@phdthesis{Kasaragod2022,
  author    = {Kasaragod, Vikram Babu},
  title     = {Biochemical and Structural Basis for the Moonlighting Function of Gephyrin},
  doi       = {10.25972/OPUS-14307},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143077},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Neurons are specialized cells dedicated to transmit the nerve impulses throughout the human body across specialized structures called synapses. At the synaptic terminals, a crosstalk between multiple macromolecules regulates the structure and function of the presynaptic nerve endings and the postsynaptic recipient sites. Gephyrin is the central organizer at inhibitory postsynaptic specializations and plays a crucial role in the organization of these structures by anchoring GABAA receptors (GABAAR) and glycine receptors (GlyR) to the postsynaptic membrane. This 93 kDa protein features an N-terminal G domain and a C-terminal E domain and the latter interacts directly with the intracellular loop between transmembrane helices 3 and 4 of certain subunits of the GlyRs and GABAARs. Biochemical and structural analyses have already provided valuable insights into the gephyrin-GlyR interaction. Interestingly, biochemical studies on the gephyrin-GABAAR interaction demonstrated that the GABAARs also depend on the same binding site as the GlyRs for the interaction with the gephyrin, but the molecular basis for this receptor specific interaction of gephyrin was still unknown. Co-crystal structures of GephE-GABAAR α3- derived peptides with supporting biochemical data presented in this study deciphered the receptor-specific interactions of gephyrin in atomic detail. In its moonlighting function, gephyrin also catalyzes the terminal step of the evolutionarily conserved molybdenum cofactor biosynthesis. Molybdenum, an essential transition element has to be complexed with a pterin-based cofactor resulting in the formation of the molybdenum cofactor (Moco). Moco is an essential component at the active site of all molybdenum-containing enzymes with the exception of nitrogenase. Mutations in enzymes involved in this pathway lead to a rare yet severe disease called Moco deficiency, which manifest itself in severe neurodevelopmental abnormalities and early childhood death. Moco biosynthesis follows a complex multistep pathway, where in the penultimate step, the N-terminal G domain of gephyrin activates the molybdopterin to form an adenylated molybdopterin intermediate. In the terminal step, this intermediate is then transferred to the C-terminal E domain of gephyrin, which catalyzes the metal insertion and deadenylation reaction to form active Moco. Previous biochemical and structural studies provided valuable insights into the penultimate step of the Moco biosynthesis but the terminal step remained elusive. Through the course of my dissertation, I crystallized the C-terminal E domain in the apo-form as well as in complex with ADP and AMP. These structures shed lightonto the deadenylation reaction and the formation of a ternary E-domain-ADP-Mo/W complex and thus provide structural insight into the metal insertion mechanism. Moreover, the structures also provided molecular insights into a mutation leading to Moco deficiency. Finally, ternary complexes of GephE, ADP and receptor-derived peptides provided first clues regarding the integration of gephyrin's dual functionality. In summary, during the course of the dissertation I was able to derive high resolution structural insights into the interactions between gephyrin and GABAARs, which explain the receptor-specific interaction of gephyrin and, furthermore, these studies can be extended in the future to understand GABAAR subunit-specific interactions of gephyrin. Finally, the understanding of Moco biosynthesis shed light on the molecular basis of the fatal Moco deficiency.},
  subject      = {Gephyrin},
  language  = {en}
}
@phdthesis{Wencker2022,
  author    = {Wencker, Freya Dorothea Ruth},
  title     = {The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover},
  doi       = {10.25972/OPUS-20712},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207124},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5'-untranslated region (5'-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes ('read-through'). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5'-region of the met mRNA. The met mRNA is degraded from its 5'-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5'-end (metI and metC) and a longer half-life towards the 3'-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon ('ON') or prevented operon transcription ('OFF'), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as 'OFF' mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum W{\"u}rzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Lisowski2022,
