http://hdl.handle.net/10900/42133 Mon, 09 Nov 2026 15:54:09 GMT 2026-11-09T15:54:09Z http://hdl.handle.net/10900/185305 Genetic Engineering of Uridyl Peptide Antibiotic Biosynthetic Gene Clusters for Improved Production Koutsandrea, Panagiota-Hanna Pseudomonas aeruginosa ist ein gramnegatives Bakterium, welches durch eine WHO-Klassifizierung als Problemkeim eingestuft wurde und zu schweren nosokomialen Infektionen, vorwiegend in immunsupprimierten Patienten, führt. Die Antibiotikaklasse der Uridylpeptide, bestehend aus den Untergruppen der Mureidomycine, Pacidamycine, Napsamycine und Sansanmycine, zeichnet sich durch selektive antipseudomonale Aktivität aus. Daher stellen sie eine vielversprechende Wirkstoffklasse dar, mit deren Weiterentwicklung sich diese Arbeit befasst hat. Zu diesem Zweck wurden die Biosynthese-Gencluster der Pacidamycine und Napsamycine in Streptomyceten durch molekularbiologische Methoden modifiziert, sodass die Produktion erwünschter Antibiotika erhöht und die unerwünschter Derivate reduziert wurde. Thu, 05 Nov 2026 00:00:00 GMT http://hdl.handle.net/10900/185305 2026-11-05T00:00:00Z http://hdl.handle.net/10900/185302 Functional Analysis of CNGA3 Missense Variants Associated with Achromatopsia Using an Aequorin-Based Bioassay Solaki, Maria The CNGA3 gene encodes the major subunit of the cyclic nucleotide-gated ion channel (CNG channel) in cone photoreceptors and is one of the major disease-causative genes for achromatopsia. Most CNGA3 variants are missense variants, the majority of which have not been functionally characterized, thereby significantly hampering the establishment of a definitive genetic diagnosis in achromatopsia patients. However, an objective assessment of the pathogenicity of CNGA3 variants is essential with regard to potential gene therapies. The aim of this study was to: i. establish a CNGA3 variant database and to provide an expert-based assessment of sequence variants in the CNGA3 gene, ii. develop a medium-throughput bioassay to test the functionality of CNGA3 channels carrying functionally yet uncharacterized missense variants versus wild-type channels; and iii. to incorporate the obtained functional data into the existing ACMG/AMP classification enabling revision or confirmation of the classification. In this doctoral thesis, the CNGA3 variant spectrum was substantially expanded to a total of 316 sequence alterations. From these, 244 variants could be interpreted as “likely disease-causing” following extensive in silico variant assessment, ACMG/AMP-based classification, and manual/expert-based variant categorization. The predominant variant type among the “likely disease-causing” variants were missense substitutions displaying the highest frequency of 66.8%. Furthermore, I have developed an aequorin-based luminescence bioassay that enables the assessment of the function of mutated CNGA3 channels by quantifying the CNGA3 channel-mediated calcium influx in a cell culture system, thereby facilitating ACMG/AMP-based re-classification of these variants. Using this approach, I functionally analyzed a substantial number of 150 missense substitutions in the CNGA3 gene, 55 of which had previously been classified as variants of uncertain significance (VUS). Of the 150 missense substitutions analyzed, 25 exhibited channel function characteristics similar to those of the wild-type channels, and were considered “functionally normal”. In contrast, the remaining 125 missense substitutions displayed (severely) impaired or absent channel function, thus classifying them as “functionally abnormal”. These functional data enabled the ACMG/AMP-based re-classification of 52 out of 55 VUS as benign, likely benign, or likely pathogenic. This resulted in a VUS re-classification rate of 94.5%. In summary, the expansion of the CNGA3 variant spectrum, functional evaluation, and subsequent ACMG/AMP-based variant re-classification presented in this doctoral thesis will greatly facilitate the clinical variant interpretation with respect to the analyzed CNGA3 missense variants ensuring a molecular genetic diagnosis for CNGA3-associated achromatopsia patients, hereby identifying patients eligible for future gene therapeutic trials and treatments. Mon, 09 Nov 2026 00:00:00 GMT http://hdl.handle.net/10900/185302 2026-11-09T00:00:00Z http://hdl.handle.net/10900/185189 Synthese und Entwicklung von Replikationsprotein A Inhibitoren zur Überwindung der Therapieresistenz bei Krebserkrankungen Schmidberger, Gregor Dissertation ist gesperrt bis 25. November 2027 !; Das Replikationsprotein A ist das wichtigste ssDNA-bindende Protein in Eukaryoten. Es schützt die DNA vor Nukleasen und macht die einzelsträngige DNA zugänglich für weitere Proteine in DNA-Stoffwechselprozessen. Da es kein funktionell redundantes Protein in menschlichen Zellen gibt, kann eine Hemmung nicht durch Kompensation oder Evolution umgangen werden, was ein Hauptgrund für die Resistenzentwicklung bei Tumorzellen ist. Beim Zusammenbau des RPA-Heterotrimers zeigte RPA3 