Aspergillus fumigatus ist die häufigste Ursache für Lungeninfektionen in immunsuppri-mierten Patienten. Virulenzfaktoren sind häufig an Kontrollmechanismen für Entwick-lung gekoppelt, welche im verwandten Modellorganismus Aspergillus nidulans entdeckt wurden. Diese Arbeit präsentiert die Charakterisierung des F-box Proteins Fbx15 in A. fumigatus, welches einen starken Einfluss auf die Entwicklung in A. nidulans hat. Die Deletion von fbx15 resultierte in starken Wachstumsdefekten unter vielen Stress induzie-renden Bedingungen, welche klassische Virulenz Faktoren beinhalten, wie erhöhte Tem-peratur, oxidativer Stress und Aminosäuremangel, während das Wachstum unter Stan-dardbedingungen nicht beeinflusst war. Oxidativer Stress induziert eine transiente Erhöhung der fbx15 Expression, welche nach 40 Minuten zu einer dreifach erhöhten Pro-teinmenge führte. Fbx15 ist ein stabiles F-box Protein mit einer Halbwertszeit von 90 Minuten. Generell funktionieren F-box Proteine als Substratadapter für SCF-E3-Ubiquitin-Ligasen. Fbx15 liegt unter normalen Bedingungen phosphoryliert vor und in-teragiert mit der Skp1/A Untereinheit des SCF-Komplexes, vorzugsweise in kleineren Subpopulationen im Zytoplasma. Phosphoryliertes Fbx15 wird bevorzugt in SCF-Komplexe eingebaut. Oxidativer Stress führt zu einer schnellen Dephosphorylierung von Fbx15. Fbx15 Varianten, welche nicht phosphoryliert werden können, interagieren mit Skp1/A primär im Kern. Fbx15 rekrutiert drei Untereinheiten des COP9-Signalosoms und Proteine welche in Transkription, Translation, Signalübertragung, Morphologie oder Stoffwechsel involviert sind. Fbx15 bindet die Ssn6/F Untereinheit des konservierten Ssn6/SsnF-Tup1/RcoA Co-Repressors und wird für dessen Kernlokalisation benötigt. Dephosphoryliertes Fbx15 interagiert mit Ssn6/F im Kern und eine Fbx15-Ssn6/F be-dingte Genrepression wird für die Reduzierung der Gliotoxin-Biosynthese benötigt. fbx15 Deletionsstämme sind nicht in der Lage immunsupprimierte Mäuse in einem Model für invasive Aspergillose zu infizieren, was eine essentielle Funktion von Fbx15 für die Viru-lenz bestätigt. Diese Arbeit zeigt, dass Fbx15 nicht nur Teil von SCF-E3-Ubiquitin-Ligasen sein kann, sondern eine zweite neue molekulare Funktion aufweist, welche die physische Interaktion mit der Co-Repressor Untereinheit Ssn6/F und dessen Lokalisa-tionskontrolle beinhaltet. Diese duale Funktion resultiert in einer essentiellen Funktion von Fbx15 für die Kontrolle der oxidativen Stressantwort, des Sekundärmetabolismus und der Virulenz im opportunistischen Humanpathogen A. fumigatus.

Raman spectroscopy offers much better spectral selectivity but its usage has been limited by its poor sensitivity. The discovery of surface-enhanced Raman scattering (SERS) effect, which results in increased sensitivities of up to 108-fold for some compounds, has eliminated this drawback. A new SERS active substrate was developed in this study. Silver nanoparticle-doped polyvinyl alcohol (PVA) coated SERS substrate prepared through chemical and electrochemical reduction of silver particles dispersed in the polymer matrix. Performances of the substrates were evaluated with some biologically important compounds. The specific detection of DNA has gained significance in recent years since increasingly DNA sequences of different organisms are being assigned. Such sequence knowledge can be employed for identification of the genes of microorganisms or diseases. In this study, specific proteasome gene sequences were detected both label free spectrophotometric detection and SERS detection. In label free spectrophotometic detection, proteasome gene probe and complementary target gene sequence were attached to the gold nanoparticles separately. Then, the target and probe oligonucleotide-modified gold solutions were mixed for hybridization and the shift in the surface plasmon absorption band of gold nanoparticles were followed. SERS detection of specific nucleic acid sequences are mainly based on hybridization of DNA targets to complementary probe sequences, which are labelled with SERS active dyes. In this study, to show correlation between circulating proteasome levels and disease state we suggest a Raman spectroscopic technique that uses SERGen probes. This novel approach deals with specific detection of elevated or decreased levels of proteasome genes&rsquo / transcription in patients as an alternative to available enzyme activity measurement methods. First, SERGen probes were prepared using SERS active labels and specific proteasome gene sequences. Then DNA targets to complementary SERGen probe sequences were hybridized and SERS active label peak was followed.

Most patients diagnosed with cancer will receive systematic chemotherapy at some point during their illness, which almost always cause severe side effects for the patients such as, anemia, nausea and vomiting. The problems with today’s chemotherapy is not only that it cause severe side effects, but also that the cancer may develop resistance to the therapy, which is why the development of a new type of therapeutic agent is in dire need. The ubiquitin proteasome system (UPS) is a vital machinery for the cancer cells to maintain protein homeostasis, which also make them vulnerable to any disruption of this system. In recent years, a new technology has been developed that utilize the UPS by chemically bringing an E3 ubiquitin ligase into close proximity of a protein of choice and tagging the protein with ubiquitin for degradation. This technology is called proteolysis targeting chimera (PROTAC). In this project, we managed to theoretically develop a new type of cancer therapeutic agent, that utilize the PROTAC system together with the first-in-class proteasome regulatory subunit, POH1, inhibitor Capzimin as a warhead. By using Capzimin as a warhead it should be possible to polyubiquitinate POH1, and thus induce proteotoxic stress in the cancer cells to trigger apoptosis. This theoretically developed drug is therefore called Capzimin-PROTAC, which should be able to trigger apoptosis in cancer cells, and at the same time being relatively safe to normal healthy cells.

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