Refine
Has Fulltext
- yes (554)
Is part of the Bibliography
- yes (554) (remove)
Year of publication
- 2018 (554) (remove)
Document Type
- Journal article (365)
- Doctoral Thesis (156)
- Preprint (22)
- Conference Proceeding (3)
- Other (2)
- Working Paper (2)
- Book (1)
- Book article / Book chapter (1)
- Habilitation (1)
- Master Thesis (1)
Language
- English (554) (remove)
Keywords
- Parton Distributions (23)
- Hadron-Hadron scattering (experiments) (16)
- Positronen-Emissions-Tomografie (16)
- Extension (14)
- PET (14)
- Decay (13)
- boron (12)
- Squark (11)
- ++ (10)
- positron emission tomography (9)
Institute
- Physikalisches Institut (83)
- Theodor-Boveri-Institut für Biowissenschaften (81)
- Graduate School of Life Sciences (64)
- Klinik und Poliklinik für Nuklearmedizin (34)
- Institut für Anorganische Chemie (22)
- Institut für Psychologie (20)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (19)
- Medizinische Klinik und Poliklinik II (17)
- Institut für Informatik (15)
- Institut für Mathematik (14)
Sonstige beteiligte Institutionen
- Johns Hopkins School of Medicine (15)
- Johns Hopkins University School of Medicine (5)
- Department of Biomedical Imaging, National Cerebral and Cardiovascular Research Center, Suita, Japan (2)
- Division of Medical Technology and Science, Department of Medical Physics and Engineering, Course of Health Science, Osaka University Graduate School of Medicine, Suita Japan (2)
- Institut for Molecular Biology and CMBI, Department of Genomics, Stem Cell Biology and Regenerative Medicine, Leopold-Franzens-University Innsbruck, Innsbruck, Austria (2)
- International Max Planck Research School Molecular Biology, University of Göttingen, Germany (2)
- Johns Hopkins School of Medicine, The Russell H Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA (2)
- ACC GmbH Analytical Clinical Concepts (1)
- BMBF (1)
- Boehringer Ingelheim Pharma GmbH & Co. KG (1)
ResearcherID
- J-8841-2015 (1)
Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis.
The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds.
To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations.
In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.
Historical maps are fascinating documents and a valuable source of information for scientists of various disciplines. Many of these maps are available as scanned bitmap images, but in order to make them searchable in useful ways, a structured representation of the contained information is desirable.
This book deals with the extraction of spatial information from historical maps. This cannot be expected to be solved fully automatically (since it involves difficult semantics), but is also too tedious to be done manually at scale.
The methodology used in this book combines the strengths of both computers and humans: it describes efficient algorithms to largely automate information extraction tasks and pairs these algorithms with smart user interactions to handle what is not understood by the algorithm. The effectiveness of this approach is shown for various kinds of spatial documents from the 16th to the early 20th century.
The success of semantic systems has been proven over the last years.
Nowadays, Linked Data is the driver for the rapid development of ever new intelligent systems.
Especially in enterprise environments semantic systems successfully support more and more business processes.
This is especially true for after sales service in the mechanical engineering domain.
Here, service technicians need effective access to relevant technical documentation in order to diagnose and solve problems and defects.
Therefore, the usage of semantic information retrieval systems has become the new system metaphor.
Unlike classical retrieval software Linked Enterprise Data graphs are exploited to grant targeted and problem-oriented access to relevant documents.
However, huge parts of legacy technical documents have not yet been integrated into Linked Enterprise Data graphs.
Additionally, a plethora of information models for the semantic representation of technical information exists.
The semantic maturity of these information models can hardly be measured.
This thesis motivates that there is an inherent need for a self-contained semantification approach for technical documents.
This work introduces a maturity model that allows to quickly assess existing documentation.
Additionally, the approach comprises an abstracting semantic representation for technical documents that is aligned to all major standard information models.
The semantic representation combines structural and rhetorical aspects to provide access to so called Core Documentation Entities.
A novel and holistic semantification process describes how technical documents in different legacy formats can be transformed to a semantic and linked representation.
The practical significance of the semantification approach depends on tools supporting its application.
This work presents an accompanying tool chain of semantification applications, especially the semantification framework CAPLAN that is a highly integrated development and runtime environment for semantification processes.
The complete semantification approach is evaluated in four real-life projects: in a spare part augmentation project, semantification projects for earth moving technology and harvesting technology, as well as an ontology population project for special purpose vehicles.
