@phdthesis{Wittmann2014,
  author    = {Wittmann, Katharina},
  title     = {Adipose Tissue Engineering - Development of Volume-Stable 3-Dimensional Constructs and Approaches Towards Effective Vascularization},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107196},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Adipose tissue defects and related pathologies still represent major challenges in reconstructive surgery. Based on to the paradigm 'replace with alike', adipose tissue is considered the ideal substitute material for damaged soft tissue [1-3]. Yet the transfer of autologous fat, particularly larger volumes, is confined by deficient and unpredictable long term results, as well as considerable operative morbidity at the donor and recipient site [4-6], calling for innovative treatment options to improve patient care. With the aim to achieve complete regeneration of soft tissue defects, adipose tissue engineering holds great promise to provide functional, biologically active adipose tissue equivalents. Here, especially long-term maintenance of volume and shape, as well as sufficient vascularization of engineered adipose tissue represent critical and unresolved challenges [7-9]. For adipose tissue engineering approaches to be successful, it is thus essential to generate constructs that retain their initial volume in vivo, as well as to ensure their rapid vascularization to support cell survival and differentiation for full tissue regeneration [9,10]. Therefore, it was the ultimate goal of this thesis to develop volume-stable 3D adipose tissue constructs and to identify applicable strategies for sufficient vascularization of engineered constructs. The feasibility of the investigated approaches was verified by translation from in vitro to in vivo as a critical step for the advancement of potential regenerative therapies. For the development of volume-stable constructs, the combination of two biomaterials with complementary properties was successfully implemented. In contrast to previous approaches in the field using mainly non-degradable solid structures for mechanical protection of developing adipose tissue [11-13], the combination of a cell-instructive hydrogel component with a biodegradable porous support structure of adequate texture was shown advantageous for the generation of volume-stable adipose tissue. Specifically, stable fibrin hydrogels previously developed in our group [14] served as cell carrier and supported the adipogenic development of adipose-derived stem cells (ASCs) as reflected by lipid accumulation and leptin secretion. Stable fibrin gels were thereby shown to be equally supportive of adipogenesis compared to commercial TissuCol hydrogels in vitro. Using ASCs as a safe source of autologous cells [15,16] added substantial practicability to the approach. To enhance the mechanical strength of the engineered constructs, porous biodegradable poly(ε caprolactone)-based polyurethane (PU) scaffolds were introduced as support structures and shown to exhibit adequately sized pores to host adipocytes as well as interconnectivity to allow coherent tissue formation and vascularization. Low wettability and impaired cell attachment indicated that PU scaffolds alone were insufficient in retaining cells within the pores, yet cytocompatibility and differentiation of ASCs were adequately demonstrated, rendering the PU scaffolds suitable as support structures for the generation of stable fibrin/PU composite constructs (Chapter 3). Volume-stable adipose tissue constructs were generated by seeding the pre-established stable fibrin/PU composites with ASCs. Investigation of size and weight in vitro revealed that composite constructs featured enhanced stability relative to stable fibrin gels alone. Comparing stable fibrin gels and TissuCol as hydrogel components, it was found that TissuCol gels were less resilient to degradation and contraction. Composite constructs were fully characterized, showing good cell viability of ASCs and strong adipogenic development as indicated by functional analysis via histological Oil Red O staining of lipid vacuoles, qRT-PCR analysis of prominent adipogenic markers (PPARγ, C/EBPα, GLUT4, aP2) and quantification of leptin secretion. In a pilot study in vivo, investigating the suitability of the constructs for transplantation, stable fibrin/PU composites provided with a vascular pedicle gave rise to areas of well-vascularized adipose tissue, contrasted by insufficient capillary formation and adipogenesis in constructs implanted without pedicle. The biomaterial combination of stable fibrin gels and porous biodegradable PU scaffolds was thereby shown highly suitable for the generation of volume-stable adipose tissue constructs in vivo, and in addition, the effectiveness of immediate vascularization upon implantation to support adipose tissue formation was demonstrated (Chapter 4). Further pursuing the objective to investigate adequate vascularization strategies for engineered adipose tissue, hypoxic preconditioning was conducted as a possible approach for in vitro prevascularization. In 2D culture experiments, analysis on the cellular level illustrated that the adipogenic potential of ASCs was reduced under hypoxic conditions when applied in the differentiation phase, irrespective of the oxygen tension encountered by the cells during expansion. Hypoxic treatment of ASCs in 3D constructs prepared from stable fibrin gels similarly resulted in reduced adipogenesis, whereas endothelial CD31 expression as well as enhanced leptin and vascular endothelial growth factor (VEGF) secretion indicated that hypoxic treatment indeed resulted in a pro-angiogenic response of ASCs. Especially the observed profound regulation of leptin production by hypoxia and the dual role of leptin as adipokine and angiogenic modulator were considered an interesting connection advocating further study. Having confirmed the hypothesis that hypoxia may generate a pro-angiogenic milieu inside ASC-seeded constructs, faster vessel ingrowth and improved vascularization as well as an enhanced tolerance of hypoxia-treated ASCs towards ischemic conditions upon implanatation may be expected, but remain to be verified in rodent models in vivo (Chapter 5). Having previously been utilized for bone and cartilage engineering [17-19], as well as for revascularization and wound healing applications [20-22], stromal-vascular fraction (SVF) cells were investigated as a novel cell source for adipose tissue engineering. Providing cells with adipogenic differentiation as well as vascularization potential, the SVF was applied with the specific aim to promote adipogenesis and vascularization in engineered constructs in vivo. With only basic in vitro investigations by Lin et al. addressing the SVF for adipose repair to date [23], the present work thoroughly investigated SVF cells for adipose tissue construct generation in vitro, and in particular, pioneered the application of these cells for adipose tissue engineering in vivo. Initial in vitro experiments compared SVF- and ASC-seeded stable fibrin constructs in different medium compositions employing preadipocyte (PGM-2) and endothelial cell culture medium (EGM-2). It was found that a 1:1 mixture of PGM-2 and EGM-2, as previously established for co-culture models of adipogenesis [24], efficiently maintained cells with adipogenic and endothelial potential in SVF-seeded constructs in short and long-term culture setups. Observations on the cellular level were supported by analysis of mRNA expression of characteristic adipogenic and endothelial markers. In preparation of the evaluation of SVF-seeded constructs under in vivo conditions, a whole mount staining (WMS) method, facilitating the 3D visualization of adipocytes and blood vessels, was successfully established and optimized using native adipose tissue as template (Chapter 6). In a subcutaneous nude mouse model, SVF cells were, for the first time in vivo, elucidated for their potential to support the functional assembly of vascularized adipose tissue. Investigating the effect of adipogenic precultivation of SVF-seeded stable fibrin constructs in vitro prior to implantation on the in vivo outcome, hormonal induction was shown beneficial in terms of adipocyte development, whereas a strong vascularization potential was observed when no adipogenic inducers were added. Via histological analysis, it was proven that the developed structures were of human origin and derived from the implanted cells. Applying SVF cells without precultivation in vitro but comparing two different fibrin carriers, namely stable fibrin and TissuCol gels, revealed that TissuCol profoundly supported adipose formation by SVF cells in vivo. This was contrasted by only minor SVF cell development and a strong reduction of cell numbers in stable fibrin gels implanted without precultivation. Histomorphometric analysis of adipocytes and capillary structures was conducted to verify the qualitative results, concluding that particularly SVF cells in TissuCol were highly suited for adipose regeneration in vivo. Employing the established WMS technique, the close interaction of mature adipocytes and blood vessels in TissuCol constructs was impressively shown and via species-specific human vimentin staining, the expected strong involvement of implanted SVF cells in the formation of coherent adipose tissue was confirmed (Chapter 7). With the development of biodegradable volume-stable adipose tissue constructs, the application of ASCs and SVF cells as two promising cell sources for functional adipose regeneration, as well as the thorough evaluation of strategies for construct vascularization in vitro and in vivo, this thesis provides valuable solutions to current challenges in adipose tissue engineering. The presented findings further open up new perspectives for innovative treatments to cure soft tissue defects and serve as a basis for directed approaches towards the generation of clinically applicable soft tissue substitutes.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Ahmed2014,
  author    = {Ahmed, Arabe},
  title     = {Assessing particle deposition in a representative in vitro model of the rat respiratory tract},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-104912},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The aim of this thesis was to develop an in vitro model (IVR) of the rat lung for the purpose of investigating the deposition of drug particles in the rat airways. The model attempted to account for the affect of drug product characteristics and physiological parameters on deposition in the lungs. In addition, the model outputs were compared with in vivo lung deposition results from live rats and in silico predictions using published computer model of lung deposition in pre-clinical species. Initial work focussed on developing an aerosol exposure system capable of dosing small rodent to a range of airborne test materials. The system consists of two main parts; a fluidised bed aerosol generator and connection of the generator output to a nose only exposure chamber capable of accommodating 12 small animals in a single layer. In addition, an aerodynamic particle spectrometer (APS) was installed for continuously measuring the size distribution and airborne concentration of aerosol particles generated in the exposure chamber. System validation showed acceptable degree of variation of the test material tested, Fluorescent Microspheres (FMS) throughout the exposure chamber (CV < 15.0\%). Particle size (MMAD ± GSD) using the APS was shown to be stable throughout the exposure periods. The IVR model developed in this project was based on a number of euthanased (n=7), female Sprague-Dawley rats (weight: 372 ± 56 g), which underwent high-resolution micro-CT scans. The physical model consisted of five sub sections; Extra-Thoracic region containing the snout and nasophyarynx, trachea-bronchial region containing the trachea, bronchi, and bronchioles. All sections of the model were attached to one another in numerical order and housed within a containment unit. At the rear end of the cast, a flexible diaphragm was attached in order to collect the fraction of inhaled particles exiting the TB section and possibly reaching the lung, referred to as the Post-TB section. A study was conducted to assess the influence of inhalation parameters such as the breathing frequency and tidal volume on total and regional dose distribution using FMS as test material. The major finding of this study was the demonstration of the model sensitivity to changes in breathing parameters especially respiratory frequency, where the data showed increased deposition in the peripheral regions of the model with decreased respiratory frequency. Other studies assessed the effect of particle characteristics on deposition on the IVR model, such as particle size, dose increase and formulation changes. The results assessing particle size effect showed a slightly higher deposition levels for the 4µm sized particles versus 2µm sized particles in the head region; 90.8 ± 3.6\% and 88.2 ± 6.6\%. However, this difference did not reach statistical significance (P> 0.05) probably due to the polydispersity of aerosolised FMS particles. In addition, the regional deposition analysis showed an increased lung peripheral deposition with the smaller particles. In addition, the model was shown to be sensitive to changes in formulation composition mediated by inclusion of MgSt. The next stage of work was to validate the model in terms of comparison with lung deposition for in vivo rats. For lung deposition comparison, the absolute amount deposited in the IVR lung model (expressed as µg/kg) was shown to have a reasonably strong correlation with in vivo lung concentration measures (µg/kg); R2= 0.66, P < 0.05. Compounds were predicted well and within 2-folds of the measured lung deposition values. However, knowing the variability in biological systems and the multiple components required to estimate lung doses, predictions within 2-fold of the measured values would seem reasonable In terms of comparison with in silico model predictions using MPPD, similar deposition levels were noted between the two models, particularly when the data was expressed as percentage of total particles inhaled. The data showed the highest deposition levels were noted in the head region (> 80\%) and less than 5.0\% deposition for the peripheral lung fractions. With regards to using the