  author    = {Lisowski, Clivia},
  title     = {Maturation of the \(Salmonella\) containing vacuole is compromised in G1 arrested host cells},
  doi       = {10.25972/OPUS-18523},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185239},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The interaction of bacterial pathogens and the human host is a complex process that has shaped both organisms on a molecular, cellular and population level. When pathogenic bacteria infect the human body, a battle ensues between the host immune system and the pathogen. In order to escape an immune response and to colonize the host, pathogenic bacteria have developed diverse virulence strategies and some pathogens even replicate within host cells. For survival and propagation within the dynamic environment of a host cell, these bacteria interfere with the regulation of host pathways, such as the cell cycle, for their own benefit. The intracellular pathogen Salmonella Typhimurium invades eukaryotic cells and resides and replicates in a modified vacuolar compartment in which it is protected from the innate immune response. To this end, it employs a set of virulence factors that help to invade cells (SPI-1 effectors) and to hijack and modify the host endolysosomal system, in order to stabilize and mature its vacuolar niche (SPI-2 effectors). Previous studies have shown that Salmonella arrests host cells in G2/M phase and that Salmonella infected cells progress faster from G1 into S phase, suggesting that the G1 phase is disadvantageous for Salmonella infection. In fact, it has already been observed that Salmonella replication is impaired in G1 arrested cells. However, the reason for this impairment remained unclear. The current study addressed this question for the first time and revealed that the highly adapted, intracellular lifestyle of Salmonella is drastically altered upon G1 arrest of the host cell. It is shown that proteasomal degradation in G1 arrested cells is delayed and endolysosomal and autophagosomal trafficking is compromised. Accordingly, processing of lysosomal proteins is insufficient and lysosomal activity is decreased; resulting in uneven distribution and accumulation of endolysosomes and autophagosomes, containing undegraded cargo. The deregulation of these cellular signaling pathways affects maturation of the Salmonella containing vacuole (SCV). For the first time it is shown that acidification of SCVs is impaired upon G1 arrest. Thus, an important environmental factor for the switch from SPI-1 to SPI-2 gene expression is missing and the SPI-2 system is not activated. Consequently, targeting and modification of host cell structures by SPI-2 effectors e.g. recruitment of endolysosomal membrane proteins, like LAMP1, or exchange of endosomal cargo, is compromised. In addition, degradation of Salmonella SPI-1 effectors by the host proteasome is delayed. Their prolonged presence sustained the recruitment of early endosomes and contributed to the SCV remaining in an early, vulnerable maturation stage. Finally, it was shown that SCV membrane integrity is compromised; the early SCV ruptures and bacteria are released into the cytoplasm. Depending on the host cell type, SPI-2 independent, cytoplasmic replication is promoted. This might favor bacterial spreading, dissemination into the tissue and provide an advantage in host colonization. Overall, the present study establishes a link between host cell cycle regulation and the outcome of Salmonella infection. It fills the gap of knowledge as to why the host cell cycle stage is of critical importance for Salmonella infection and sheds light on a key aspect of host-pathogen interaction.},
  subject      = {Salmonella Typhimurium},
  language  = {en}
}
@phdthesis{Wallstabe2022,
  author    = {Wallstabe, Lars},
  title     = {Development and preclinical evaluation of tumour-reactive T cells expressing a chemically programmable chimeric antigen receptor},
  doi       = {10.25972/OPUS-17907},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179071},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The genetic modification of T cells for the expression a chimeric antigen receptor (CAR) endows them with a new specificity for an antigen. Adoptive immunotherapy with CD19-CAR T cells has achieved high rates of sustained complete remissions in B cell malignancies. However, the downregulation or loss of the targeted antigen after mono-specific CAR T cell therapy, e.g. against CD19 or CD22, has been reported. Targeting multiple antigens on tumour cells, sequentially or simultaneously, could overcome this limitation. Additionally, targeting multiple antigens with CAR T cells could drive the translation from hematologic malignancies to prevalent solid cancers, which often express tumour-associated