Three additional case studies underline the broad applicability of the presented ideas.
The thesis describes the development of new synthetic strategies towards planar nanometer-sized and electron-deficient polycyclic aromatic dicarboximides, which are rather unexplored compared with the large variety of electron-rich polycyclic aromatic hydrocarbons and nanographenes. Thus, new polycyclic aromatic systems containing a different number of dicarboximide groups were designed since this class of compounds has revealed its significance in the past due to a range of desirable molecular properties and its high thermal and photochemical stability. The synthetic concept towards these systems includes different C–C coupling techniques that were combined within coupling cascade reactions. Therefore, this thesis provides new insights into the reactivity of aromatic substrates and elucidates mechanistic aspects of C–C coupling cascade reactions to facilitate the precise design of new and desirable materials based on polycyclic aromatic dicarboximides. Furthermore, structure-property relationships as well as the optical and electrochemical properties were investigated by UV/Vis absorption and fluorescence spectroscopy and cyclic or square wave voltammetry. Insights into the molecular structures in the solid state were obtained by single-crystal X-ray analysis. In subsequent studies, highly electron-deficient perylene bisimides and their reduced species have been investigated in detail. Thus, core-functionalized perylene bisimides were synthesized and UV/Vis absorption spectroscopy, spectroelectrochemistry and cyclic or square wave voltammetry were used to determine their optical properties and the stability of the individual reduced species.
We demonstrate two-quantum (2Q) coherent two-dimensional (2D)electronic spectroscopy using a shot-to-shot-modulated pulse shaper and fluorescence detection. Broadband collinear excitation is realized with the supercontinuum output of an argon-filled hollow-core fiber, enabling us to excite multiple transitions simultaneously in the visible range. The 2Q contribution is extracted via a three-pulse sequence with 16-fold phase cycling and simulated employing cresyl violet as a model system. Furthermore, we report the first experimental realization of one-quantum−two-quantum (1Q-2Q) 2D spectroscopy, offering less congested spectra as compared with the 2Q implementation. We avoid scattering artifacts and nonresonant solvent contributions by using fluorescence as the observable. This allows us to extract quantitative information about doubly excited states that agree with literature expectations. The high sensitivity and background-free nature of fluorescence detection allow for a general applicability of this method to many other systems.
The rise of automated driving will fundamentally change our mobility in the near future. This thesis specifically considers the stage of so called highly automated driving (Level 3, SAE International, 2014). At this level, a system carries out vehicle guidance in specific application areas, e.g. on highway roads. The driver can temporarily suspend from monitoring the driving task and might use the time by engaging in so called non-driving related tasks (NDR-tasks). However, the driver is still in charge to resume vehicle control when prompted by the system. This new role of the driver has to be critically examined from a human factors perspective.
The main aim of this thesis was to systematically investigate the impact of different NDR-tasks on driver behavior and take-over performance. Wickens’ (2008) architecture of multiple resource theory was chosen as theoretical framework, with the building blocks of multiplicity (task interference due to resource overlap), mental workload (task demands), and aspects of executive control or self-regulation. Specific adaptations and extensions of the theory were discussed to account for the context of NDR-task interactions in highly automated driving.
Overall four driving simulator studies were carried out to investigate the role of these theoretical components. Study 1 showed that drivers focused NDR-task engagement on sections of highly automated compared to manual driving. In addition, drivers avoided task engagement prior to predictable take-over situations. These results indicate that self-regulatory behavior, as reported for manual driving, also takes place in the context of highly automated driving. Study 2 specifically addressed the impact of NDR-tasks’ stimulus and response modalities on take-over performance. Results showed that particularly visual-manual tasks with high motoric load (including the need to get rid of a handheld object) had detrimental effects. However, drivers seemed to be aware of task specific distraction in take-over situations and strictly canceled visual-manual tasks compared to a low impairing auditory-vocal task. Study 3 revealed that also the mental demand of NDR-tasks should be considered for drivers’ take-over performance. Finally, different human-machine-interfaces were developed and evaluated in Simulator Study 4. Concepts including an explicit pre-alert (“notification”) clearly supported drivers’ self-regulation and achieved high usability and acceptance ratings.
Overall, this thesis indicates that the architecture of multiple resource theory provides a useful framework for research in this field. Practical implications arise regarding the potential legal regulation of NDR-tasks as well as the design of elaborated human-machine-interfaces.