IVR model to assess the relationship between dose, particle size and efficacy, an in vivo study using FP with different particle sizes (2.0 and 4.0 µm) but same doses ( 100 and 1000 µg/kg). This study demonstrated that exposure of rat to FP powder resulted in a dose-dependent inhibition of neutrophils in BAL fluids. However, a clear difference in neutrophils suppression was demonstrated for equivalent doses but different particle sizes of FP, where the smaller FP particles (2.0 µm) induced a greater level of neutrophils suppression in comparison with larger FP particles (4.0 µm). In addition, a reasonably good correlation for the relationship between lung deposition in the IVR model and a neutrophils suppression level was demonstrated. Furthermore this data support the hypothesis that regional deposition is an important determinant in efficacy. Therefore, this suggests that the IVR model may be a useful as a tool to describe in vivo efficacy with in vitro data. However, further studies should be conducted to evaluate the validity of this model and relationship. The IVR model has a number of important limitations. First, the model is based on scans up to generation four of the rat respiratory tract as this represented the limits of the micro-CT scanning technology at the time of this study. Therefore deposition in the deeper region of the lung may not be reflected precisely in the IVR model. Second, the regional deposition data generated using the model tended to show an overestimation of deposition in head region and an underestimation of deposition in the peripheral regions of the lung, in comparison with in vivo lung deposition data. Third, the current model does not take into account lung clearance. However, the amount of the drug present in the in vivo lungs is dependent on numerous physiological processes such as dissolution, passive or active absorption into the systemic circulation, binding to lung tissue and mucociliary clearance. Consequently, the results generated using this IVR model for drug molecules with high lung clearance rate should be treated with some caution. Future work extending this research could go in a number of directions. In this research, a representative model of the rat respiratory tract was constructed from analysis of imaging data from a number of euthanised Sprague-Dawley rats. This model represented the "average respiratory tract" in terms of dimensions of Sprague-Dawley rats. However, there is considerable variability in the airway dimensions between rats. This variability encompasses a number of factors such as the strains of rats, sex and age, and disease state. Thus, it may be possible to produce a small number of airway models to represent small and large rats and scaled to represent the extrathoracic and peripheral regions based on literature reports of their dimensions in different rat populations. This approach will then enable the effect of intersubject airway dimensions for different rat populations on aerosol deposition to be thoroughly examined. In addition, due to the limitation of the micro-CT technology used to construct the physical IVR model, detailed morphology only up to generation 4 were captured. However, recent advances in MRI technology, such as the use of in situ-MRI based scanning technology have enabled rat airway morphometry to be extended to 16 airway generation. This coupled with improvements in the resolutions of rapid-prototyping process means it may be possible to construct a rat model that reflects the in vivo lung morphology more accurately, and thus enable greater understanding of the link between aerosol deposition and airway geometry. In conclusion, a model cast of the rat lung was developed and validated to allow the deposition of inhaled particles in the rat lung to be investigated. The model may be used to estimate the lung concentration in vivo rats in preference to exposure concentration measurements based on filter samples which have been shown to be a poor indicator of the lung concentration immediately after exposure. In addition, the model has the potential to be used along with live rats in an inhalation rig in pulmonary pharmaceutics research and may facilitate in development of inhaled formulations to target specific regions within the lung as well as screening of inhaled drugs in preclinical setting.},
  subject      = {Ratte},
  language  = {en}
}
@phdthesis{Jarzina2022,
  author    = {Jarzina, Sebastian Oskar},
  title     = {Assessment of systemic toxicity in vitro using the Adverse Outcome Pathway (AOP) concept: nephrotoxicity due to receptor-mediated endocytosis and lysosomal overload and inhibition of mtDNA polymerase-ɣ as case studies},
  doi       = {10.25972/OPUS-26484},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-264842},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The US National Research Council (NRC) report "Toxicity Testing in the 21st Century: A Vision and a strategy (Tox21)", published in 2007, calls for a complete paradigm shift in tox-icity testing. A central aspect of the proposed strategy includes the transition from apical end-points in in vivo studies to more mechanistically based in vitro tests. To support and facilitate the transition and paradigm shift in toxicity testing, the Adverse Outcome Pathway (AOP) concept is widely recognized as a pragmatic tool. As case studies, the AOP concept was ap-plied in this work to develop AOPs for proximal tubule injuries initiated by Receptor-mediated endocytosis and lysosomal overload and Inhibition of mtDNA polymerase-. These AOPs were used as a mechanistic basis for the development of in vitro assays for each key event (KE). To experimentally support the developed in vitro assays, proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) were treated with model compounds. To measure the dis-turbance of lysosomal function in the AOP - Receptor-mediated endocytosis and lysosomal overload, polymyxin antibiotics (polymyxin B, colistin, polymyxin B nonapeptide) were used as model compounds. Altered expression of lysosomal associated membrane protein 1/2 (LAMP-1/2) (KE1) and cathepsin D release from lysosomes (KE2) were determined by im-munofluorescence, while cytotoxicity (KE3) was measured using the CellTiter-Glo® cell via-bility assay. Importantly, significant differences in polymyxin uptake and susceptibility be-tween cell lines were observed, underlining the importance of in vitro biokinetics to determine an appropriate in vitro point of departure (PoD) for risk assessment. Compared to the in vivo situation, distinct expression of relevant transporters such as megalin and cubilin on mRNA and protein level in the used cell lines (RPTEC/TERT1 and NRK-52E) could not be con-firmed, making integration of quantitative in vitro to in vivo extrapolations (QIVIVE) neces-sary. Integration of QIVIVE by project partners of the University of Utrecht showed an im-provement in the modelled biokinetic data for polymyxin B. To assess the first key event, (KE1) Depletion of mitochondrial DNA, in the AOP - Inhibition of mtDNA polymerase-, a RT-qPCR method was used to determine the mtDNA copy number in cells treated with mod-el compounds (adefovir, cidofovir, tenofovir, adefovir dipivoxil, tenofovir disoproxil fumarate). Mitochondrial toxicity (KE2) was measured by project partners using the high-content imaging technique and MitoTracker® whereas cytotoxicity (KE3) was determined by CellTiter-Glo® cell viability assay. In contrast to the mechanistic hypothesis underlying the AOP - Inhibition of mtDNA polymerase-, treatment with model compounds for 24 h resulted in an increase rather than a decrease in mtDNA copy number (KE1). Only minor effects on mitochondrial toxicity (KE2) and cytotoxicity (KE3) were observed. Treatment of RPT-EC/TERT1 cells for 14 days showed only a slight decrease in mtDNA copy number after treatment with adefovir dipivoxil and tenofovir disoproxil fumarate, underscoring some of the limitations of short-term in vitro systems. To obtain a first estimation for risk assessment based on in vitro data, potential points of departure (PoD) for each KE were calculated from the obtained in vitro data. The most common PoDs were calculated such as the effect concentra-tion at which 10 \% or 20_\% effect was measured (EC10, EC20), the highest no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), the benchmark 10 \% (lower / upper) concentrations (BMC10, BMCL10, BMCU10) and a modelled non-toxic con-centration (NtC). These PoDs were then compared with serum and tissue concentrations de-termined from in vivo studies after treatment with therapeutic / supratherapeutic doses of the respective drugs in order to obtain a first estimate of risk based on in vitro data. In addition, AOPs were used to test whether the quantitative key event relationships between key events allow prediction of downstream effects and effects on the adverse outcome (AO) based on measurements of an early key event. Predictions of cytotoxicity from the mathematical rela-tionships showed good concordance with measured cytotoxicity after treatment with colistin and polymyxin b nonapeptide. The work also revealed uncertainties and limitations of the ap-plied strategy, which have a significant impact on the prediction and on a risk assessment based on in vitro results.},
  language  = {en}
}
@phdthesis{Kappenberger2024,
  author    = {Kappenberger, Jeannette Sarah},
  title     = {Biochemical characterization of the TFIIH translocase XPB from \(Chaetomium\) \(thermophilum\)},
  doi       = {10.25972/OPUS-24409},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244096},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {DNA repair and gene expression are two major cellular processes that are fundamental for the maintenance of biological life. Both processes require the enzymatic activity of the super family 2 helicase XBP, which is an integral subunit of the general transcription factor TFIIH. During transcription initiation, XPB catalyzes the initial melting of promoter DNA enabling RNA polymerase II to engage with the coding DNA strand and start gene transcription. In nucleotide excision repair, XPB acts in concert with the other TFIIH helicase XPD causing strand separation around a lesion site. Mutations within the genes encoding XPB or other TFIIH subunits are associated with different cancer types as well as with the autosomal recessive disorders Xeroderma Pigmentosum and trichothiodystrophy and rarely combined features of Xeroderma Pigmentosum and Cockayne syndrome. In the last few years, great progress has been made towards unraveling the structure of TFIIH and its individual subunits including XPB. These structural insights tremendously improved our understandings with respect to the molecular interactions within this intriguing protein complex. However, the underlying regulation mechanisms that functionally control XPB during transcription and repair remained largely elusive. We thus executed the biochemical characterization of this protein to investigate the functional network that regulates XPB within the scaffold of TFIIH. Due to their enhanced stability compared to the human proteins, we utilized the proteins that originate from the thermophilic fungus Chaetomium thermophilum for this purpose as a model organism for eukaryotic TFIIH. The present work provides novel insights into the enzymatic function and regulation of XPB. We could show that both, DNA and the TFIIH subunit p52 stimulate XPB's ATPase activity and that the p52-mediated activity is further boosted by p8, another subunit within TFIIH. Surprisingly, DNA can activate XPB's ATPase activity to a greater extent than its TFIIH interaction partners p52/p8, but when both, i.e. p52/p8 and DNA are present at the same time, p52 dominates the activation and the enzymatic speed is maintained at the level observed through the sole activation of p52/p8. We thus defined p52 as the master regulator of XPB that simultaneously activates and represses XPB's enzymatic activity. Based on a correlative mutagenesis study of the main interface between p52 and XPB that was set into context with recent structural data, a model for the p52-mediated activation and speed limitation of XPB's ATPase was proposed. The research on XPB's ATPase was expanded with the investigation of the inhibition mechanism of XPB's ATPase via the natural compound Triptolide. Furthermore, we investigated XPB's DNA translocase function and could observe that XPB can only perform its translocase movement when it is fully incorporated into core TFIIH and this translocase movement is further enhanced by the nucleotide excision repair factor XPA. Fluorescence polarization measurements with nucleotide analogues revealed that XPB displays the highest affinity towards DNA in the ADP + Pi bound state and its binding is weakened when ADP is bound or the nucleotide is dissociated from the enzyme, suggesting a movement on the DNA during the distinct states of the ATPase cycle. Finally, the well-known and highly conserved RED motif was found to be the crucial element in XPB to enable this translocase movement. Combined, the data presented in this work provide novel insights into the intricate regulation network that controls XPB's enzymatic activity within TFIIH and furthermore show that XPB's enzymatic activity is tightly controlled by various factors.},
  subject      = {DNS-Reparatur},
  language  = {en}
}
@phdthesis{Englbrecht2014,
  author    = {Englbrecht, Clemens},
