@phdthesis{Janz2024,
  author    = {Janz, Anna},
  title     = {Human induced pluripotent stem cells (iPSCs) in inherited cardiomyopathies: Generation and characterization of an iPSC-derived cardiomyocyte model system of dilated cardiomyopathy with ataxia (DCMA)},
  doi       = {10.25972/OPUS-24096},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240966},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The emergence of human induced pluripotent stem cells (iPSCs) and the rise of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology innovated the research platform for scientists based on living human pluripotent cells. The revolutionary combination of both Nobel Prize-honored techniques enables direct disease modeling especially for research focused on genetic diseases. To allow the study on mutation-associated pathomechanisms, we established robust human in vitro systems of three inherited cardiomyopathies: arrhythmogenic cardiomyopathy (ACM), dilated cardiomyopathy with juvenile cataract (DCMJC) and dilated cardiomyopathy with ataxia (DCMA). Sendai virus vectors encoding OCT3/4, SOX2, KLF4, and c-MYC were used to reprogram human healthy control or mutation-bearing dermal fibroblasts from patients to an embryonic state thereby allowing the robust and efficient generation of in total five transgene-free iPSC lines. The nucleofection-mediated CRISPR/Cas9 plasmid delivery in healthy control iPSCs enabled precise and efficient genome editing by mutating the respective disease genes to create isogenic mutant control iPSCs. Here, a PKP2 knock-out and a DSG2 knock-out iPSC line were established to serve as a model of ACM. Moreover, a DNAJC19 C-terminal truncated variant (DNAJC19tv) was established to mimic a splice acceptor site mutation in DNAJC19 of two patients with the potential of recapitulating DCMA-associated phenotypes. In total eight self-generated iPSC lines were assessed matching internationally defined quality control criteria. The cells retained their ability to differentiate into cells of all three germ layers in vitro and maintained a stable karyotype. All iPSC lines exhibited a typical stem cell-like morphology as well as expression of characteristic pluripotency markers with high population purities, thus validating the further usage of all iPSC lines in in vitro systems of ACM, DCMA and DCMJC. Furthermore, cardiac-specific disease mechanisms underlying DCMA were investigated using in vitro generated iPSC-derived cardiomyocytes (iPSC-CMs). DCMA is an autosomal recessive disorder characterized by life threatening early onset cardiomyopathy associated with a metabolic syndrome. Causal mutations were identified in the DNAJC19 gene encoding an inner mitochondrial membrane (IMM) protein with a presumed function in mitochondrial biogenesis and cardiolipin (CL) remodeling. In total, two DCMA patient-derived iPSC lines (DCMAP1, DCMAP2) of siblings with discordant cardiac phenotypes, a third isogenic mutant control iPSC line (DNAJC19tv) as well as two control lines (NC6M and NC47F) were directed towards the cardiovascular lineage upon response to extracellular specification cues. The monolayer cardiac differentiation approach was successfully adapted for all five iPSC lines and optimized towards ventricular subtype identity, higher population purities and enhanced maturity states to fulfill all DCMA-specific requirements prior to phenotypic investigations. To provide a solid basis for the study of DCMA, the combination of lactate-based metabolic enrichment, magnetic-activated cell sorting, mattress-based cultivation and prolonged cultivation time was performed in an approach-dependent manner. The application of the designated strategies was sufficient to ensure adult-like characteristics, which included at least 60-day-old iPSC-CMs. Therefore, the novel human DCMA platform was established to enable the study of the pathogenesis underlying DCMA with respect to structural, morphological and functional changes. The disease-associated protein, DNAJC19, is constituent of the TIM23 import machinery and can directly interact with PHB2, a component of the membrane bound hetero-oligomeric prohibitin ring complexes that are crucial for phospholipid and protein clustering in the IMM. DNAJC19 mutations were predicted to cause a loss of the DnaJ interaction domain, which was confirmed by loss of full-length DNAJC19 protein in all mutant cell lines. The subcellular investigation of DNAJC19 demonstrated a nuclear restriction in mutant iPSC-CMs. The loss of DNAJC19 co-localization with mitochondrial structures was accompanied by enhanced fragmentation, an overall reduction of mitochondrial mass and smaller cardiomyocytes. Ultrastructural analysis yielded decreased mitochondria sizes and abnormal cristae providing a link to defects in mitochondrial