In this thesis stability and robustness properties of systems of functional differential equations which dynamics depends on the maximum of a solution over a prehistory time interval is studied. Max-operator is analyzed and it is proved that due to its presence such kind of systems are particular case of state dependent delay differential equations with piecewise continuous delay function. They are nonlinear, infinite-dimensional and may reduce to one-dimensional along its solution. Stability analysis with respect to input is accomplished by trajectory estimate and via averaging method. Numerical method is proposed.
Fluorescence microscopy has become one of the most important techniques for the imaging of biological cells and tissue, since the technique allows for selective labeling with fluorescent molecules and is highly suitable for low-light applications down to the single molecule regime. The methodological requirements are well-defined for studying membrane receptors within a highly localized nanometer-thin membrane. For example, G-protein-coupled receptors (GPCRs) are an extensively studied class of membrane receptors that represent one of the most important pharmaceutical targets. Ligand binding and GPCR activation dynamics are suspected to take place at the millisecond scale and may even be far faster. Thus, techniques that are fast, selective, and live-cell compatible are required to monitor GPCR dynamics. Fluorescence resonance energy transfer (FRET) and total internal reflection fluorescence microscopy (TIRF-M) are methods of choice to monitor the dynamics of GPCRs selectively within the cell membrane.
Despite the remarkable success of these modalities, there are limitations. Most importantly, inhomogeneous illumination can induce imaging artifacts, rendering spectroscopic evaluation difficult. Background signal due to scattering processes or imperfect labeling can hamper the signal-to-noise, thus limiting image contrast and acquisition speed. Careful consideration of the internal physiology is required for FRET sensor design, so that ligand binding and cell compatibility are well-preserved despite the fluorescence labeling procedures. This limitation of labeling positions leads to very low signal changes in FRET-based GPCR analysis. In addition, microscopy of these systems becomes even more challenging in single molecule or low-light applications where the accuracy and temporal resolution may become dramatically low. Fluorescent labels should therefore be brighter, protected from photobleaching, and as small as possible to avoid interference with the binding kinetics. The development of new fluorescent molecules and labeling methods is an ongoing process. However, a complete characterization of new labels and sensors takes time. So far, the perfect dye system for GPCR studies has not been found, even though there is high demand.
Thus, this thesis explores and applies a different approach based on improved illumination schemes for TIRF-M as well as metal-coated coverslips to enhance fluorescence and FRET efficiency. First, it is demonstrated that a 360° illumination scheme reduces typical TIRF artifacts and produces a much more homogenously illuminated field of view. Second, membrane imaging and FRET spectroscopy are improved by metal coatings that are used to modulate the fluorescent properties of common fluorescent dyes. Computer simulation methods are used to understand the underlying photophysics and to design the coatings. Third, this thesis explores the operational regime and limitations of plasmonic approaches with high sectioning capabilities. The findings are summarized by three publications that are presented in the results section of this work. In addition, the theory of fluorescence and FRET is explained, with particular attention to its emission modulations in the vicinity of metal-dielectric layers. Details of the instrumentation, computer simulations, and cell culture are described in the method section. The work concludes with a discussion of the findings within the framework of recent technological developments as well as perspectives and suggestions for future approaches complete the presented work.
Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis.
First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level.
Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice.
In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation.
Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.
G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology.
Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored.
In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 % could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.
Alzheimer’s disease is a complex network of several pathological hallmarks. These characteristics always occur concomitantly and cannot be taken as distinct features of the disease. While there are hypotheses trying to explain the origin and progression of the illness, none of them is able to pinpoint a definitive cause. This fact challenges researchers not to focus on one individual hallmark but, bearing in mind the big picture, target two or more indications at once. This work, therefore, addresses two of the major characteristics of AD: the cholinergic hypothesis and neurotoxic oxidative stress. The former was achieved by targeting the postsynaptic muscarinic M1 acetylcholine receptor to further investigate its pharmacology, and the latter with the synthesis of neuroprotective natural antioxidant hybrids.