  title     = {Biochemische Rekonstitution und funktionelle Charakterisierung der Zusammenlagerungsmaschinerie spleißosomaler U snRNPs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Das Spleißen von pr{\"a}-mRNAs stellt in der Expression eukaryontischer Gene einen essentiellen Reifungsschritt dar. Erst durch das exakte Entfernen von nicht-kodierenden Introns und Verbinden der kodierenden Exons kann die genetische Information am Ribosom in funktionelle Proteine umgesetzt werden. Spleißen wird durch das Spleißosom katalysiert, welches sich aus den small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 und U6 und einer großen Anzahl weiterer Proteinfaktoren zusammensetzt. Die snRNPs bestehen aus einer Uridin-reichen snRNA, spezifischen und generellen (Sm-)Proteinen. Die Sm-Proteine B/B`, D1, D2, D3, E, F, und G bilden einen heptameren Ring um die sog. Sm-Bindungsstelle der snRNAs. W{\"a}hrend die Zusammenlagerung von Sm-Proteinen mit der RNA in vitro spontan ablaufen kann, wird dieser Prozess in vivo von zwei makromolekularen Proteinkomplexen assistiert, die als PRMT5- bzw. SMN-Komplex bezeichnet werden. Der PRMT5-Komplex (bestehend aus PRMT5, WD45 und pICln) agiert in der fr{\"u}hen Phase der Zusammenlagerung. Seine Hauptfunktion ist die symmetrische Dimethylierung der Sm-Proteine und die Stabilisierung von Sm-Proteinkomplexen durch das Chaperon pICln in zwei Intermediaten. Einhergehend mit dieser Aktivit{\"a}t werden auch Aggregation bzw. unspezifische Wechselwirkungen der Sm-Proteine mit RNAs verhindert. In der sp{\"a}ten Phase der Zusammenlagerung l{\"o}st der SMN-Komplex (bestehend aus SMN, Gemin2-8 und unrip) pICln-Intermediate auf, wobei dieser die Sm-Proteine en bloc {\"u}bernimmt und sie auf die snRNA {\"u}bertr{\"a}gt. W{\"a}hrend dieser Reaktion wird pICln aus den Komplexen verdr{\"a}ngt. Ein Fehlen des SMN-Proteins, einer Schl{\"u}sselkomponente des SMN-Komplexes, f{\"u}hrt zur autosomal rezessiven Erbkrankheit `Spinale Muskelatrophie` (SMA) wobei der Schweregrad der Krankheit invers mit der Menge an funktionellem SMN-Protein korreliert. Es wird vermutet, dass eine gest{\"o}rte snRNP-Biogenese die Ursache der SMA ist. In der vorliegenden Arbeit sollte die U snRNP-Zusammenlagerungsmaschinerie aus rekombinanten Bausteinen rekonstituiert werden und so funktionellen und strukturellen Studien zug{\"a}nglich gemacht werden. Folgende Resultate wurden in dieser Arbeit erhalten: 1) Im ersten Teil der Arbeit wurde eine experimentelle Strategie etabliert, welche die Rekonstitution des humanen SMN-Komplexes aus rekombinanten Untereinheiten erlaubte. Entscheidend hierf{\"u}r war die Definition von Subkomplexen aufgrund einer Protein-Interaktionskarte. Die Subkomplexe konnten separat hergestellt und anschließend zum Gesamtkomplex vereinigt werden. 2) Die erfolgreiche Etablierung eines rekonstitutiven Systems erlaubte eine detaillierte biochemische Charakterisierung des SMN-Komplexes. Es konnte gezeigt werden, dass der rekombinante Komplex alle f{\"u}r die Biogenese von U snRNPs n{\"o}tigen Schritte bewerkstelligen konnte. Dies schließt sowohl die {\"U}bernahme der Sm-Proteine aus den pICln-Intermediaten als auch das Verdr{\"a}ngen des Chaperons pICln und die {\"U}bertragung der Sm-Proteine auf die snRNAs ein. 3) Durch die Reduzierung des SMN-Gesamtkomplexes um Gemin3-5 auf einen SMN-Pentamer konnte dieser als ein funktioneller Kernbereich identifiziert werden, der die einzelnen Schritte der U snRNP-Biogenese vergleichbar mit dem gesamten Komplex bewerkstelligen konnte. Zudem agierte dieser reduzierte Komplex als notwendiger und ausreichender Spezifit{\"a}tsfaktor der RNP-Zusammenlagerung. 4) Das rekombinante System erm{\"o}glichte erstmals SMN-Komplexe mit SMA-pathogenen Mutationen herzustellen und einer eingehenden funktionellen und strukturellen Untersuchung zu unterziehen. Die detaillierte Analyse der SMA-verursachenden Punktmutation SMN(E134K) offenbarte spezifische Defekte im Zusammenlagerungsprozess und damit Einblicke in die Pathophysiologie der Krankheit. Mit der im Rahmen dieser Arbeit etablierten Rekonstitution des rekombinanten SMN-Komplexes wurde die Grundlage f{\"u}r die detaillierte biochemische und strukturbiologische Untersuchung der Zusammenlagerungsmaschinerie spleißosomaler U snRNPs gelegt. Dieses experimentelle System wird auch bei der Aufdeckung der biochemischen Defekte hilfreich sein, die zur neuromuskul{\"a}ren Krankheit SMA f{\"u}hren.},
  subject      = {Small nuclear RNP},
  language  = {de}
}
@phdthesis{Meininger2022,
  author    = {Meininger, Markus},
  title     = {Calcium hydroxide as antibacterial implant coating},
  doi       = {10.25972/OPUS-26112},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261122},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {In modern medicine hip and knee joint replacement are common surgical procedures. However, about 11 \% of hip implants and about 7 \% of knee implants need re-operations. The comparison of implant registers revealed two major indications for re-operations: aseptic loosening and implant infections, that both severely impact the patients' health and are an economic burden for the health care system. To address these problems, a calcium hydroxide coating on titanium was investigated in this thesis. Calcium hydroxide is a well-known antibacterial agent and used with success in dentistry. The coatings were applied with electrochemically assisted deposition, a versatile tool that combines easiness of process with the ability to coat complex geometries homogeneously. The pH-gradient during coating was investigated and showed the surface confinement of the coating process. Surface pre-treatment altered the surface morphology and chemistry of the titanium substrates and was shown to affect the morphology of the calcium hydroxide coatings. The influence of the coating parameters stirring speed and current pulsing were examined in various configurations and combinations and could also affect the surface morphology. A change in surface morphology results in a changed adhesion and behavior of cells and bacteria. Thus, the parameters surface pre-treatment, stirring speed and current pulsing presented a toolset for tailoring cellular response and antibacterial properties. Microbiological tests with S. aureus and S. epidermidis were performed to test the time-dependent antibacterial activity of the calcium hydroxide coatings. A reduction of both strains could be achieved for 13 h, which makes calcium hydroxide a promising antibacterial coating. To give insight into biofilm growth, a protocol for biofilm staining was investigated on titanium disks with S. aureus and S. epidermidis. Biofilm growth could be detected after 5 days of bacterial incubation, which was much earlier than the 3 weeks that are currently assumed in medical treatment. Thus, it should be considered to treat infections as if a biofilm were present from day 5 on. The ephemeral antibacterial properties of calcium hydroxide were further enhanced and prolonged with the addition of silver and copper ions. Both ionic modifications significantly enhanced the bactericidal potential. The copper modification showed higher antibacterial effects than the silver modification and had a higher cytocompatibility which was comparable to the pure calcium hydroxide coating. Thus, copper ions are an auspicious option to enhance the antibacterial properties. Calcium hydroxide coatings presented in this thesis have promising antibacterial properties and can easily be applied to complex geometries, thus they are a step in fighting aseptic loosening and implant infections.},
  subject      = {Calciumhydroxid},
  language  = {en}
}
@phdthesis{Steinbacher2015,
  author    = {Steinbacher, Andreas Edgar},
  title     = {Circular dichroism and accumulative polarimetry of chiral femtochemistry},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116500},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {This work brings forward successful implementations of ultrafast chirality-sensitive spectroscopic techniques by probing circular dichroism (CD) or optical rotation dispersion (ORD). Furthermore, also first steps towards chiral quantum control, i.e., the selective variation of the chiral properties of molecules with the help of coherent light, are presented. In the case of CD probing, a setup capable of mirroring an arbitrary polarization state of an ultrashort laser pulse was developed. Hence, by passing a left-circularly polarized laser pulse through this setup a right-circularly polarized laser pulse is generated. These two pulse enantiomers can be utilized as probe pulses in a pump--probe CD experiment. Besides CD spectroscopy, it can be utilized for anisotropy or ellipsometry spectroscopy also. Within this thesis, the approach is used to elucidate the photochemistry of hemoglobin, the oxygen transporting protein in mammalian blood. The oxygen loss can be triggered with laser pulses as well, and the results of the time-resolved CD experiment suggest a cascade-like relaxation, probably through different spin states, of the metallo-porphyrins in hemoglobin. The ORD probing was realized via the combination of common-path optical heterodyne interferometric polarimetry and accumulative femtosecond spectroscopy. Within this setup, on the one hand the applicability of this approach for ultrafast studies was demonstrated explicitly. On the other hand, the discrimination between an achiral and a racemic solution without prior spatial separation was realized. This was achieved by inducing an enantiomeric excess via polarized femtosecond laser pulses and following its evolution with the developed polarimeter. Hence, chiral selectivity was already achieved with this method which can be turned into chiral control if the polarized laser pulses are optimized to steer an enhancement of the enantiomeric excess. Furthermore, within this thesis, theoretical prerequisites for anisotropy-free pump--probe experiments with arbitrary polarized laser pulses were derived. Due to the small magnitude of optical chirality-sensitve signals, these results are important for any pump--probe chiral spectroscopy, like the CD probing presented in this thesis. Moreover, since for chiral quantum control the variation of the molecular structure is necessary, the knowledge about rearrangement reactions triggered by photons is necessary. Hence, within this thesis the ultrafast Wolff rearrangement of an α-diazocarbonyl was investigated via ultrafast photofragment ion spectroscopy in the gas phase. Though the compound is not chiral, the knowledge about the exact reaction mechanism is beneficial for future studies of chiral compounds.},
  subject      = {Ultrakurzzeitspektroskopie},
  language  = {en}
}
@unpublished{WohlgemuthMiyazakiTsukadaetal.2017,
  author    = {Wohlgemuth, Matthias and Miyazaki, Mitsuhiko and Tsukada, Kohei and Weiler, Martin and Dopfer, Otto and Fujii, Masaaki and Mitrić, Roland},
  title     = {Deciphering environment effects in peptide bond solvation dynamics by experiment and theory},
  series = {Physical Chemistry Chemical Physics},
  journal   = {Physical Chemistry Chemical Physics},
  doi       = {10.1039/C7CP03992A},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159483},
  year      = {2017},
  abstract  = {Most proteins work in aqueous solution and the interaction with water strongly affects their structure and function. However, experimentally the motion of a specific single water molecule is difficult to trace by conventional methods, because they average over the heterogeneous solvation structure of bulk water surrounding the protein. Here, we provide a detailed atomistic picture of the water rearrangement dynamics around the -CONH- peptide linkage in the two model systems formanilide and acetanilide, which simply differ by the presence of a methyl group at the peptide linkage. The combination of picosecond pump-probe time-resolved infrared spectroscopy and molecular dynamics simulations demonstrates that the solvation dynamics at the molecular level is strongly influenced by this small structural difference. The effective timescales for solvent migration triggered by ionization are mainly controlled by the efficiency of the kinetic energy redistribution rather than the shape of the potential energy surface. This approach provides a fundamental understanding of protein hydration and may help to design functional molecules in solution with tailored properties.},
  language  = {en}
}
@phdthesis{HorvatCsotigebHorvat2021,
  author    = {Horvat-Cs{\´o}ti [geb. Horvat], Sonja},
  title     = {Development of Nanocarriers for Treatment and Diagnostics of Aspergillosis},
  doi       = {10.25972/OPUS-23821},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-238218},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {This thesis aimed to evaluate the possibility to use nanoparticles as antifungal drug carriers as well as their potential application in screening and diagnostics of invasive aspergillosis. The interaction of nanogels, superparamagnetic iron oxide nanoparticles (SPIOs) and gold nanoparticles (GNP) with fungal-specific polysaccharides, cells and biofilms was investigated. Firstly, it was evaluated how the charge of nanogels influence their interaction with fungal cells. Linear poly(glycidol)s (pG) and poly(2-methyl-2-oxazoline) (pMOx) polymers were synthesized and further functionalized with thiol groups for preparation of redox responsive nanogels. Results showed that negatively charged nanogels were internalized by the fungi to a much greater extent than positively charged ones. Furthermore, it was investigated how amphiphilicity of polymers used for preparation of nanogels influences nanogel-fungi interaction. It was concluded that nanogels prepared from polymers with degree of functionalization of 10\% had the strongest interaction, regardless the length of the alkyl chain. Moreover, amphotericin B-loaded nanogels had a higher antifungal effect and lower toxicity towards mammalian cells than the free drug. In addition, inverse nanoprecipitation of thiol functionalized pGs was shown to be successful for preparation of nanogels with narrow size distribution. It was also demonstrated that crosslinking of the polymeric coating in hydrogel-like network with thiol functionalized pGs improved the SPIOs imaging performance. Finally, it was investigated whether GNPs could be used as model particles for the assessment of targeting to fungi. Fc dectin-1 was conjugated covalently to GNPs decorated with pGs, and binding affinity towards β-glucans was tested by surface plasmon resonance. In summary, this thesis demonstrated evidence for the potential of pG nanogels and pG coated nanoparticles for antifungal therapy and diagnostics of fungal infections caused by A. fumigatus.},
  subject      = {Therapeutisches System},
  language  = {en}
}
@phdthesis{Kaiser2014,
  author    = {Kaiser, Conrad},
