular replication is dispensable. Moreover, staphopain A contributed to efficient colonization of the lung in a murine pneumonia model. In conclusion, this work identified at least two independent cell death pathways activated by intracellular S. aureus. While initially staphopain A mediates S. aureus-induced host cell killing, cytosolic Ca2+-overload follows later and leads to the final demise of the host cell.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}