The first aim was the design and synthesis of dualsteric agonists of the muscarinic M1 acetylcholine receptor. Activation of this receptor was previously shown to improve AD pathologies like the formation of Aβ and NFTs and protect against oxidative stress and caspase activation. Selectively targeting the M1 receptor is difficult as subtypes M1 – M5 of the muscarinic AChRs largely share the same orthosteric binding pocket. Orthosteric ligands are thus unsuitable for selective activation of one specific subtype. Secondary, allosteric binding sites are more diverse between subtypes. Allosteric ligands are, however, in most cases dependent on an orthosteric ligand to cause downstream signals. Dualsteric ligands thus utilize the characteristics of both orthosteric and allosteric ligands in form of a message-address concept. Bridged by an alkylene-linker, the allosteric part ensures selectivity, whereas the orthosteric moiety initiates receptor activation. Two sets of compounds were synthesised in this sense. In both cases, the orthosteric ligand carbachol is connected to an allosteric ligand via linkers of different chain length. The first set utilizes the selective allosteric M1 agonist TBPB, the second set employs the selective M1 positive allosteric modulator BQCA. Six compounds were obtained in twelve-step syntheses each. For each one, a reference compound lacking the carbachol moiety was synthesised. The dualsteric ligands 1a-c and 2a c were tested in the IP1 assay. The assay revealed that the TBPB-dualsterics 1 are not able to activate the receptor, whereas the respective TBPB-alkyl reference compounds 27 gave signals depending on the length of the alkylene-linker, suggesting allosteric partial agonism of alkyl compounds 27 and no dualsteric binding of the putatively dualsteric compounds 1. The dualsteric BQCA molecules 2, however, activated the receptor as expected. Efficacy of the C5 linked compound 2b was the highest, yet C3 and C8 compounds (2a and 2c) also showed partial agonism. In this case, the reference compounds 31 showed no receptor activation, implying the intended dualsteric binding mode of the BQCA-carbachol compounds 2. Further investigations will be conducted by the working group of Dr. Christian Tränkle at the Department of Pharmacology at the University of Bonn to confirm binding modes and determine affinities as well as selectivity of the synthesised dualsteric compounds.
The second project dealt with the design, synthesis and biological evaluation of neuroprotective esters of the flavonolignan silibinin. While silibinin is already a potent antioxidant, it has been observed that the 7-OH group has a pro-oxidative character, making this position attractive for functionalisation. In order to obtain more potent antioxidants, the pro-oxidative position was esterified with other antioxidant moieties like ferulic acid 35 and derivatives thereof. Seventeen esters of silibinin 32, including pure diastereomers of 7 O feruloylsilibinin (43a and 43b) and a cinnamic acid ester of 2,3-dehydrosilibinin 46, were synthesised by regioselective esterification using acyl chlorides under basic conditions. The physicochemical antioxidant properties were assessed in the FRAP assay. This assay revealed no improvement of the antioxidant properties except for 7-O-dihydrosinapinoylsilibinin 39b. These results, however, do not correlate with the neuroprotective properties determined in the HT-22 hippocampal neuronal cell model. The assay showed overadditive neuroprotective effects of the esters exceeding those of its components and equimolar mixtures with the most potent compounds being 7-O-cinnamoylsilibinin 37a, 7-O-feruloylsilibinin 38a and the acetonide-protected caffeic acid ester 40a. These potent Michael system bearing compounds may be considered as “PAINS”, but the assays used to assess antioxidant and neuroprotective activities were carefully chosen to avoid false positive readouts. The most potent compounds 37a and 38a, as well as the diastereomers 43a and 43b, were further studied in assays related to AD. In vitro ischemia, inhibition of microglial activation, PC12 cell differentiation and inhibition of Aβ42 and τ protein aggregation assays showed similar results in terms of overadditive effects of the synthesised esters. Moreover, the diastereomers 43a and 43b showed differences in their activities against oxytosis (glutamate-induced apoptosis), inhibition of Aβ42 and τ protein aggregation, and PC12 cell differentiation. The stereospecific effect or mode of action against Aβ42 and τ protein aggregation is more pronounced than that of silybin A (32a) and silybin B (32b) reported in literature and needs to be elucidated in future work. Stability measurements in cell culture medium revealed that the esters do not only get hydrolysed but are partially oxidised to their respective 2,3-dehydrosilibinin esters. Because dehydrosilibinin 45 itself is described as a more potent antioxidant than silibinin 32, 7 O