  title     = {Donor-Bridge-Acceptor Systems with Varying Bridge Units for the Investigation of Intramolecular and Intermolecular Electron Transfer Processes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97614},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Within this study, the influence of the energetics of the bridge unit on electron transfer (ET) in an electrode-bridge-donor system was investigated in a monolayer environment. This was realized by specifically designing molecules containing ferrocene carboxylic ester donors and hydroquinone derivatives as bridge units and by using a gold electrode as acceptor. The energetics of the hydroquinone derivatives was adjusted by synthetically varying its substituents with the intention of changing the ET speed and mechanisms. Thereby the choice of the substituents was based on the literature known half-wave potentials of similar solvated hydroquinone derivatives and successively confirming them by conducting cyclic voltammetry on the actual bridge units synthesized. Then, a synthetic pathway, which accommodated the limited stability of the integrated terminal ferrocene carbon acid ester, was developed and successfully employed. This was followed by developing a procedure for preparing very dense and highly ordered monolayers from the target molecules on self-made gold microelectrodes. For the electrochemical investigations, several electrolyte solutions were tested until one, which ensured low susceptibility of the characterization setup towards slight changes of the electrode arrangement and measurement parameters while ensuring sufficient stability of the monolayers, was found. Furthermore, a new, commercially available potentiostat was established for the impedance measurements, which reduced the stress on the monolayers during the electrochemical characterizations in comparison to the equipment used in many former studies. Regarding the determination of the ET rates, the data analysis protocol for the impedance measurements developed by Creager et al. was slightly adapted to allow analysis of the investigated monolayers despite their non-ideal behavior. In addition, the influence of changes to the electrical parameters of the impedance scans was investigated to minimize the error in the acquired data. The electrochemical analysis of the monolayers by conducting cyclic voltammetry on MA, MB and MC prepared from A, B and C confirmed the accomplishment of near ideal surface coverage and exceptionally high order. The surface coverages of MB and MC were, probably due to the space filled by the substituents on their bridge units, slightly lower than those of MA. Furthermore, the shape of the redox waves of the ferrocene carboxylic acid redox center in the voltammogram of MA showed a broadening and a shift towards higher potentials, which was assigned to electrostatic interference of oxidized terminal redox centers due to the especially dense packing. However, in the voltammogram of MB, no sharp redox waves of the bridge units, as predicted by the analysis of preliminary monolayers of the same type with low surface coverage, were present. This was attributed to the different and varying microenvironment of the bridge units deeply embedded within high-density monolayers. In detail, the different degree of shielding of each individual bridge unit from counter ions and solvent molecules probably resulted in the half wave potential being shifted to varying higher potentials, thus preventing the formation of sharp redox waves. In addition, electrostatic effects of oxidized bridge units could have enhanced this effect. This leads to the conclusion that the half-wave potentials of fully solvated bridge units determined by the cyclic voltammetry are not suited to predict the energetics of the oxidized bridge states embedded within the prepared high density monolayers. Finally, the monolayers were successfully analyzed by impedance spectroscopy, which showed that the ET rate of MA is slightly higher than that of MB, and both are higher than that of MC. All of the values were, according to literature, in the expected region considering the length and degree of conjugation of the backbone. However, this picture is relativized when considering the targeted energetic alignment of the bridge units. According to the predicted very small energy gap between the oxidized states of the donor and the bridge unit in MB, a domination of the hopping mechanism should have led to a several orders of magnitude higher ET rate than in MA and MC. That this was not the case was attributed to the underestimation of the energy of the oxidized bridge states by utilizing cyclic voltammetry of the fully solvated bridge units (see above). According to the small differences of the ET rates the superexchange process was assumed to be the dominating mechanism not only in MA and MC but also in MB. However, even when shifted, the predicted energetic order of the oxidized bridge states should have led to a moderately decreasing ET rate from MB over MA to MC. The reason for the actual ET rate in MA being slightly higher than in MB might be found in the electrostatic interference of the terminal redox centers in MA (see above). In conclusion, the targeted model systems were prepared and the ET rates were successfully determined. However, the problems concerning the relative energetic positioning of the involved states within the dense monolayers prevented the specific alteration of the speed and mechanism of the ET. The reason for this can be probably found in the high density and order of the monolayers prepared within this work, which hamper the intrusion of the components of the electrolyte solutions. This various degree of stabilization for the individual bridge units by counter ions and solvent molecules leads to the energy of the oxidized bridge states being splitted and shifted towards higher potentials with respect to fully solvated bridge units. This effect might be further enhanced by electrostatics of neighboring already oxidized bridge states. All this makes the predetermination of the energetics of the embedded bridge units extremely difficult. On one hand, this behavior can be considered an obstacle and could probably be circumvented by designing molecules with bulky anchor groups and rigid molecular backbones, which would ensure perpendicular arrangement to the surface and full exposure of the bridge and terminal redox centers to the solvent molecules and counter ions. On the other hand, monolayers which completely embed integral redox centers might open up the opportunity to study the effects of microenvironments similar to those in solid state materials. Regarding mixed valence compounds, the present study focuses on bistriarylamine radical cation F∙+, which contains the [3.3]paracyclophane bridge unit. The results were compared to the, except for the bridge units, identical literature known compounds G∙+ and N∙+ with [2.2]paracyclophane and p-xylene bridges respectively. This led to the conclusion that slightly different bridge units can induce substantial changes to the internal reorganization energy. This is especially noteworthy since it is usually believed that structural adaption limited to the redox centers taking part in the charge transfer dominates the internal reorganization energy. Furthermore, the application of the two-state Mulliken-Hush approach shows that compounds F∙+ and G∙+ have near identical couplings and similar thermal barriers. Confirmation of the latter finding as well as near identical thermal electron transfer rates for both compounds were provided via a cooperation project by Grampp et al. in which these values were directly extracted from temperature dependent electron paramagnetic resonance measurements. These results are quite unexpected since the "through-space" distances of the stacked pi-systems in the paracyclophane bridges differ significantly. They are well within the sum of the van der Waals radii in G∙+ and barely within them in compound F∙+. In addition, these findings weaken the common assumption of the ethylene bridges in G∙+ substantially adding to the electronic coupling, since then, in F∙+, due to its propylene linkers, the coupling should be substantially reduced. Finally, relying on the fact that the electronic couplings are only three times higher and the thermal electron transfer rates are only one order of magnitude higher for N∙+ than for compounds F∙+ and G∙+ shows that intermolecular electron transfer in solid state materials can remain efficient, if the interacting pi-systems stay within the sum of van der Waals radii of their carbons. Concerning the donor-acceptor dyads, the current investigation centers on triarylamine-cyclophane-naphtalene diimide (TAA-CP-NDI) compounds which display almost complete photoinduced charge separation. Furthermore, their singlet charge separated states show lifetimes of hundreds of nanoseconds, which is rarely found in such simple dyads. In the present case they can be attributed to the particular amount of electronic coupling V (on the order of 100 cm^-1), which is brought about by incorporation of the smallest model systems for pi-stacks, the CPs, together with the nodes on the NDI lowest unoccupied molecular orbital, which electronically decouples the central NDI from its nitrogen substituents. In agreement with studies of [2.2]- and [3.3]paracyclophane bridged mixed valence compounds (see above), the cycolphane bridged dyads show very similar electronic coupling when dealing with ground state processes like charge recombination. However, when investigating excited state processes, like charge separation in the TAA-CP-NDI dyads, one has to bear in mind that the CP orbitals are involved in the formation of intermediate states that likely possess charge transfer character. In this case, the [2.2]paracyclophane bridge obviously induces a stronger coupling than the [3.3]paracyclophane. Another interesting property of the dyads studied here is the substantial population of the triplet charge separated (CS) state of ca. one third regarding both CS states, which is brought about by singlet-triplet interconversion from the singlet CS state. Thus, the triplet CS state with a lifetime of several microseconds acts as a kind of buffer for the CS state before recombining to the ground state and, thus, leads to distinctly prolonged overall lifetimes of the charge separated states. Thus it can be concluded that the intersystem crossing and charge recombination (CR) processes of the CS states are governed by a delicate balance of a large electronic coupling V and a large exchange interaction 2J (both with regard to systems containing a through-space pathway). The latter appears to be induced by second order interaction with a local triplet state lying close in energy to the CS state. This balance results in slow CR- and singlet-triplet- interconversion rates, which differ only by one order of magnitude. Compared to the many NDI containing dyads studied so far, these features of the dyads studied here are, to the best of our knowledge, unique. Especially the combination of high quantum yield of charge separation, long lifetimes and high energy of the charge separated state make the investigated systems interesting for practical applications. Furthermore, the presented unraveling of the underlying mechanisms is of substantial value for the future design of dyads for practical applications regarding the implementation and adjustment of these favorable properties.},
  subject      = {Elektronentransfer},
  language  = {en}
}
@phdthesis{He2024,
  author    = {He, Feng},
  title     = {Drug Discovery based on Oxidative Stress and HDAC6 for Treatment of Neurodegenerative Diseases},
  doi       = {10.25972/OPUS-25349},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-253497},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Most antioxidants reported so far only achieved limited success in AD clinical trials. Growing evidences suggest that merely targeting oxidative stress will not be sufficient to fight AD. While multi-target directed ligands could synergistically modulate different steps in the neurodegenerative process, offering a promising potential for treatment of this complex disease. Fifteen target compounds have been designed by merging melatonin and ferulic acid into the cap group of a tertiary amide HDAC6 inhibitor. Compound 10b was screened as the best hybrid molecule exhibit potent HDAC6 inhibition and potent antioxidant capacity. Compound 10b also alleviated LPS-induced microglia inflammation and led to a switch from neurotoxic M1 to the neuroprotective M2 microglial phenotype. Moreover, compound 10b show pronounced attenuation of spatial working memory and long-term memory damage in an in vivo AD mouse model. Compound 10b can be a potentially effective drug candidate for treatment of AD and its druggability worth to be further studied. We have designed ten novel neuroprotectants by hybridizing with several common antioxidants, including ferulic acid, melatonin, lipoic acid, and trolox. The trolox hybrid compound exhibited the most potent neuroprotective effects in multiple neuroprotection assays. Besides, we identified the synergistic effects between trolox and vitamin K derivative, and our trolox hybrid compound showed comparable neuroprotection with the mixture of trolox and vitamin K derivative. We have designed and synthesized 24 quinone derivatives based on five kinds of different quinones including ubiquinone, 2,3,5-trimethyl-1,4-benzoquinone, memoquin, thymoquinone, and anthraquinone. Trimethylbenzoquinone and thymoquinone derivatives showed more potent neuroprotection than other quinones in oxytosis assay. Therefore, trimethylbenzoquinone and thymoquinone derivatives can be used as lead compounds for further mechanism study and drug discovery for treatment of neurodegenerative disease. We designed a series of photoswitchable HDAC inhibitors, which could be effective molecular tools due to the high spatial and temporal resolution. In total 23 target compounds were synthesized and photophysicochemically characterized. Azoquinoline-based compounds possess more thermally stable cis-isomers in buffer solution, which were further tested in enzyme-based HDAC inhibition assay. However, none of those tested compounds show significant differences in activities between trans-isomers and corresponding cis-isomers.},
  subject      = {Alzheimerkrankheit},
  language  = {en}
}
@phdthesis{Zieschang2014,
  author    = {Zieschang, Fabian},