antigens heterogeneously. We hypothesised that expression of a universal CAR, which can be programmed with hapten-like molecules, could endow T cells with specificities for multiple antigens. In this study we introduce a novel chemically programmable CAR (cpCAR) based on monoclonal antibody h38C2. Our data show, that cpCARs form a reversible chemical bond to molecules containing a diketone-group and therefore can be programmed to acquire multiple specificities. We programmed cpCAR T cells with hapten-like compounds against integrins αvβ3 and α4β1 as well as the folate receptor. We observed tumour cell lysis, IFN ɣ and IL-2 production and proliferation of programmed cpCAR T cells against tumour cells expressing the respective target antigen in vitro. As a reference to cpCARs programmed against αvβ3, we further introduced novel conventional αvβ3-CARs. These CARs, based on humanised variants of monoclonal antibody LM609 (hLM609), directly bind to integrin αvβ3 via their scFv. The four αvβ3-CAR constructs comprised either an scFv with higher affinity (hLM609v7) or lower affinity (hLM609v11) against αvβ3 integrin and either a long (IgG4 hinge, CH2, CH3) or short (IgG4 hinge) extracellular spacer. We selected the hLM609v7-CAR with short spacer, which showed potent anti-tumour reactivity both in vitro and in a murine xenograft model, for comparison with the cpCAR programmed against αvβ3. Our data show specific lysis of αvβ3-positive tumour cells, cytokine production and proliferation of both hLM609-CAR T cells and cpCAR T cells in vitro. However, conventional hLM609-CAR T cells mediated stronger anti-tumour effects compared to cpCAR T cells in the same amount of time. In line with the in vitro data, complete destruction of tumour lesions in a murine melanoma xenograft model was only observed for mice treated with conventional αvβ3-CAR T cells. Collectively, we introduce a cpCAR, which can be programmed against multiple tumour antigens, and hLM609-CARs specific for the integrin αvβ3. The cpCAR technology bears the potential to counteract current limitations, e.g. antigen loss, of current monospecific CAR T cell therapy. Targeting αvβ3 integrin with CAR T cells could have clinical applications in the treatment of solid malignancies, because αvβ3 is not only expressed on a variety of solid malignancies, but also on tumour-associated vasculature and fibroblast.},
  subject      = {Tumorimmunologie},
  language  = {en}
}
@phdthesis{Chen2022,
  author    = {Chen, Mengjia},
  title     = {Right Ventricular Dysfunction contributes to Left Ventricular Thrombus Formation in Patients post Anterior Myocardial Infarction},
  doi       = {10.25972/OPUS-20414},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204149},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Our current data demonstrate that besides the known risk factors, including apical aneurysm, reduced left ventricular longitudinal systolic function (MAPSE) and advanced diastolic dysfunction, Right ventricular dysfunction as determined by reduced tricuspid annular plane systolic excursion (TAPSE) or right ventricular fractional area change (RV_FAC) is independently associated with left ventricular thrombus formation in acute anterior myocardial infarction patients, especially in the setting of anterior myocardial infarction without the formation of an apical aneurysm. This study suggests that besides left ventricular abnormalities, right ventricular dysfunction likewise contributes LVT formation in patients with acute anterior myocardial infarction.},
  subject      = {Thrombus},
  language  = {en}
}
@phdthesis{Heib2022,
  author    = {Heib, Tobias},
  title     = {The role of Rho GTPases in megakaryopoiesis and thrombopoiesis},
  doi       = {10.25972/OPUS-21664},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216645},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Platelets, derived from megakaryocytes (MKs) in the bone marrow (BM), are small, anucleated cells that circulate in the bloodstream and are critical for thrombosis and hemostasis. Megakaryo- and subsequent thrombopoiesis are highly orchestrated processes involving the differentiation and maturation of MKs from hematopoietic stem cells (HSCs), after which MKs are able to release platelets into the bloodstream, a process termed proplatelet formation (PPF). Here, the MK penetrates the endothelial lining and releases cytoplasmic portions (proplatelets) into the blood stream, which finally mature into platelets within the ciruculation. PPF requires an extensive crosstalk as well as a tight regulation of the MK cytoskeleton, in which small GTPases of the Rho family, such as RhoA and Cdc42 are critically involved and play opposing roles. MK and platelet specific Cdc42 or RhoA-deficiency in mice results in macrothrombocytopenia. Moreover, RhoA