biogenesis and CL remodeling. Preliminary data on CL profiles revealed longer acyl chains and a more unsaturated acyl chain composition highlighting abnormities in the phospholipid maturation in DCMA. However, the assessment of mitochondrial function in iPSCs and dermal fibroblasts revealed an overall higher oxygen consumption that was even more enhanced in iPSC-CMs when comparing all three mutants to healthy controls. Excess oxygen consumption rates indicated a higher electron transport chain (ETC) activity to meet cellular ATP demands that probably result from proton leakage or the decoupling of the ETC complexes provoked by abnormal CL embedding in the IMM. Moreover, in particular iPSC-CMs presented increased extracellular acidification rates that indicated a shift towards the utilization of other substrates than fatty acids, such as glucose, pyruvate or glutamine. The examination of metabolic features via double radioactive tracer uptakes (18F-FDG, 125I-BMIPP) displayed significantly decreased fatty acid uptake in all mutants that was accompanied by increased glucose uptake in one patient cell line only, underlining a highly dynamic preference of substrates between mutant iPSC-CMs. To connect molecular changes directly to physiological processes, insights on calcium kinetics, contractility and arrhythmic potential were assessed and unraveled significantly increased beating frequencies, elevated diastolic calcium concentrations and a shared trend towards reduced cell shortenings in all mutant cell lines basally and upon isoproterenol stimulation. Extended speed of recovery was seen in all mutant iPSC-CMs but most striking in one patient-derived iPSC-CM model, that additionally showed significantly prolonged relaxation times. The investigations of calcium transient shapes pointed towards enhanced arrhythmic features in mutant cells comprised by both the occurrence of DADs/EADs and fibrillation-like events with discordant preferences. Taken together, new insights into a novel in vitro model system of DCMA were gained to study a genetically determined cardiomyopathy in a patient-specific manner upon incorporation of an isogenic mutant control. Based on our results, we suggest that loss of full-length DNAJC19 impedes PHB2-complex stabilization within the IMM, thus hindering PHB-rings from building IMM-specific phospholipid clusters. These clusters are essential to enable normal CL remodeling during cristae morphogenesis. Disturbed cristae and mitochondrial fragmentation were observed and refer to an essential role of DNAJC19 in mitochondrial morphogenesis and biogenesis. Alterations in mitochondrial morphology are generally linked to reduced ATP yields and aberrant reactive oxygen species production thereby having fundamental downstream effects on the cardiomyocytes` functionality. DCMA-associated cellular dysfunctions were in particular manifested in excess oxygen consumption, altered substrate utilization and abnormal calcium kinetics. The summarized data highlight the usage of human iPSC-derived CMs as a powerful tool to recapitulate DCMA-associated phenotypes that offers an unique potential to identify therapeutic strategies in order to reverse the pathological process and to pave the way towards clinical applications for a personalized therapy of DCMA in the future.},
  subject      = {Induzierte pluripotente Stammzelle},
  language  = {en}
}
@phdthesis{Winkler2024,
  author    = {Winkler, Jana},
  title     = {Einfluss von D-β-Hydroxybutyrat auf Stoffwechsel und Interaktion mit Chemo-/Strahlentherapie bei triple negativen Mamma-Karzinom Zellen},
  doi       = {10.25972/OPUS-34704},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-347044},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Das triple negative Mamma-Karzinom stellt eine Tumorart dar, welche besonders junge Frauen betrifft und eine schlechte Prognose aufweist. Unterst{\"u}tzende und pro- gnoseverbessernde Therapien sind deshalb Gegenstand aktueller Forschung. Eine m{\"o}gliche unterst{\"u}tzende Therapie stellt hierbei die ketogene Di{\"a}t dar. Diese Arbeit untersuchte die Fragestellung, ob β-Hydroxybutyrat (3OHB), welches als Hauptme- tabolit unter ketogener Di{\"a}t oder beim Fasten erh{\"o}ht ist, Einfluss auf das Zellwachs- tum triple-negativer Brustkrebszellen aus{\"u}bt. Außerdem wurde eruiert, ob 3OHB die {\"u}blichen Behandlungsformen - Chemotherapie und Strahlentherapie - positiv oder negativ beeinflusst. In vitro wurden Versuche mit drei triple-negativen Brust- krebszellreihen unter m{\"o}glichst physiologischen Bedingungen durchgef{\"u}hrt. Hierbei konnte durch 3OHB weder ein wachstumsf{\"o}rdernder noch ein wachstumshemmender Effekt beobachtet werden. Genauso zeigte sich bei den Chemo- oder Strahlenthera- pieversuchen keine durch 3OHB induzierte Wechselwirkung. In vivo durchgef{\"u}hrte Studien {\"u}ber den Einfluss einer ketogenen Di{\"a}t finden sich nur vereinzelt. Um be- lastbare Daten zu erhalten werden deshalb weitere Studien in Zukunft vonn{\"o}ten sein. Eine ketogene Di{\"a}t k{\"o}nnte hierbei im Rahmen eines multimodalen Therapie- konzeptes eine unterst{\"u}tzende Rolle spielen, wof{\"u}r erste Einzelfallstudien Hinweise geben},