  title     = {Energy and Electron Transfer Studies of Triarylamine-based Dendrimers and Cascades},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {In this work the synthesis of dendritic macromolecules and small redox cascades was reported and studies of their energy and electron transfer properties discussed. The chromophores in the dendrimers and the redox cascades are linked via triazoles, which were built up by CuAAC. Thereby, a synthetic concept based on building blocks was implemented, which allowed the exchange of all basic components. Resulting structures include dendrimers composed exclusively of TAAs (G1-G3), dendrimers with an incorporated spirobifluorene core (spiro-G1 and spiro-G2) and the donor-acceptor dendrimer D-A-G1, in which the terminal groups are exchanged by NDIs. Furthermore, a series of model compounds was synthesised in order to achieve a better understanding of the photophysical processes in the dendrimers. A modification of the synthetic concept for dendrimers enabled the synthesis of a series of donor-acceptor triads (T-Me, T-Cl and T-CN) consisting of two TAA donors and one NDI acceptor unit. The intermediate TAA chromophore ensured a downhill redox gradient from the NDI to the terminal TAA, which was proved by cyclic voltammetry measurements. The redox potential of the intermediate TAA was adjusted by different redox determining substituents in the "free" p-position of the TAA. Additionally, two dyads (Da and Db) were synthesised which differ in the junction of the triazole to the TAA or the NDI, respectively. In these cascades a nodal-plane along the N-N-axes in the NDI and a large twist angle between the NDI and the N-aryl substituent guaranteed a small electronic coupling. The photophysical investigations of the dendrimers focused on the homo-energy transfer properties in the TAA dendrimers G1-G3. Steady-state emission spectroscopy revealed that the emission takes place from a charge transfer state. The polar excited state resulted in a strong Stokes shift of the emission, which in turn led to a small spectral overlap integral between the absorption of the acceptor and the emission of the donor in the solvent relaxed state. According to the F{\"o}rster theory, the overlap integral strongly determines the energy transfer rate. Fluorescence up-conversion measurements showed a strong and rapid initial fluorescence anisotropy decay and a much slower decrease on the longer time scale. The experiment revealed a fast energy transfer in the first 2 ps followed by a much slower energy hopping. Time resolved emission spectra (TRES) of the model compound M indicated a solvent relaxation on the same time scale as the fast energy transfer. The F{\"o}rster estimation of energy transfer rates in G1 explains fast energy transfer in the vibrotionally relaxed state before solvent relaxation starts. Thereby, the emission spectrum of G1 in cyclohexane served as the time zero spectrum. Thus, solvent relaxation and fast energy transfer compete in the first two ps after excitation and it is crucial to discriminate between energy transfer in the Franck-Condon and in the solvent relaxed state. Furthermore, this finding demonstrates that fast energy transfer occurs even in charge transfer systems where a large Stokes shift prevents an effective spectral overlap integral if there is a sufficient overlap integral in before solvent relaxation. Energy transfer upon excitation was also observed in the spiro dendrimers spiro-G1 and spiro-G2 and identified by steady-state emission anisotropy measurements. It was assumed that the energy in spiro-G1 is completely distributed over the entire molecule while the energy in spiro-G2 is probably distributed over only one individual branch. This finding was based on a more polarised emission of spiro-G2 compared to spiro-G1. This issue has to be ascertained by e.g. time resolved emission anisotropy measurements in further energy transfer studies. Concerning the electron transfer properties of TAA-triazole systems the radical cations of G1-G2, spiro-G1 and spiro-G2 and of the model compound M were investigated by steady-state absorption spectroscopy. Experiments showed that the triazole bridge exhibits small electronic communication between the adjacent chromophores but still possesses sufficient electronic coupling to allow an effective electron transfer from one chromophore to the other. Due to the high density of chromophores, their D-A-D structure and their superficial centrosymmetry, the presented dendrimers are prospective candidates for two-photon absorption applications. The dyads, triads and the donor-acceptor dendrimer D-A-G1 were investigated regarding their photoinduced electron transfer properties and the effects that dominate charge separation and charge recombination in these systems. The steady-state absorption spectra of all cascades elucidated a superposition of the absorption characteristics of the individual subunits and spectra indicated that the chromophores do not interact in the electronic ground state. Time resolved transient absorption spectroscopy of the cascades was performed in the fs- and ns-time regime in MeCN and toluene as solvent. Measurements revealed that upon with 28200 cm-1 (355) nm and 26300 cm-1 (380 nm), respectively, an electron is transferred from the TAA towards the NDI unit yielding a CS state. In the triads at first a CS1 state is populated, in which the NDI is reduced and the intermediate TAA1 is oxidised. Subsequently, an additional electron transfer from the terminal TAA2 to TAA1 led to the fully CS2 state. Fully CS states of the dyads and triads exhibit lifetimes in the ns-time regime. In contrast for Db in MeCN, a lifetime of 43 ps was observed for the CS state together with the population of a 3NDI state. The signals of the other CS states decay biexponentially, which is a result of the presence of the 1CS and the 3CS states. While magnetic field dependent measurements of Db did not show an effect due to the large singlet-triplet splitting, T-CN exhibited a strong magnetic field dependence which is an evidence for the 1CS/3CS assignment. Further analysis of the singlet-triplet dynamics are required and are currently in progress. Charge recombination occurred in the Marcus inverted region for compounds solved in toluene and in the Marcus normal region for MeCN as solvent. However, a significant inverted region effect was observed only for Db. Triads are probably characterised by charge recombination rates in the inverted and in the normal region near to the vertex of the Marcus parabola. Hence the inverted region effect is not pronounced and the rate charge recombination rates are all in the same magnitude. However, compared to the charge recombination rate of Db the enlarged spatial distance between the terminal TAA and the NDI in the fully CS2 states in the triads resulted in reduced charge recombination rates by ca. one order of magnitude. More important than a small charge recombination rate is an overall lifetime of the CS states and this lifetime can significantly be enhanced by the population of the 3CS state. The reported results reveal that a larger singlet-triplet splitting in the dyads led to a CS state lifetime in the us time regime while a lifetime in the ns-time regime was observed in cases of the triads. Moreover, the singlet-triplet splitting was found to be solvent dependent in the triads, which is a promising starting point for further investigations concerning singlet-triplet splitting. The donor-acceptor dendrimer D-A-G1 showed similar characteristics to the dyads. The generation of a CS state is assumed due to a clear NDI radical anion band in the transient absorption spectrum. Noteworthy, the typical transient absorption band of the TAA radical cation is absent for D A-G1 in toluene. Bixon-Jortner analysis yielded a similar electronic coupling in D-A-G1 compared to the dyads. However, the charge recombination rate is smaller than of Db due to a more energetic CS state, which in the inverted region slows down charge recombination. In combination a singlet-triplet splitting similar to the dyads prolongs the CS state lifetime up to 14 us in diluted solution. Both effects result in an even better performance of D-A-G1 concerning energy conversion. D A-G1 is therefore a promising key structure for further studies on light harvesting applications. In a prospective study a second generation donor-acceptor dendrimer D-A-G2 might be an attractive structure accessible by "click reaction" of 13 and 8. D-A-G2 is expected to exhibit a downhill oriented gradient of CS states as assumed from the CV studies on G1-G3.},
  subject      = {Sternpolymere},
  language  = {en}
}
@phdthesis{Voelker2013,
  author    = {V{\"o}lker, Michael},
  title     = {Entwicklung, Charakterisierung und Anwendung neuer In-vitro-Methoden zur Untersuchung des Fremdstoffmetabolismus und der Inhibition fremdstoffmetabolisierender Enzyme},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-99434},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Arzneistoffe werden nach ihrer Applikation durch verschiedene fremdstoff-metabolisierende Enzyme des Organismus biochemisch ver{\"a}ndert. Durch eine Hemmung dieser Enzyme, z. B. durch Grapefruitsaft oder einen gleichzeitig eingenommenen Arzneistoff, kann es insbesondere bei Arzneistoffen mit geringer therapeutischer Breite, wie z. B. Theophyllin oder Phenprocoumon, zu gef{\"a}hrlichen Nebenwirkungen kommen. Besonders gef{\"a}hrdet sind multimorbide Patienten, die eine Therapie mit einer Vielzahl von Arzneimitteln erhalten. Um den Metabolismus von neuen Wirkstoffen und deren Interaktionspotential zu untersuchen, werden u. a. In-vitro-Experimente mit Zellfraktionen oder einzelnen Enzymen durchgef{\"u}hrt. Bei Inhibitionsassays wird der Einfluss von Arzneistoffen auf die Umsetzung eines Testsubstrates untersucht. Ein Großteil dieser Arbeit besch{\"a}ftigt sich daher mit der Entwicklung von Methoden, mit denen die Inhibition wichtiger fremd-stoffmetabolisierender Enzyme, wie Cytochrom-P450-Enzyme (CYP-Enzyme), Glutathion-S-Transferasen (GSTs) und Carboxylesterasen (CES), untersucht werden kann. Dabei wurde auch eine Charakterisierung der Testsubstrate vorgenommen. Dar{\"u}ber hinaus wurden die Bioaktivierung von Clopidogrel und die Bildung von reaktiven Metaboliten untersucht. Aufgrund aktueller Diskussionen {\"u}ber die Interaktion zwischen Clopidogrel und Omeprazol wurde in dieser Arbeit die Bioaktivierung von Clopidogrel mit Hilfe von LC/MS/MS-Analysen und rekombinanten CYP-Enzymen sowie humanen Lebermikrosomen untersucht. Aufgrund der Instabilit{\"a}t des aktiven Metaboliten wurde in den inkubierten Proben eine Derivatisierung mit Dimedon durchgef{\"u}hrt. Die Untersuchungen zeigten, dass die Umwandlung zum 2-Oxo-Clopidogrel durch mehrere CYP-Enzyme erfolgt. Neben CYP2C19 sind CYP1A2, CYP2B6, CYP2C9 und CYP3A4 beteiligt. Anhand von selektiven Inhibitoren konnte CYP3A4 f{\"u}r die Bildung des aktiven Metaboliten aus 2-Oxo-Clopidogrel identifiziert werden. Neben der Biotransformation durch CYP-Enzyme wird haupts{\"a}chlich der Carbons{\"a}ureester des Clopidogrels hydrolysiert. Untersuchungen mit humanen Subzellfraktionen und rekombinanten Carboxylesterasen zeigen, dass die Esterhydrolyse durch CES1 katalysiert wird. Des Weiteren wurde der Metabolismus von Omeprazol untersucht. Es stellte sich heraus, dass die 5-Hydroxylierung und die 5-O-Demethylierung haupts{\"a}chlich durch CYP2C19 und CYP2D6 erfolgen. Dabei besitzt Omeprazol die h{\"o}chste Affinit{\"a}t zu CYP2C19. Die Bildung von Omeprazolsulfon wird hingegen nur durch CYP3A4 katalysiert. Mit Hilfe etablierter CYP-Inhibitionsassays wurde der Einfluss von Clopidogrel und Omeprazol auf neun verschiedene CYP-Enzyme untersucht. Durch Clopidogrel wurden CYP2B6 (IC50 = 6 nM), CYP2C19 (IC50 = 0.4 µM) und CYP1A2 (IC50 = 2.8 µM) gehemmt. Omeprazol inhibiert v. a. CYP2C19 (IC50 = 2 µM) und CYP3A4 (IC50 = 17 µM). Im Folgenden wurde auch der Einfluss von Omeprazol auf die Bildung von 2-Oxo-Clopidogrel untersucht. Die Bioaktivierung wurde allerdings erst bei einer Omeprazol-Konzentration von mehr als 10 µM beeinflusst. Am st{\"a}rksten wurde dabei CYP2C19 (IC50 ca. 100 µM) gehemmt. Aufgrund der recht schwachen Inhibition von CYP2C19 durch Omeprazol und der Tatsache, dass mehrere CYP-Enzyme die Bildung von 2-Oxo-Clopidogrel katalysieren, l{\"a}sst sich der Wirkungsverlust von Clopidogrel bei einer gleichzeitigen Einnahme von Omeprazol anhand der Ergebnisse der In-vitro-Versuche nicht durch eine Hemmung von CYP2C19 erkl{\"a}ren. Eine bisher nur wenig bei In-vitro-Interaktionsstudien untersuchte Klasse fremdstoffmetabolisierender Enzyme sind die Carboxylesterasen (CES), die v. a. bei der Bioaktivierung von Esterprodrugs eine wichtige Rolle spielen. F{\"u}r die Entwicklung von Inhibitionsassays wurden zun{\"a}chst verschiedene Modellsubstrate ausgew{\"a}hlt. Nach Inkubation dieser Substrate mit humanen Subzellfraktionen und rekombinanten Carboxylesterase-Enzymen wurden die Metaboliten mit Hilfe einer HPLC/UV-Analyse quantifiziert. Es zeigte sich, dass Methyl-4-nitrobenzoat und Mycophenolatmofetil selektiv durch CES1 hydrolysiert werden. Die Hydrolyse von Phenylacetat, p-Nitrophenylacetat und 4-Methylumbelliferylacetat wurde durch alle verwendeten Enzyme katalysiert. Dar{\"u}ber hinaus konnte eine Hydrolyse der aus Boswellia-Arten (Weihrauch) stammenden 3-O-Acetyl-11-keto--boswellias{\"a}ure durch CES2 beobachtet werden. Aufgrund der bei den meisten Modellsubstraten auftretenden Instabilit{\"a}t im Inkubationspuffer war eine Korrektur mit Hilfe von Blindproben erforderlich. Die Hydrolyse konnte durch Erniedrigung des pH-Wertes des Inkubationspuffers von 7.4 auf 6.5 und durch die Zugabe von Essigs{\"a}ure zur Stoppl{\"o}sung verlangsamt werden. Anschließend wurde die Beeinflussung der Hydrolyse von p-Nitrophenylacetat durch Pflanzenextrakte untersucht. Es zeigte sich, dass zahlreiche Extrakte die Esterasen aus der humanen Leber hemmten und die Aktivit{\"a}t bei einer Extraktkonzentration von 25-50 µg/ml weit unterhalb von 50 \% lag. Die Inhibition von CES durch Pflanzenextrakte stellt daher ein bisher unbekanntes Risiko f{\"u}r Arzneimittelinteraktionen dar. Cytochrom-P450-Enzyme (CYP-Enzyme) sind die wichtigste Gruppe fremdstoff-metabolisierender Enzyme. Zur Untersuchung der Beeinflussung dieser Enzyme durch neue Wirkstoffe werden daher standardm{\"a}ßig In-vitro-Interaktionsstudien durchgef{\"u}hrt. Von der Food and Drug Administration (FDA) wurden daher f{\"u}r jedes CYP-Enzym verschiedene Arzneistoffe als Testsubstrate vorgeschlagen. Zus{\"a}tzlich kommen bei solchen Untersuchungen Modellsubstrate zum Einsatz, deren Metaboliten fluoreszieren und die somit f{\"u}r ein Hochdurchsatz-Screening mit Hilfe von Mikrotiterplatten verwendet werden k{\"o}nnen. In dieser Arbeit wurde eine Reihe von Modellsubstanzen (Cumarin- und Harman-Derivate) auf ihre Eignung als Substrate f{\"u}r CYP-Inhibitionsassays untersucht. Nach der Entwicklung von Methoden zur Detektion der Metaboliten, die durch LC/MS/MS-Analysen oder durch HPLC/Fluoreszenzanalysen erfolgte, wurden die CYP-Enzyme identifiziert, die an der Umsetzung der Substrate beteiligt sind und mit Hilfe von CYP-Enzymen und humanen Lebermikrosomen wurden die Km-Werte der Substrate bestimmt. Die Untersuchungen zur Stabilit{\"a}t der CYP-Enzyme {\"u}ber 60 min zeigten, dass diese bei 37 °C stark an Aktivit{\"a}t verlieren, insbesondere CYP1A2. F{\"u}r eine maximale Umsetzungsgeschwindigkeit war eine NADPH-Konzentration von 1 mM ausreichend. Die Untersuchung von 14 Standardsubstraten ergab, dass die Mehrheit selektiv durch das entsprechende CYP-Enzym umgesetzt wird. Die Amodiaquin-N-deethylierung, die Tolbutamidhydroxylierung, die Chlorzoxazon-6-hydroxylierung und die 4-Nitrophenol-2-hydroxylierung wurden durch mehrere CYP-Enzyme katalysiert. Als Positivkontrollen f{\"u}r die Inhibitionsassays und zur Identifizierung der am Metabolismus beteiligten CYP-Enzyme werden von der FDA verschiedene Inhibitoren vorgeschlagen. Da nicht zu allen Inhibitoren Daten {\"u}ber deren Isoenzymselektivit{\"a}t vorliegen, wurde mit Hilfe der Assays die inhibitorische Aktivit{\"a}t von zw{\"o}lf Inhibitoren auf neun verschiedene CYP-Enzyme untersucht. Alle Inhibitoren hemmten das jeweilige angegebene CYP-Enzym. Bei Furafyllin (CYP1A2), Tranylcypromin (CYP2A6), Clopidogrel (CYP2B6), Montelukast (CYP2C8), Sulfaphenazol (CYP2C9), Chinidin (CYP2D6) und Ketoconazol (CYP3A4) konnte eine Konzentration ermittelt werden, bei der nur ein CYP-Enzym gehemmt wird. F{\"u}r Quercetin, Nootkaton, Diethyldithiocarbamat, Sertralin und Ticlopidin wurde eine Inhibition mehrerer CYP-Enzyme festgestellt. Mit Hilfe der CYP-Inhibitionsassays wurden Extrakte lebertoxischer Arzneipflanzen, wie z. B. Tussilago farfara (Huflattich) oder Chelidonium majus (Sch{\"o}llkraut), untersucht. Alle Extrakte hemmten konzentrationsabh{\"a}ngig die CYP-Enzyme, am st{\"a}rksten die Enzyme der Subfamilie CYP2C. Als In-vitro-Substrate f{\"u}r CYP-Inhibitionsassays werden aufgrund ihrer starken Fluoreszenz h{\"a}ufig Cumarin-Derivate eingesetzt. In dieser Arbeit wurden daher 18 O-alkylierte bzw. O-benzylierte Derivate von 7-Hydroxycumarin, 7-Hydroxy-4-methylcumarin und 7-Hydroxy-4-trifluormethylcumarin synthetisiert und die Umsetzung durch verschiedene CYP-Enzyme mit Hilfe der zuvor optimierten LC/LC/Fluoreszenz-basierten Assays untersucht. An der O-Desalkylierung der Cumarin-Derivate waren haupts{\"a}chlich CYP1A2, CYP2B6 und im geringeren Ausmaß CYP2C19, CYP2D6 und CYP2E1 beteiligt. Die h{\"o}chste Affinit{\"a}t besaßen die Substrate zu CYP1A2. Debenzylierungen wurden neben CYP1A2 haupts{\"a}chlich durch CYP3A4 katalysiert. Die h{\"o}chsten Umsetzungsgeschwindigkeiten wurden f{\"u}r die Debenzylierung von 7-Benzyloxy-4-methylcumarin (BMC, 14 pmol/pmol P450/min) und 7-Benzyloxy-4-trifluormethylcumarin (BFC, 9 pmol/pmol P450/min) beobachtet. F{\"u}r 7-Methoxy-4-trifluormethylcumarin (MFC) war die Umsetzungs¬geschwindigkeit f{\"u}r die O-Demethylierung mit CYP1A2 und CYP2B6 im Vergleich zu CYP2C9 deutlich h{\"o}her. MFC und 7-Ethoxy-4-trifluormethylcumarin (EFC) eignen sich daher v. a. f{\"u}r Inhibitionsuntersuchungen von CYP2B6. Bei den untersuchten 7 Alkyloxycumarinen handelt es sich in allen F{\"a}llen nicht um selektive CYP-Substrate. Sie k{\"o}nnen demnach nicht f{\"u}r Inhibitionsuntersuchungen mit humanen Lebermikrosomen verwendet werden. Ein Einsatz f{\"u}r Simultanbestimmungen der Hemmung mehrerer CYP-Enzyme in einem Versuch (Cocktail-Assay) ist aus diesem Grund ebenfalls nicht m{\"o}glich. Durch LC/MS-Analysen nach Inkubation der Cumarin-Derivate mit humanen Lebermikrosomen zeigte sich, dass neben den entsprechenden O Desalkylmetaboliten mehrere Hydroxymetaboliten entstehen und der O Desalkylmetabolit insbesondere bei Derivaten mit l{\"a}ngeren Alkylsubstituenten nicht der Hauptmetabolit ist. Ein Ziel der Arbeitsgruppe ist es zudem, neue In-vitro-Substrate zur Untersuchung der Inhibition von CYP-Enzymen mit besseren enzymkinetischen und analytischen Eigenschaften zu entwickeln. Grundstruktur hierf{\"u}r ist das -Carbolin, da -Carbolin-Derivate eine starke Fluoreszenz aufweisen. Von dem Naturstoff Harmin ist bekannt, dass dieser durch CYP1A2, CYP2C9, CYP2C19 und CYP2D6 O-demethyliert wird. Durch Modifizierung der Harman-Struktur sollte die CYP-Isoenzymselektivit{\"a}t f{\"u}r die O-Dealkylierung gesteigert werden und Substrate f{\"u}r weitere CYP-Enzyme erhalten werden. Hierf{\"u}r wurden in der Arbeitsgruppe u. a. 2-Benzyl-7-benzyloxyharman (BBH), 2-Benzyl-7-methoxyharman (BMH), 7-Methoxy-9-(4-carboxybenzyl)harman (MCBH) und 2-Methyl-7-methoxyharman (MMH) hergestellt. In dieser Arbeit wurden LC/LC/Fluoreszenz- und LC/MS/MS-Methoden zur Quantifizierung der aus diesen Derivaten entstehenden O-Desalkylmetaboliten entwickelt und die Substrate charakterisiert. Die Einf{\"u}hrung von Benzylsubstituenten an der phenolischen Hydroxylgruppe von Harmol (BBH) f{\"u}hrte zum Metabolismus durch CYP3A4 und die Substitution mit einem Carboxybenzylrest am Indolstickstoff (MCBH) verst{\"a}rkte die Selektivit{\"a}t zu den Enzymen der Subfamilie 2C. Durch die Methylierung des Pyridin-Stickstoffs des Harmins (MMH) wurde ein selektives Substrat f{\"u}r CYP2D6 erhalten, weshalb bei dieser Substanz auch humane Lebermikrosomen verwendet werden k{\"o}nnen. Durch die im Vergleich zu anderen CYP2D6-Substraten erhaltene hohe Umsetzungsgeschwindigkeit l{\"a}sst sich die Proteinkonzentration minimieren. F{\"u}r die {\"u}berwiegend an der O-Dealkylierung der Substrate beteiligten CYP-Enzyme wurden die Km-Werte ermittelt. Bei der Untersuchung von verschiedenen CYP-Inhibitoren zeigte sich, dass mit diesen Substraten vergleichbare IC50-Werte, wie mit den Standardsubstraten, erhalten werden. Die Harman-Derivate k{\"o}nnen daher zur Untersuchung der Inhibition wichtiger CYP-Enzyme eingesetzt werden und bieten eine Alternative zu den bisher vorhandenen Fluoreszenz-Substraten. Durch die Einstellung des pH-Wertes im Anschluss an die Inkubation lassen sich die Metaboliten ebenfalls fluorimetrisch in der Mikrotiterplatte detektieren und k{\"o}nnen f{\"u}r ein Hochdurchsatz-Screening eingesetzt werden. Allerdings m{\"u}ssen die Fluoreszenzeigenschaften weiter verbessert werden, um eine kontinuierliche Bestimmung w{\"a}hrend der Inkubation zu erm{\"o}glichen. In der pharmazeutischen Industrie besteht ein großes Interesse an der Detektion von reaktiven Metaboliten, um eine potentielle Lebertoxizit{\"a}t von neuen Wirkstoffen vorhersagen zu k{\"o}nnen. Hierf{\"u}r werden die Testsubstanzen mit humanen Lebermikrosomen inkubiert und die reaktiven Metaboliten mit Glutathion abgefangen. Zur Optimierung der LC/MS/MS-Analysen wurde in dieser Arbeit die Fragmentierung solcher Addukte anhand von Standardsubstanzen untersucht. Bei allen untersuchten Glutathion-Addukten trat eine Abspaltung der Pyroglutamins{\"a}ure bei positiver Polarit{\"a}t mit einer vergleichbaren Signalintensit{\"a}t auf, weshalb eine Detektion dieses Fragmentes durch einen Neutral-Loss-Scan am besten geeignet erschien. Mit Hilfe der Screening-Methode wurden zuerst Arzneistoffe untersucht, von denen reaktive Metaboliten bekannt sind. F{\"u}r die Bioaktivierung von Clozapin konnten CYP1A2, CYP2D6 und CYP3A4 identifiziert werden, w{\"a}hrend die Toxifizierung von Paracetamol haupts{\"a}chlich durch CYP1A2 und CYP3A4 erfolgte. Auff{\"a}llig war, dass mit steigender Paracetamolkonzentration keine S{\"a}ttigung der Umsetzung auftrat. Durchgef{\"u}hrte Molek{\"u}lver{\"a}nderungen am Glutathion, wie die Einf{\"u}hrung eines Dansylrestes oder eines Biotins, f{\"u}hrten zu keiner deutlichen Verbesserung der Detektion der reaktiven Metaboliten. Dar{\"u}ber hinaus zeigte sich, dass bei den markierten GSH-Derivaten die Umsetzung durch GSTs erheblich reduziert ist. Mit der Screening-Methode wurden allerdings viele falsch positive Signale erhalten, so dass diese nicht f{\"u}r eine Untersuchung von Extrakten lebertoxischer Pflanzen eingesetzt werden konnte. F{\"u}r eine eindeutige und schnelle Identifizierung der Signale als Glutathion-Addukte ist daher die hochaufl{\"o}sende Massenspektrometrie erforderlich. Eine weitere Klasse fremdstoffmetabolisierender Enzyme sind die Glutathion-S-Transferasen (GSTs), {\"u}ber deren Inhibition durch Arzneistoffe und Pflanzenextrakte in der Literatur nur wenige Daten vorliegen. Zur Entwicklung von Inhibitionsassays wurden die in der Literatur beschriebenen Substrate 1-Chlor-2,4-dinitrobenzol, 4 Nitrochinolin-N-oxid, 1,2-Dichlor-4-nitrobenzol und 4-Nitrobenzylchlorid verwendet. Die Detektion der Metaboliten erfolgte im Gegensatz zu der h{\"a}ufig eingesetzten Photometrie mit Hilfe der HPLC/UV- bzw. einer LC/MS/MS-Analyse. F{\"u}r die Kalibrierung wurden zun{\"a}chst die entsprechenden Glutathionkonjugate aus den Substraten synthetisiert. Bei den durchgef{\"u}hrten diskontinuierlichen Assays stellte die h{\"a}ufig auftretende nichtenzymatische Reaktion der Substrate mit Glutathion ein Problem dar. Durch die Erniedrigung des pH-Wertes des Inkubationspuffers von 7.4 auf 6.5 und der Senkung der Inkubationstemperatur von 37 °C auf 25 °C konnte die nichtenzymatische Reaktion w{\"a}hrend der Inkubation erheblich verlangsamt werden. Die nichtenzymatische Reaktion nach der Inkubation konnte durch Zugabe von Oxidationsmitteln gestoppt werden. Von den getesteten humanen Lebersubzell¬fraktionen besaß die cytosolische Fraktion bei allen Substraten die h{\"o}chste Aktivit{\"a}t. Im Rahmen der Assayentwicklung wurde die Glutathion-, die Proteinkonzentration und die Inkubationszeit optimiert. Es wurden die Km- und Vmax-Werte f{\"u}r die Umsetzung der Substrate ermittelt. Als Positivkontrolle diente das ebenfalls synthetisierte Glutathionkonjugat der Etacryns{\"a}ure, f{\"u}r das die IC50-Werte mit jedem Substrat bestimmt wurden. Dabei konnte ein Einfluss des pH-Wertes des Inkubationspuffers und der Inkubationstemperatur auf die gemessene inhibitorische Aktivit{\"a}t beobachtet werden. Anschließend wurde ein Screening von Arzneistoffen, ausgew{\"a}hlten Naturstoffen und etwa 50 Pflanzenextrakten auf eine Inhibition der GSTs in humanem Lebercytosol mit 1-Chlor-2,4-dinitrobenzol, das am schnellsten von allen Substraten umgesetzt wurde, durchgef{\"u}hrt. Von den getesteten Naturstoffen fiel eine ausgepr{\"a}gte Hemmung durch Biflavonoide auf. Nahezu alle untersuchten Pflanzenextrakte hemmten die GSTs. Eine starke Inhibition der GSTs zeigten Extrakte aus Cinnamomum cassia (Zimt), die sich als nicht-kompetitiv herausstellte. Weiterhin wurde eine starke Hemmung der Extrakte gerbstoffhaltiger Pflanzen, wie z. B. Hamamelis virginiana (virginische Zaubernuss) oder Krameria triandra (Ratanhia), beobachtet. Hier resultierten IC50-Werte zwischen 5 und 30 µg/ml. Ein Vergleich verschiedener Methoden zur Detektion des Metaboliten 2,4 Dinitrophenyl-S-glutathion zeigte, dass die Photometrie f{\"u}r die Untersuchung der Inhibition von Pflanzenextrakten aufgrund der St{\"o}rung durch die Pflanzenmatrix ungeeignet ist. Mit Hilfe der verwendeten HPLC/UV- sowie der LC/MS/MS-Analyse konnte der Metabolit selektiv erfasst werden und reproduzierbare Ergebnisse f{\"u}r die Inhibition der GSTs durch Pflanzenextrakte erzielt werden. Neben den GSTs wurde auch die Beeinflussung der Glutathionreduktase (GR) in dieser Arbeit untersucht. Hierf{\"u}r wurde ein HPLC-basierter Assay entwickelt, bei dem das reduzierte Glutathion mit 5,5´-Dithiobis(2-nitrobenzoes{\"a}ure) derivatisiert und das entstandene gemischte Disulfid aus Glutathion und 5-Thio-2-nitrobenzoes{\"a}ure quantifiziert wurde. Zur Untersuchung der Inhibition durch Pflanzenextrakte wurde humanes Lebercytosol verwendet, das von allen humanen Lebersubzellfraktionen die h{\"o}chste Aktivit{\"a}t besaß. Im Vergleich zu den GSTs wurde die GR durch die {\"u}berwiegende Zahl der ausgew{\"a}hlten Pflanzenextrakte kaum gehemmt. Eine nennenswerte Inhibition der GR konnte nur bei Extrakten von Juglans regia (Walnuss) beobachtet werden. Fazit In dieser Arbeit wurden eine Reihe von In-vitro-Methoden zur Untersuchung der Inhibition von CYP-Enzymen und weiteren fremdstoffmetabolisierenden Enzymen, wie CES oder GSTs, entwickelt. Aufgrund der dabei angewendeten selektiven HPLC-basierten Quantifizierung der Metaboliten durch UV-, Fluoreszenz- oder MS-Detektion k{\"o}nnen mit diesen Methoden auch Proben mit komplexer Matrix untersucht werden. F{\"u}r alle Assays wurden die Inkubationsbedingungen optimiert und die enzymkinetischen Parameter vieler Substrate ermittelt. Dar{\"u}ber hinaus wurden wichtige Erkenntnisse {\"u}ber die Isoenzymselektivit{\"a}t dieser Substrate gewonnen. Die Eignung der Assays wurde anhand von Standardinhibitoren bewiesen. Schließlich wurde die inhibitorische Aktivit{\"a}t von zahlreichen Pflanzenextrakten bestimmt, deren Auswirkung auf fremdstoffmetabolisierende Enzyme bisher unbekannt war. Die in dieser Arbeit beschriebenen Methoden k{\"o}nnen f{\"u}r die Untersuchung des Metabolismus von Arzneistoffen und der Inhibition fremdstoffmetabolisierender Enzyme, die f{\"u}r eine Zulassung neuer Wirkstoffe erforderlich ist, routinem{\"a}ßig eingesetzt werden.},
  subject      = {Xenobiotikum},
  language  = {de}
}
@phdthesis{Stuckensen2016,
  author    = {Stuckensen, Kai},
