Inhalational anthrax is an acute infectious disease caused by exposure of the lungs to B. anthracis spores. Alveolar macrophages engulf spores causing them to germinate to the vegetative form of B. anthracis, which secretes edema toxin (ET)and lethal toxin (LT). The pathogenesis of inhalational anthrax is characterized by flu-like symptoms, respiratory distress, meningitis and shock, which is fatal in almost all cases.

The mechanism behind the respiratory distress is not well understood. Therefore, our goal was to determine the effects of lethal toxin in the human lung epithelium. To

study alterations in a more physiological setting, we developed a differentiated, polarized lung epithelial system. Lethal toxin exposure disrupted the lung barrier

function and wound healing. Assembly defects of junction proteins and additional multicellular junction sites resulted in a higher permeability. Pretreatment with

keratinocyte growth factor (KGF) and dexamethasone increased the viability, resulting in the rescue of the permeability changes.

Upon LT treatment, a more rigid cytoskeleton was observed, evidenced by enhanced actin stress fiber formations and tubulin stabilization. Cytoskeleton and adhesion

alterations prevented the epithelial cells from polarization, directed migration, and wound healing. The MAPK pathway and Cdc42 activity might be partially responsible for these motility defects.

Lethal toxin is known to induce rapid cell death in murine macrophages. In contrast, human epithelial cells are more resistant to the cytotoxic effect of LT. By following

the growth of epithelial cells after LT treatment, we observed inhibited cell proliferation due to a cell cycle arrest in the G1 phase.

Surprisingly, biotinylated lethal factor did not induce cytotoxicity in murine macrophages. This is not due to an internalization or proteolytic activity defect; instead changes in the mitochondrial potential and proteasome activity were observed. Biotinylated LT did not reduce proteasome activity as seen in LT treated cells and caused hypopolarization of the mitochondria. However, it is possible that biotinylation of lethal toxin could prevent interaction of LT with proteins that induce cell death.

The major challenge for anthrax treatment is to find a treatment, which can act faster, is easy to use and can bring patient out of the dangerous physiological state

in late pathogenesis. Our study has implications in saving the viability and barrier function of lung epithelial cells. One can devise better dosage and delivery of KGF

and dexamethasone as treatment modality for post anthrax exposure to reduce respiratory distress. Furthermore, overcoming the cell cycle arrest by the development of a drug would reduce the damage of lung epithelial cells and induce proliferation. The discovery that biotinylated LT is non-toxic to murine macrophages could revolutionize treatment of anthrax infection. Exploring the types of posttranslational modifications of LT that decrease toxicity and finding the mechanism behind it might, lead to therapies that directly counteract the effects of the lethal toxin in vivo.

Kohlenhydrate gehören zu den Biomolekülen mit dem mengenmäßig größten Anteil in der Natur, jedoch wurden sie lange Zeit fast ausschließlich als Energiespeicher, Stütz- und Gerüstsubstanzen betrachtet. Die in den letzten Jahren stetig wachsenden Erkenntnisse ihrer essentiellen Bedeutung in wichtigen biologischen Prozessen und die damit verbundenen vielversprechenden Anwendungsmöglichkeiten als Impfstoffe und Medikamente gegen eine Reihe von Krankheiten wie zum Beispiel Krebs, Aids, Diabetes oder Alzheimer führen jedoch zu einem immer größer werdenden Interesse dieser Stoffklasse.

Die vorliegende Dissertation gibt einen umfassenden und systematischen Einblick in die Koordinationsmöglichkeiten von Kohlenhydraten an Palladium(II). Dabei erweist sich Palladium(II) in besonderem Maße als ein Zentralatom, das in der Lage ist, die Isomeren- und Konformerenverteilung von Monosacchariden drastisch zu verschieben und so Formen zugänglich zu machen, die ohne Komplexierung nur schwer oder gar nicht nachweisbar sind. Palladium(II) ermöglicht nicht nur stabile Komplexe sowohl mit Pyranosen als auch mit Furanosen in den verschiedensten Bindungsmodi aufzubauen, sondern dient darüber hinaus, durch die in Abhängigkeit des jeweiligen Bindungsmodus auftretenden charakteristischen CIS-Werte der 13C-NMR-Signale, als „Sonde“ für die Koordination an ein bestimmtes Kohlenhydratisomer. Die erhaltenen Ergebnisse erweitern grundlegend die Kenntnisse der Koordinationschemie auf diesem Gebiet und können auch aus pharmazeutischer und katalytischer Sicht von Bedeutung sein.

Synthesis of ribosomal RNA by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. In this thesis a reproducible large-scale purification protocol for Pol I from S. cerevisiae could be developed. Crystals were obtained, diffraction to < 4 Å could be recorded, however, the enormously complex non-crystallographic symmetry impeded structure solution.

Switching to cryo-electron microscopy, the structure of the complete 14-subunit enzyme could be solved to 12 Å resolution, a homology model for the core enzyme could be generated, and the crystal structure of the subcomplex A14/43 could be solved. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor, and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3’-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2, and apparently enables rRNA proofreading and 3’-end trimming.

This study characterized a panel of NSCLC cell lines as well as a stably transfected cell model expressing wild-type and mutated EGFR variants in terms of response to the EGFR-targeting drugs, gefitinib and cetuximab. The examinations support the notion that response to gefitinib is neither exclusive nor strictly determined by the presence of EGFR kinase mutations. Yet, cells expressing EGFR kinase domain mutations tended to generally respond better to treatments with gefitinib compared to those with wild-type EGFR. On the other hand, preliminary studies suggesting that cellular sensitivity to cetuximab is determined by factors other than EGFR kinase domain mutations could be substantiated through a robust set of data. Moreover, several promising candidate genes differentially expressed in gefitinib sensitive and resistant NSCLC cell lines were revealed by a global mRNA expression analyses. In addition, though statistically questionable, several biologically interesting genes that are possibly involved in determining in vitro response of NSCLC cells to cetuximab have been postulated. In this work, four cancer cell models, which are long-term exposed to gefitinib or cetuximab were established and characterized in terms of gain-of-resistance towards EGFR-targeting compounds, as well as in regard to biological and molecular alterations caused by long-term treatments. It was found that gefitinib long-term treatment of primary sensitive A431 cells confered growth-resistance to this TKI, but not to cetuximab. This observation may have clinical implications for patients that relapsed on gefitinib as it suggests that they might still profit from cetuximab therapy. On the other hand long-term exposure of A431 cells to cetuximab did not render cells resistant to neither the antibody nor gefitinib in regard to in vitro growth-inhibition. Furthermore, it appeared that long-term gefitinib-treated A431 cells downregulate overall EGFR levels, while long-term cetuximab exposed cells displayed decreased EGFR surface levels but constant overall expression. In addition, a candidate-based approach identified genes that are differentially expressed in cancer cells with primary or secondary resistance to gefitinib or cetuximab. Finally, this study for the first time provided evidence that cetuximab may block metastasis-facilitating epithelial-mesenchymal-transition (EMT) in an epithelial cell line. Moreover, it was suggested that activation of the HGFR may compensate for cetuximab-mediated blockage of EGFR. This was also reflected by an increased response of this population towards treatment with HGFR inhibitors.