deficient mice displayed a frequent mislocalization of entire MKs into BM sinusoids, a finding that was reverted upon concomitant lack of Cdc42 but accompanied by an aggravated macrothrombocytopenia. Whether receptors are involved in the process of transendothelial MK migration, however, remained unclear. In the first part of this thesis, a centrifugation-based method ('spin isolation') to harvest murine BM cells was established, which not only reduces experimental time, costs and animals but is also highly suitable for studies on primary and in vitro cultured BM-derived cells. The spin isolation was used particularly for MK studies during the course of the thesis. In the second part of this thesis, a MK- and platelet-specific RhoA/Cdc42 double-deficiency was shown to result in reduced expression of a variety of MK-specific glycoproteins and cytoskeletal regulators of importance during MK maturation and PPF, a phenotype culminating in virtually abolished platelet biogenesis. We thus uncovered that RhoA/Cdc42-regulated gene expression is a prerequisite for cytoplasmic MK maturation, but dispensable for endomitosis. In the third part of this thesis we analyzed mice double-deficient in RhoA and prominent MK receptors which are potentially involved in the regulation of PPF in the BM environment. We were able to show that integrins as well as the inhibitory receptor G6b-B are dispensable for transendothelial migration of RhoA-deficient MKs. Surprisingly however, the myelofibrosis and concomitant osteosclerosis observed in G6b-B single-deficient mice was attenuated in RhoA/G6b B double-deficient mice, thus implying an important role of RhoA during myelofibrotic disease progression. BM transplantation experiments furthermore revealed that not only the macrothrombocytopenia but also the transmigration of RhoA-deficient MKs is due to cell-intrinsic defects and not related to possible Pf4-Cre off-target effects in non-hematopoietic cells. In the last part of this study we demonstrated that the new approach for MK- and platelet-specific gene ablatation using Gp1ba-Cre deleter mice is associated with intrinsic MK defects and in addition results in insufficient depletion of RhoA compared to the Pf4-Cre model, positioning the latter still as the gold standard for studying MK biology.},
  subject      = {Megakaryozyt},
  language  = {en}
}
@phdthesis{Guentzel2022,
  author    = {G{\"u}ntzel, Paul Mathias},
  title     = {Bioinspired Ion Pairs Transforming Poorly Water-soluble Compounds into Protic Ionic Liquids and Deep Eutectic Solvents},
  doi       = {10.25972/OPUS-21980},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-219806},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Microbial, mammalian and plant cells produce and contain secondary metabolites, which typically are soluble in water to prevent cell damage by crystallization. The formation of ion pairs, e.g. with carboxylic acids or mineral acids, is a natural blueprint to keep basic metabolites in solution. It was aimed at showing whether the mostly large carboxylates form soluble protic ionic liquids (PILs) with basic natural products resulting in enhanced aqueous solubility. Furthermore, their supramolecular pattern in aqueous solution was studied. Thereby, naturally occurring carboxylic acids were identified being appropriate counterions for natural basic compounds and facilitate the formation of PILs with their beneficial characteristics, like improved dissolution rate and enhanced apparent solubility.},
  subject      = {Ionic Liquids},
  language  = {en}
}
@phdthesis{Toksabay2022,
  author    = {Toksabay, Sinem},
  title     = {Synthesis and on surface self assembly properties of pi extended tribenzotriquinacenes},
  doi       = {10.25972/OPUS-24573},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245734},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Tribenzotriquinacene (TBTQ) is a polycyclic aromatic framework with a particularly rigid, C3v symmetrical, bowl-shaped core bearing three mutually fused indane wings. It has been discussed as a defect center for a nanographene by Kuck and colleagues. Therefore, extended TBTQ structures are promising models for saturated defect structures in graphene and graphene like molecules and could be used to investigate the role of defects for the electronic properties of graphene. With this motivation, three different pi-extended TBTQ derivatives have been synthesized in this work. Several different Scholl reaction conditions were tried to obtain fully annulated product of hexaphenyl substituted TBTQ. The desired benzannulated TBTQ derivative could not be obtained due to unfavourable electron density in the respective positions of the molecule and increased reactivity of the bay position of the precursor. As an another method