  title     = {Fabrication of hierarchical cell carrier matrices for tissue regeneration by directional solidification},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145510},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The key hypothesis of this work represented the question, if mimicking the zonal composition and structural porosity of musculoskeletal tissues influences invading cells positively and leads to advantageous results for tissue engineering. Conventional approaches in tissue engineering are limited in producing monolithic "scaffolds" that provide locally variating biological key signals and pore architectures, imitating the alignment of collagenous fibres in bone and cartilage tissues, respectively. In order to fill this gap in available tissue engineering strategies, a new fabrication technique was evolved for the production of scaffolds to validate the hypothesis. Therefore, a new solidification based platform procedure was developed. This process comprises the directional solidification of multiple flowable precursors that are "cryostructured" to prepare a controlled anisotropic pore structure. Porous scaffolds are attained through ice crystal removal by lyophilisation. Optionally, electrostatic spinning of polymers may be applied to provide an external mesh on top or around the scaffolds. A consolidation step generates monolithic matrices from multi zonal structures. To serve as matrix for tissue engineering approaches or direct implantation as medical device, the scaffold is sterilized. An Adjustable Cryostructuring Device (ACD) was successively developed; individual parts were conceptualized by computer aided design (CAD) and assembled. During optimisation, a significant performance improvement of the ACDs accessible external temperature gradient was achieved, from (1.3 ± 0.1) K/mm to (9.0 ± 0.1) K/mm. Additionally, four different configurations of the device were made available that enabled the directional solidification of collagenous precursors in a highly controlled manner with various sample sizes and shapes. By using alginate as a model substance the process was systematically evaluated. Cryostructuring diagraphs were analysed yielding solidification parameters, which were associated to pore sizes and alignments that were determined by image processing. Thereby, a precise control over pore size and alignment through electrical regulation of the ACD could be demonstrated. To obtain tissue mimetic scaffolds for the musculoskeletal system, collagens and calcium phosphates had to be prepared to serve as raw materials. Extraction and purification protocols were established to generate collagen I and collagen II, while the calcium phosphates brushite and hydroxyapatite were produced by precipitation reactions. Besides the successive augmentation of the ACD also an optimization of the processing steps was crucial. Firstly, the concentrations and the individual behaviour of respective precursor components had to be screened. Together with the insights gained by videographic examination of solidifying collagen solutions, essential knowledge was gained that facilitated the production of more complex scaffolds. Phenomena of ice crystal growth during cryostructuring were discussed. By evolutionary steps, a cryostructuring of multi-layered precursors with consecutive anisotropic pores could be achieved and successfully transferred from alginate to collagenous precursors. Finally, very smooth interfaces that were hardly detectable by scanning electron microscopy (SEM) could be attained. For the used collagenous systems, a dependency relation between adjustable processing parameters and different resulting solidification morphologies was created. Dehydrothermal-, diisocyanate-, and carbodiimide- based cross linking methods were evaluated, whereby the "zero length" cross linking by carbodiimide was found to be most suitable. Afterwards, a formulation for the cross linking solution was elaborated, which generated favourable outcomes by application inside a reduced pressure apparatus. As a consequence, a pore collapse during wet chemical cross linking could be avoided. Complex monolithic scaffolds featuring continuous pores were fabricated that mimicked structure and respective composition of different areas of native tissues by the presence of biochemical key stimulants. At first, three types of bone scaffolds were produced from collagen I and hydroxyapatite with appropriate sizes to fit critical sized defects in rat femurs. They either featured an isotropic or anisotropic porosity and partly also contained glycosaminoglycans (GAGs). Furthermore, meniscus scaffolds were prepared by processing two precursors with biomimetic contents of collagen I, collagen II and GAGs. Here, the pore structures were created under boundary conditions, which allowed an ice crystal growth that was nearly orthogonal to the external temperature gradient. Thereby, the preferential alignment of collagen fibres in the natural meniscus tissue could be mimicked. Those scaffolds owned appropriate sizes for cell culture in well plates or even an authentic meniscus shape and size. Finally, osteochondral scaffolds, sized to either fit well plates or perfusion reactors for cell culture, were fabricated to mimic the composition of subchondral bone and different cartilage zones. Collagen I and the resorbable calcium phosphate brushite were used for the subchondral zone, whereas the cartilage zones were composed out of collagen I, collagen II and tissue mimetic contents of GAGs. The pore structure corresponded to the one that is dominating the volume of natural osteochondral tissue. Energy dispersive X-ray spectroscopy (EDX) and SEM were used to analyse the composition and pore structure of the individual scaffold zones, respectively. The cross section pore diameters were determined to (65 ± 25) µm, (88 ± 35) µm and(93 ± 42) µm for the anisotropic, the isotropic and GAG containing isotropic bone scaffolds. Furthermore, the meniscus scaffolds showed pore diameters of (93 ± 21) µm in the inner meniscus zone and (248 ± 63) µm inside the outer meniscus zone. Pore sizes of (82 ± 25) µm, (83 ± 29) µm and (85 ± 39) µm were present inside the subchondral, the lower chondral and the upper chondral zone of osteochondral scaffolds. Depending on the fabrication parameters, the respective scaffold zones were also found to feature a specific micro- and nanostructure at their inner surfaces. Degradation studies were carried out under physiological conditions and resulted in a mean mass loss of (0.52 ± 0.13) \%, (1.56 ± 0.10) \% and (0.80 ± 0.10) \% per day for bone, meniscus and osteochondral scaffolds, respectively. Rheological measurements were used to determine the viscosity changes upon cooling of different precursors. Micro computer tomography (µ-CT) investigations were applied to characterize the 3D microstructure of osteochondral scaffolds. To obtain an osteochondral scaffold with four zones of tissue mimetic microstructure alignment, a poly (D, L-lactide-co-glycolide) mesh was deposited on the upper chondral zone by electrostatic spinning. In case of the bone scaffolds, the retention / release capacity of bone morphogenetic protein 2 (BMP-2) was evaluated by an enzyme linked immunosorbent assay (ELISA). Due to the high presence of attractive BMP binding sites, only less than 0.1 \% of the initially loaded cytokine was released. The suitability of combining the cryostructuring process with 3D powder printed calcium phosphate substrates was evaluated with osteochondral scaffolds, but did not appear to yield more preferable results than the non-combined approach. A new custom build confined compression setup was elaborated together with a suitable evaluation procedure for the mechanical characterisation under physiological conditions. For bone and cartilage scaffolds, apparent elastic moduli of (37.6 ± 6.9) kPa and (3.14 ± 0.85) kPa were measured. A similar behaviour of the scaffolds to natural cartilage and bone tissue was demonstrated in terms of elastic energy storage. Under physiological frequencies, less than 1.0 \% and 0.8 \% of the exerted energy was lost for bone and cartilage scaffolds, respectively. With average relaxation times of (0.613 ± 0.040) sec and (0.815 ± 0.077) sec, measured for the cartilage and bone scaffolds, they respond four orders of magnitude faster than the native tissues. Additionally, all kinds of produced scaffolds were able to withstand cyclic compression at un-physiological frequencies as high as 20 Hz without a loss in structural integrity. With the presented new method, scaffolds could be fabricated whose extent in mimicking of native tissues exceeded the one of scaffolds producible by state of the art methods. This allowed a testing of the key hypothesis: The biological evaluation of an anisotropic pore structure in vivo revealed a higher functionality of immigrated cells and led finally to advantageous healing outcomes. Moreover, the mimicking of local compositions in combination with a consecutive anisotropic porosity that approaches native tissue structures could be demonstrated to induce zone specific matrix remodelling in stem cells in vitro. Additionally, clues for a zone specific chondrogenic stem cell differentiation were attained without the supplementation of growth factors. Thereby, the hypothesis that an increased approximation of the hierarchically compositional and structurally anisotropic properties of musculoskeletal tissues would lead to an improved cellular response and a better healing quality, could be confirmed. With a special focus on cell free in situ tissue engineering approaches, the insights gained within this thesis may be directly transferred to clinical regenerative therapies.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@article{BossertdeBruinGoetzetal.2016,
  author    = {Bossert, Nelli and de Bruin, Donny and G{\"o}tz, Maria and Bouwmeester, Dirk and Heinrich, Doris},
  title     = {Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications},
  series = {Scientific Reports},
  volume    = {6},
  journal   = {Scientific Reports},
  number    = {37897},
  doi       = {10.1038/srep37897},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167482},
  year      = {2016},
  abstract  = {DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications.},
  language  = {en}
}
@phdthesis{Bothe2021,
  author    = {Bothe, Sebastian Helmut},
  title     = {Fragmentbasiertes Design von p97-Liganden: Identifizierung von Startstrukturen zur Entwicklung von Protein-Protein-Interaktionsinhibitoren f{\"u}r die SHP-Bindestelle der AAA+ ATPase p97},
  doi       = {10.25972/OPUS-23911},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-239112},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Die AAA+ ATPase p97 ist ein essenzielles Protein, das an einer Vielzahl zellul{\"a}rer Prozesse beteiligt ist und eine Schl{\"u}sselrolle in der Protein-Hom{\"o}ostase spielt. Die funktionale Diversit{\"a}t von p97 beruht auf der Interaktion zahlreicher unterschiedlicher Kofaktoren, die vorwiegend an die N-Dom{\"a}ne von p97 binden. Aufgrund seiner Bedeutung in der Regulierung diverser physiologischer und pathologischer Prozesse stellt p97 eine interessante Zielstruktur f{\"u}r die Entwicklung neuer Wirkstoffe dar, die insbesondere in der Krebstherapie von Bedeutung sein k{\"o}nnte. Bekannte p97-Inhibitoren greifen vor allem die ATPase-Funktion des Proteins an. Ein neuer pharmakologischer Ansatz stellt die Inhibierung der Kofaktorbindung an die N-Dom{\"a}ne dar. Ein solcher Protein-Protein-Interaktionsinhibitor w{\"a}re nicht nur von therapeutischem Interesse, sondern h{\"a}tte auch einen besonderen Nutzen f{\"u}r die Entschl{\"u}sselung molekularer und zellul{\"a}rer Funktionen von p97-Kofaktoren. In dieser Arbeit wurde ein fragmentbasierter Ansatz f{\"u}r die Identifizierung von chemischen Startstrukturen f{\"u}r die Entwicklung eines Protein-Protein- Interaktionsinhibitors verfolgt. Als Zielstruktur wurde die SHP-Bindestelle in der N-Dom{\"a}ne gew{\"a}hlt. Die Identifizierung von Liganden erfolgte sowohl durch computergest{\"u}tzte Methoden (insbesondere virtuelles Screening und Molekulardynamik-Simulationen) als auch experimentell durch biophysikalische Techniken (wie Biolayer-Interferometrie, R{\"o}ntgenstrukturanalyse und ligandbasierte NMR-Techniken). Die Grundlage des computerbasierten Designs stellte eine Analyse der bekannten Kristallstrukturen der p97-Komplexe mit den SHP-Motiven der Kofaktoren UFD1 und Derlin-1 dar. Dar{\"u}ber hinaus dienten Molekulardynamik-Simulationen der Analyse der Wassereigenschaften innerhalb der SHP-Bindestelle. Darauf aufbauend wurden verschiedene Pharmakophormodelle entwickelt, die die Grundlage des im Anschluss durchgef{\"u}hrten virtuellen Screenings und Dockings bildeten. Anhand der Ergebnisse von Molekulardynamik-Simulationen wurden zehn Verbindungen f{\"u}r die experimentelle Validierung ausgew{\"a}hlt. Hiervon konnten zwei Fragmente in STD-NMR- und Biolayer-Interferometrie-Experimenten als Liganden best{\"a}tigt werden. In einem parallel durchgef{\"u}hrten biophysikalischen Fragmentscreening mittels Biolayer-Interferometrie wurden unter mehr als 650 Verbindungen 22 identifiziert, die an die N-Dom{\"a}ne binden. 15 dieser Fragmente wurden durch einen orthogonalen STD-NMR-Assay best{\"a}tigt. F{\"u}nf dieser Verbindungen zeigten Affinit{\"a}ten mit KD-Werten kleiner 500μMund g{\"u}nstigen Ligandeffizienzen. Des Weiteren konnte die Bindungskinetik und Affinit{\"a}t des in der Literatur als p97-Inhibitor berichteten Naturstoffes Xanthohumol bestimmt und eine Bindung an die N-Dom{\"a}ne best{\"a}tigt werden. Zur Identifizierung m{\"o}glicher Bindestellen dieser f{\"u}nf Fragmente wurden mixed-solvent Molekulardynamik-Simulationen durchgef{\"u}hrt. Diese ergaben, dass alle Verbindungen die SHP-Bindestelle in der N-Dom{\"a}ne adressieren. Die Regionen fielen mit hot spots der Kofaktorwechselwirkungen zusammen und stellen somit m{\"o}gliche Ankerpunkte f{\"u}r die Weiterentwicklung dar. F{\"u}r zwei Fragmente konnten die postulierten Bindestellen mittels R{\"o}ntgenstrukturanalyse bzw. STD-NMR-Messungen an p97-Alanin-Mutanten best{\"a}tigt werden. Die erhaltene R{\"o}ntgenstruktur ist die erste p97-Struktur, die ein gebundenes Fragment an der N-Dom{\"a}ne zeigt.},
  subject      = {Arzneimitteldesign},
  language  = {de}
}
@phdthesis{Schmitz2016,
  author    = {Schmitz, Tobias},