eine stabilisierende Eigenschaft für den gesamten RPA-Komplex. Diese Eigenschaft wurde als möglicher Angriffspunkt für einen Protein-Protein-Interaktionsinhibitors identifiziert. In dieser Arbeit wurde eine bereits vielversprechende Grundstruktur weiter auf Struktur-Wirkungsbeziehungen untersucht, vor allem mit dem Ziel die Lipophilie der Verbindungen zu reduzieren. Es gelang die Grundstruktur bei Erhalt der biologischen Aktivität zu verkleinern, zu derigidisieren und den Anteil an aromatischen Resten zu verringern. Durch eine sichtbare DNA-Anreicherung im verwendeten EMSA-Testsystem bei einigen Verbindungen rückten Target-Engagement Strategien in den Vordergrund. Hierzu wurde der Inhibitor mit photoreaktiven Gruppen versehen, sodass eine Photoaffinitätsmarkierung mit dem RPA durchgeführt werden konnte. In einem weiteren Ansatz wurden 2D NMR-Experimente mit Inhibitor und dem RPA2/3 forciert. Durch das Anbringen von polaren Gruppen am Inhibitor konnte die Löslichkeit in gepufferten, wässrigen Systemen erhöht werden. Nach Inkubation mit dem modifizierten Inhibitor konnte ein teilweise entfaltetes RPA2/3 nachgewiesen werden. Dieses Ergebnis ist im Einklang mit dem postulierten Wirkmechanismus eines RPA2/3 Protein-Protein-Interaktionsinhibitors und kann für eine zukünftige Wirkstoffentwicklung zur Überwindung der Therapieresistenz bei Krebserkrankungen von Nutzen sein. Thu, 25 Nov 2027 00:00:00 GMT http://hdl.handle.net/10900/185189 2027-11-25T00:00:00Z http://hdl.handle.net/10900/185169 Development and neuronal basis of hierarchical sequences in birdsong Koparkar, Avani Prasad Communication is the cornerstone of civilization. The ability to learn and produce complex vocalizations has helped human societies to flourish. On the tree of life, humans and birds belong to a subgroup that shows sensorimotor vocal learning. Songbirds learn their songs through vocal imitation, and this behavior is controlled by a series of interconnected brain regions called the song system.Bengalese finches are a species of domesticated songbirds that are known for their sequentially variable songs, with probabilistic sequencing. Bengalese finch songs contain sequence structures such as chunks, which are syllable sequences that always appear in the same order, repeat phrases, in which a single syllable type can repeat a variable number of times and INs, which are made of introductory notes and inter-notes. Introductory notes are 1-3 syllable types which represent premotor activity and are repeated at the beginning of song bouts, while inter-notes have the same syllable types, but occur between syllables within a bout. In this thesis, I first characterize the development of higher-order sequencing in Bengalese finch song. I establish pipelines for segmentation and annotation of developmental songs of Bengalese finches. I also establish a method to identify chunks methodically. I show repeats as well as INs have monotonically decreasing repeat number distributions early in development, but repeat phrase distributions are refined over development, and become characteristically peaked. I show that sequence structures like chunks are formed due to the acquisition of syllable-to-syllable bigram transitions. Chunks can also be repeated variably in song, forming higher-order sequence structures called metarepeats. Meta-repeat number distributions also follow a developmental trajectory similar to repeat number distributions, and are also refined over development. I also compare the developmental trajectories of these phrases across birds with a common tutor and show that juveniles can copy acoustic structure and global transition patterns, while maintaining individual variability in local transition statistics. Next, I investigate the neural mechanisms behind higher-order sequencing in Bengalese finches through lesion studies. I show that the pallial nucleus mMAN (medial magnocellular nucleus of the anterior nidopallium) affects sequence entropy in song, and affects the variability of repeat phrases. I show evidence that mMAN is necessary for learned changes to flexible sequencing in song. My preliminary work shows that lesions of nucleus Nif (nucleus interfacialis of the nidopallium) suggest that Nif may also be involved in the control of sequencing variability. My work provides insights into the contributions of inputs to premotor nucleus HVC (proper name) in the control of variable sequencing in song. It also provides an important data basis on which statistical models of song syntax can be developed and refined. Bengalese finch songs display long-range dependencies between song elements, which are not well captured by simple Markov models. In the outlook, I present ongoing work on computational modeling of song syntax, and show that a hierarchical Bayesian sequence model can capture the long-range dependencies in Bengalese finch song.; Die Dissertation ist gesperrt bis zum 29. September 2028 ! Fri, 29 Sep 2028 00:00:00 GMT http://hdl.handle.net/10900/185169 2028-09-29T00:00:00Z