for benzannulation is the on-surface synthesis of graphene flakes and can be carried out using electron beams e.g. in a tunneling microscope (STM). According to our previous research, the parent system TBTQ and centro-methyl TBTQ on silver and gold surfaces showed that the gas phase deposition of these molecules gives rise to the formation of highly ordered two-dimensional assemblies with unique structural features. This shows the feasibility for the formation of defective graphene networks starting from the parent structures. Therefore, the same deposition technique was used to deposit Me-TBTQ(OAc)3Ph6, and investigate the molecular self-assembly properties directly on the surface of Cu (111). In summary, the substrate temperature dependent self-assembly of Me-TBTQ(OAc)3Ph6 molecules on Cu(111), shows the following evolution of orientations. At room temperature, molecules form dimers, which construct a higher-coverage honeycomb lattice. Furthermore, one of the acetyl group located in the bay positions of the TBTQ core is cleaved and the remaining two induce the metal-molecule interaction. It was presumed that by increasing the temperature to 393 K, the remaining acetyl and methyl groups would beeliminated from the molecular structure.In addition, the smaller TBTQ-Ph6 molecules preferably lie flat on Cu(111) crystal and allowing the molecules to settle into a C3-symmetry and form a dense hexagonal structure.},
  subject      = {Triquinacenderivate},
  language  = {en}
}
@phdthesis{Fey2022,
  author    = {Fey, Christina},
  title     = {Establishment of an intestinal tissue model for pre-clinical screenings},
  doi       = {10.25972/OPUS-24410},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244107},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed. In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the "gold standard" in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs. In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes. Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture. Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive. With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10\% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies. Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.},
  subject      = {D{\"u}nndarm},
  language  = {en}
}
@phdthesis{Mueller2022,
  author    = {M{\"u}ller, Stefan},
  title     = {Coherent Multiple-Quantum Multidimensional Fluorescence Spectroscopy},
  doi       = {10.25972/OPUS-24411},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244113},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {This thesis describes novel concepts for the measurement of the static and dynamic properties of the electronic structure of molecules and nanocrystals in the liquid phase by means of coherent fluorescence-detected spectroscopy in two and three frequency dimensions. These concepts are based on the systematic variation ("phase cycling") of a sequence of multiple time-delayed femtosecond excitation pulses in order to decode a multitude of novel nonlinear signals from the resulting phase-dependent fluorescence signal. These signals represent any permutation of correlations between zero-, one-, two-, and three-quantum coherences. To this end, two new phase-cycling schemes have been developed which can simultaneously resolve and discriminate several nonlinear signals of sixth order, including those of the fourth order of nonlinearity. By means of the sixth-order signals recorded in this work, static properties of highly excited electronic states in molecules such as their energies, transition dipole moments, and relative displacement of electronic potential surfaces, as well as dynamic properties in terms of their relaxation kinetics, can be ascertained. Furthermore, it was shown that these signals are suitable for the characterization of exciton-exciton correlations in colloidal quantum dots and for the measurement of ultrafast exciton-exciton annihilation in molecular aggregates. The experiments performed in this thesis mark an important step towards the complete characterization of the nonlinear response of quantum systems. In view of this, the concept of fluorescence-detected multiple-quantum coherence multidimensional spectroscopy introduced here offers a unified, systematic approach. In virtue of the technical advantages such as the use of a single excitation beam and the absence of nonresonant contributions, the measurement protocols developed here can be directly transferred to other incoherent observables and to sample systems in other states of matter. Furthermore, the approaches presented here can be systematically extended to higher frequency dimensions and higher orders of nonlinearity.},
  subject      = {Coherent Multidimensional Spectroscopy},
  language  = {en}
}