  title     = {Functional coatings by physical vapor deposition (PVD) for biomedical applications},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144825},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Metals are the most used materials for implant devices, especially in orthopedics, but despite their long history of application issues such as material failure through wear and corrosion remain unsolved leading to a certain number of revision surgeries. Apart from the problems associated with insufficient material properties, another serious issue is an implant associated infection due to the formation of a biofilm on the surface of the material after implantation. Thus, improvements in implant technology are demanded, especially since there is a projected rise of implants needed in the future. Surface modification methods such as physical vapour deposition (PVD), oxygen diffusion hardening and electrochemical anodization have shown to be efficient methods to improve the surfaces of metallic bulk materials regarding biomedical issues. This thesis was focused on the development of functional PVD coatings that are suitable for further treatment with surface modification techniques originally developed for bulk metals. The aim was to precisely adjust the surface properties of the implant according to the targeted application to prevent possible failure mechanisms such as coating delamination, wear or the occurrence of post-operative infections. Initially,  tantalum layers with approx 5 µm thickness were deposited at elevated substrate temperatures on cp Ti by RF magnetron sputtering. Due to the high affinity of tantalum to oxygen, these coatings are known to provide a self healing capacity since the rapid oxide formation is known to close surface cracks. Here, the work aimed to reduce the abrupt change of mechanical properties between the hard and brittle coating and the ductile substrate by creating an oxygen diffusion zone. It was found that the hardness and adhesion could be significantly increased when the coatings were treated afterwards by oxygen diffusion hardening in a two step process. Firstly, the surface was oxidized at a pressure of 6.7•10-3 mbar at 350 450 °C, followed by 1-2 h annealing in oxygen-free atmosphere at the same temperature leading to a diffusion of oxygen atoms into deeper parts of the substrate as proved by X-ray diffraction (XRD) analysis. The hereby caused mechanical stress in the crystal lattice led to an increase in Vickers hardness of the Ta layers from 570 HV to over 900 HV. Investigations into the adhesion of oxygen diffusion treated samples by Rockwell measurements demonstrated an increase of critical force for coating delamination from 12 N for untreated samples up to 25 N for diffusion treated samples. In a second approach, the development of modular targets aimed to produce functional coatings by metallic doping of titanium with biologically active agents. This was demonstrated by the fabrication of antimicrobial Ti(Ag) coatings using a single magnetron sputtering source equipped with a titanium target containing implemented silver modules under variation of bias voltage and substrate temperature. The deposition of both Ti and Ag was confirmed by X-ray diffraction and a clear correlation between the applied sputtering parameters and the silver content of the coatings was demonstrated by ICP-MS and EDX. Surface-sensitive XPS measurements revealed that higher substrate temperatures led to an accumulation of Ag in the near-surface region, while the application of a bias voltage had the opposite effect. SEM and AFM microscopy revealed that substrate heating during film deposition supported the formation of even and dense surface layers with small roughness values, which could even be enforced by applying a substrate bias voltage. Additional elution measurements using ICP-MS showed that the release kinetics depended on the amount of silver located at the film surface and hence could be tailored by variation of the sputter parameters. In a final step, the applied Ti and Ti(Ag) coatings deposited on cp Ti, stainless steel (316L) and glass substrates were subsequently nanostructured using a self-ordering process induced by electrochemical anodization in aqueous fluoride containing electrolytes. SEM analysis showed that nanotube arrays could be grown from the Ti and Ti(Ag) coatings deposited at elevated temperatures on any substrate, whereby no influence of the substrate on nanotube morphology could be observed. EDX measurements indicated that the anodization process led to the selective etching of Ti from Ti(Ag) coating. Further experiments on coatings deposited on glass surfaces revealed that moderate substrate temperatures during deposition resulting in smooth Ti layers as determined by AFM measurements, are favorable for the generation of highly ordered nanotube arrays. Such arrays exhibited superhydrophilic behavior as proved by contact angle measurements. XRD analysis revealed that the nanostructured coatings were amorphous after anodization but could be crystallized to anatase structure by thermal treatment at temperatures of 450°C.},
  subject      = {PVD-Verfahren},
  language  = {en}
}
@phdthesis{Schmitz2016,
  author    = {Schmitz, Michael},
  title     = {Functional hydrophilic polymers for chemoselective coupling},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145629},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Chemoselective poly(oxazolines) (POx) and poly[(oligo ethylene glycol) acrylates] were synthesized. An initiator was produced for the preparation of poly(oxazoline)s capable of participating in click chemistry reactions which allows the functionalization of the polymer at the α terminus which was confirmed by 1H NMR spectroscopy. The initiator was used for the polymerization of hydrophilic 2 methyl 2 oxazoline (MeOx), whereby chemoselective, alkyne functionalized polymers could be prepared for Cu-catalyzed azide-alkyne cycloaddition. The desired molecular weight could be achieved through the living, ring opening cationic polymerization and was confirmed by 1H NMR, SEC and MALDI ToF measurements. Polymers were terminated with piperidine if no further functionalization was needed, or with an ester derivate for enabling amine attachment in a subsequent step. In addition, polymers were functionalized by termination with NaN3 in order to provide the counterpart to the azide-alkyne reaction. IR spectroscopy was suitable for the azide detection. The coupling of polymers showed the reactivity and could be confirmed by SEC, 1H NMR and IR spectroscopy. The composition of cysteine functionalized POx was completed by thiol-ene chemistry. Since the commercially available iso 2 propyl 2 oxazoline is not available for the cationic polymerization, 2 butenyl and 2 decenyl 2 oxazoline (ButenOx and DecenOx) were first prepared. The synthesis of both copolymers, based on MeOx could be confirmed by 1H NMR as well as with SEC, whereby narrow distributions with dispersities of 1.06 could be achieved. The cysteine functionalization of the copolymers was enabled by the creation of a thiazolidine component which could be synthesized by acetal and formyl protection of cysteine and subsequent functionalization with a thiol. The component enabled the reaction with a polymer by thiol-ene reaction which was started by the addition of dimethoxyphenyl-acetophenone and was catalyzed by irradiation with UV light. Both copolymers, with a shorter (polymers with BuenOx) and longer (polymers with DecenOx) hydrophobic sidechain could be functionalized. 1H NMR spectroscopic analysis showed a quantitative reaction with the thiazolidine derivate. After deprotection by acidic workup the desired, cysteine functionalized polymer could be isolated. Quantification of cysteine functions was ensured by a modified TNBSA assay, whereby the thiols were first oxidized in order to confirm an independent measurement of amine functions. Both, the TNBSA assay as well as the NMR measurement showed the desired number of cysteine residues. The cytotoxicity of functionalized polymers with different compositions was tested by a luminescent cell viability assay (LCVA). Both, the amount of cysteine functions (5-10\%) in the copolymers as well as the length of the hydrophobic side chain were varied. All polymers did not show cytotoxicity up to concentrations of 10 mg∙mL-1. The cell activity and cell numbers only decreased below 50\% and 20\% respectively, when copolymers with 5\% cysteine and longer sidechains were measured, which was attributed to a contamination of the sample itself. The cooperation partner performed Native Chemical Ligation (NCL) with model peptides and purified the products by HPLC. A sterically non demanding peptide was synthesized, consisting of an aromatic amino acid and four glycine units. The aromatic unit was used for the quantification of the polymer-peptide conjugate in the 1H NMR spectroscopy. A polymer having five cysteine side chains has been fully implemented by NCL to a conjugate of one polymer with five peptides. A sterically more demanding peptide was additionally used and MALDI ToF measurements confirmed the successful conjugation. Furthermore the cysteine functionalized polymer was used for nanogel synthesis. The thiol of the cysteine function was oxidized in an inverse mini-emulsion by H2O2, resulting in nanogels (~500 nm) which could be confirmed by SEM, AFM, DLS and NTA measurements. Besides POx, oligo (ethylene glycol)acrylates (OEGA) were polymerized; by copolymerization with the reactive pentafluorophenyl acrylate (PFPA) reactive and amphiphilic polymers were obtained. The synthesis of PFPA could be confirmed spectroscopically by 1H , 19F NMR, and by FT IR. Copolymers were synthesized by RAFT polymerization with narrow dispersities. Functionalization with an amine functionalized thiazolidine led to a hydrophilic cysteine functionalized polymer after acidic deprotection. Apart from this polymer, a thioester functionalization was successfully performed by reaction of the active polymer with a cyclic amine functionalized thioester which does not release a toxic by product (such as the resulting thiol) during NCL and thus features a very high potential to replace former thioester.},
  subject      = {Konjugate},
  language  = {en}
}
@phdthesis{Waag2013,
  author    = {Waag, Thilo},
  title     = {Funktionalisierung von Nanodiamanten f{\"u}r Wirkstofftransport und Knochenersatzmaterialien},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-94597},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Ziel der vorliegenden Arbeit ist das Design, die Synthese und das anschließende Testen von Nanodiamant-Wirkstoff-Konjugaten. Daf{\"u}r m{\"u}ssen zun{\"a}chst Nanodiamanten mit geeigneten Linkersystemen funktionalisiert werden, um anschließend verschiedene pharmazeutische Wirkstoffe auf der Diamantoberfl{\"a}che zu immobilisieren. Die Wirksamkeit der so angebundenen Inhibitoren auf die verschiedenen Erreger muss anschließend in vitro und in vivo getestet werden. Auch die Art der Aufnahme der Nanodiamanten in die verschiedenen Zellen muss untersucht werden. Dazu sollen Fluoreszenzfarbstoffe, wie z.B. Oregon Green 488, auf der Diamantoberfl{\"a}che immobilisiert werden.},
  subject      = {Diamant},
  language  = {de}
}
@phdthesis{Laug2014,
  author    = {Laug, Roderich},
  title     = {Funktionsaufkl{\"a}rung von CYR61 und CTGF in mesenchymalen Stammzellen und Lungenendothelzellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98711},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Cystein rich protein 61 (CYR61/CCN1) und Connective tissue growth factor (CTGF/CCN2) stellen aufgrund ihrer Multifunktionalit{\"a}t zwei sehr interessante Vertreter aus der derzeit sechs Mitglieder umfassenden Familie der CCN-Proteine (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) dar. Seit der Entdeckung von CYR61 und CTGF konnten die {\"u}berlappenden, aber meist nicht redundanten zellspezifischen Effekte in verschiedenen Zellsystemen nachgewiesen werden. Die Einfl{\"u}sse auf zahlreiche Prozesse wie Proliferation und Migration, aber auch Angiogenese und das {\"U}berleben von Zellen lassen eine weitreichende Bedeutung im Zusammenhang mit vielen Entwicklungsprozessen vermuten, so auch der des muskuloskelettalen Systems und der Entwicklung der Lunge. In der vorliegenden Arbeit wurden f{\"u}r die n{\"a}here Charakterisierung von CYR61 und CTGF humane mesenchymale Stammzellen (hMSC) und die humane prim{\"a}re Lungenendothelzelllinie HPMEC-ST1.6R (human pulmonary microvascular endothelial cells) gew{\"a}hlt. Beide Zellsysteme sind f{\"u}r die Untersuchung der Funktionsf{\"a}higkeit in den verschiedenen Kompartimenten bestens geeignet. So ist die Zelllinie HPMEC-ST1.6R den prim{\"a}ren Endothelzellen, im Vergleich mit anderen in der Forschung eingesetzten Zelllinien, in Bezug auf spezifische Oberfl{\"a}chenmarker am n{\"a}chsten. Mesenchymale Stammzellen bilden als multipotente Zellen das R{\"u}ckrat des muskuloskelettalen Systems und sind an der Hom{\"o}ostase des menschlichen St{\"u}tz- und Bewegungsapparates maßgeblich beteiligt. Um experimentell nutzbare Konzentrationen an rekombinanten Proteinen zu erhalten, wurde ein Baculovirus-Expressionsystems gew{\"a}hlt. Nach der erfolgreichen Klonierung der CTGF/Fc-Tag Sequenz in einen Expressionsvektor konnte dies auch durch Produktion in SF21-Insektenzellen erreicht und erstmalig rekombinantes CTGF/Fc von hoher Reinheit gewonnen werden. Allerdings konnte eine best{\"a}ndige Funktionsf{\"a}higkeit der aufgereinigten Proteine mittels eines Proliferationstestes nachfolgend nur bedingt best{\"a}tigt werden. F{\"u}r die weitere Versuchsplanung, einer Untersuchung der Auswirkung von rekombinantem CTGF (rCTGF) bzw. CYR61 (rCYR61) auf die Zielzellen, musste zun{\"a}chst die zelleigene ctgf bzw. cyr61 Expression herunterreguliert werden, um einen endogenen St{\"o}reffekt auszuschließen. Durch den Einsatz spezifischer shRNAs konnte ctgf/CTGF sowohl in den hMSC-, wie auch den HPMEC-ST1.6R-Zielzellen deutlich herunterreguliert und nachfolgend eine markant reduzierte Proliferation beobachtet werden. Ein Effekt f{\"u}r die Regulation von cyr61 blieb aus. In dieser Arbeit wurden anschließend erstmals mittels Microarray-Analysen Ver{\"a}nderungen im Genexpressionsmuster der ctgf herunterregulierten hMSC- bzw. Lungenendothelzellen gegen{\"u}ber Kontrollzellen untersucht. Des Weiteren war die Auswirkung einer Behandlung von ctgf herunterregulierten Zielzellen mit rCTGF gegen{\"u}ber unbehandelten Kontrollzellen von Interesse. F{\"u}r beide Zellsysteme konnten signifikante Genregulationen nach der Behandlung mit CTGF spezifischen shRNAs gegen{\"u}ber den Kontrollzellen detektiert werden, mit interessanten Genclustern im Bereich der TGF-beta (transforming growth factor ß) Signalgebung, sowie der fokalen Adh{\"a}sion (z.B. VEGF). Eine Behandlung mit rCTGF hingegen zeigte gegen{\"u}ber den unbehandelten Kontrollzellen in der Auswertung der Microarray-Analyse keine signifikante Ver{\"a}nderung im Genexpressionsmuster. In dieser Arbeit wurden, neben einer effektiven Gewinnung von rekombinantem CTGF und der Herunterregulation der endogenen ctgf Expression, wichtige Erkenntnisse zur Biologie von CTGF (und CYR61) in mesenchymalen Stammzellen hMSC und der Lungenendothelzelllinie HPMEC-ST1.6R erlangt. Die erhaltenen Microarray-Daten bieten eine fundierte Grundlage f{\"u}r zahlreiche fortf{\"u}hrende Untersuchungen.},
  subject      = {Connective Tissue Growth Factor},
  language  = {de}
}