  subject      = {Ketogene Kost},
  language  = {de}
}
@phdthesis{Rainer2024,
  author    = {Rainer, Johannes},
  title     = {Vaskulartoxische Wirkung von Taxanen bei fortgeschrittenen Tumorerkrankungen},
  doi       = {10.25972/OPUS-35072},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350722},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Taxane (wie Paclitaxel oder Cabazitaxel) sind bew{\"a}hrte Arzneimittel in den systemischen Therapieschemata vieler b{\"o}sartiger Erkrankungen, einschließlich Brust- und Eierstockkrebs. Sie f{\"o}rdern die Stabilisierung der Mikrotubuli, was zu einem Stillstand des Zellzyklus w{\"a}hrend der Mitose f{\"u}hrt, auf den die Apoptose folgt. Neben dieser antimitotischen Wirkung von Taxanen ist seit einiger Zeit auch eine gef{\"a}ßver{\"a}ndernde Wirkung von Taxanen bekannt. K{\"u}rzlich wurde gezeigt, dass Taxane tats{\"a}chlich St{\"o}rungen in der Gef{\"a}ßarchitektur verursachen, indem sie den Kalziumeinstrom {\"u}ber TRPC6, einen unselektiven Kationenkanal, ausl{\"o}sen. Der erh{\"o}hte intrazellul{\"a}re Ca2+-Spiegel bewirkt eine Rundung der Endothelzellen, was zu einer St{\"o}rung des endothelialen Monolayers, Serumausfluss und Gef{\"a}ßkollaps f{\"u}hrt. In dieser Arbeit konzentrierten wir uns auf die Gef{\"a}ßbetten von peripheren Organen wie dem Herzen oder der Niere in Abh{\"a}ngigkeit vom Tumorstadium und der Taxol-Behandlung. Die Organe wurden mit immunhistochemischen Techniken angef{\"a}rbt, um Ver{\"a}nderungen in der Architektur und Morphologie der Blutgef{\"a}ße zu untersuchen. Wir fanden Ver{\"a}nderungen in der Morphologie der Kapillaren des Herzens und dar{\"u}ber hinaus Ver{\"a}nderungen in der Expression endothelialer Antigene in Abh{\"a}ngigkeit vom Tumorstadium, insbesondere eine zunehmende endotheliale Expression von TRPC6 in Abh{\"a}ngigkeit vom Tumorstadium. Diese Ergebnisse liefern neue Erkenntnisse f{\"u}r das Verst{\"a}ndnis der systemischen Auswirkungen maligner Erkrankungen und tragen dazu bei, Folgeerkrankungen bei Patienten mit fortgeschrittenem Krebs zu verhindern.},
  subject      = {Taxane},
  language  = {de}
}
@phdthesis{Nadernezhad2024,
  author    = {Nadernezhad, Ali},
  title     = {Engineering approaches in biofabrication of vascularized structures},
  doi       = {10.25972/OPUS-34589},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-345892},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Biofabrication technologies must address numerous parameters and conditions to reconstruct tissue complexity in vitro. A critical challenge is vascularization, especially for large constructs exceeding diffusion limits. This requires the creation of artificial vascular structures, a task demanding the convergence and integration of multiple engineering approaches. This doctoral dissertation aims to achieve two primary objectives: firstly, to implement and refine engineering methods for creating artificial microvascular structures using Melt Electrowriting (MEW)-assisted sacrificial templating, and secondly, to deepen the understanding of the critical factors influencing the printability of bioink formulations in 3D extrusion bioprinting. In the first part of this dissertation, two innovative sacrificial templating techniques using MEW are explored. Utilizing a carbohydrate glass as a fugitive material, a pioneering advancement in the processing of sugars with MEW with a resolution under 100 microns was made. Furthermore, by introducing the "print-and-fuse" strategy as a groundbreaking method, biomimetic branching microchannels embedded in hydrogel matrices were fabricated, which can then be endothelialized to mirror in vivo vascular conditions. The second part of the dissertation explores extrusion bioprinting. By introducing a simple binary bioink formulation, the correlation between physical properties and printability was showcased. In the next step, employing state-of-the-art machine-learning approaches revealed a deeper understanding of the correlations between bioink properties and printability in an extended library of hydrogel formulations. This dissertation offers in-depth insights into two key biofabrication technologies. Future work could merge these into hybrid methods for the fabrication of vascularized constructs, combining MEW's precision with fine-tuned bioink properties in automated extrusion bioprinting.},
  subject      = {3D-Druck},
  language  = {en}
}