cinnamoyl-2,3-dehydrosilibinin 46 was expected to be even more potent than its un-oxidised counterpart 37a in terms of neuroprotection. The oxytosis assay, however, showed that the neurotoxicity of 46 is much more pronounced, especially at higher concentrations, reducing its neuroprotective potential. Dehydrosilibinin esters are therefore inferior to the silibinin esters for application as neuroprotectants, because of the difficulty of their synthesis and their increased neurotoxicity. A synergistic effect of both species (silibinin and the oxidised form) might, however, be possible or even necessary for the pronounced neuroprotective effects of silibinin esters. As the dehydro-species show distinct neuroprotective properties at low concentrations, their continuous formation over time might make an essential contribution to the overall neuroprotection of the synthesised esters. Due to solubility issues for some of the ester compounds, 7-O-cinnamoylsilibinin 37a was converted into a highly soluble hemisuccinate. The vastly improved solubility of 7 O cinnamoyl-23-O-succinylsilibinin 48 was confirmed in shake-flask experiments. Contrary to expectation, stability examinations showed that the succinyl compound 48 is not cleaved to form 7-O-cinnamoylsilibinin 37a. Neuroprotection assays confirmed that 48 is not a prodrug of the corresponding ester. It was determined that the main site of hydrolysis is the 7-position, cleaving 37 to silibinin 32 and cinnamic acid thus reducing the compound’s neuroprotective effects. Nevertheless, the compound still showed neuroprotection at a concentration of 25 µM. The improved solubility might be more beneficial than the higher neuroprotection of the poorly soluble parent compound 37a in vivo. 7 O Cinnamoylsilibinin 37a was further investigated to reduce Aβ25 35 induced learning impairment in mice. While tendencies of improved short-term and long-term memory in the animals were observed, the effects are not yet statistically significant in both Y-maze and passive avoidance tests. A greater number of test subjects is necessary to ensure correctness of the preliminary results presented in this work. However, an effect of ester 37a is observable in vivo, showing blood-brain barrier penetration. The esters synthesised are a novel approach for the treatment of AD as they show strong neuroprotective effects and their hydrolysis products or metabolites are only non-toxic natural products.
In diabetic cardiomyopathy, left ventricular (LV) diastolic dysfunction is one of the earliest signs of cardiac involvement prior to the definitive development of heart failure (HF). We aimed to explore the LV diastolic function using electrocardiography (ECG)-gated \(^{18}\)F-fluorodeoxyglucose positron emission tomography (\(^{18}\)F-FDG PET) imaging beyond the assessment of cardiac glucose utilization in a diabetic rat model. ECG-gated \(^{18}\)F-FDG PET imaging was performed in a rat model of type 2 diabetes (ZDF fa/fa) and ZL control rats at age of 13 weeks (n=6, respectively). Under hyperinsulinemic-euglycemic clamp to enhance cardiac activity, \(^{18}\)F-FDG was administered and subsequently, list-mode imaging using a dedicated small animal PET system with ECG signal recording was performed. List-mode data were sorted and reconstructed into tomographic images of 16 frames per cardiac cycle. Left ventricular functional parameters (systolic: LV ejection fraction (EF), heart rate (HR) vs. diastolic: peak filling rate (PFR)) were obtained using an automatic ventricular edge detection software. No significant difference in systolic function could be obtained (ZL controls vs. ZDF rats: LVEF, 62.5±4.2 vs. 59.4±4.5%; HR: 331±35 vs. 309±24 bpm; n.s., respectively). On the contrary, ECG-gated PET imaging showed a mild but significant decrease of PFR in the diabetic rats (ZL controls vs. ZDF rats: 12.1±0.8 vs. 10.2±1 Enddiastolic Volume/sec, P<0.01). Investigating a diabetic rat model, ECG-gated \(^{18}\)F-FDG PET imaging detected LV diastolic dysfunction while systolic function was still preserved. This might open avenues for an early detection of HF onset in high-risk type 2 diabetes before cardiac symptoms become apparent.
Given points in the plane, connect them using minimum ink. Though the task seems simple, it turns out to be very time consuming. In fact, scientists believe that computers cannot efficiently solve it. So, do we have to resign? This book examines such NP-hard network-design problems, from connectivity problems in graphs to polygonal drawing problems on the plane. First, we observe why it is so hard to optimally solve these problems. Then, we go over to attack them anyway. We develop fast algorithms that find approximate solutions that are very close to the optimal ones. Hence, connecting points with slightly more ink is not hard.
I. Timing is a crucial feature in organisms that live within a variable and changing environment. Complex mechanisms to measure time are wide-spread and were shown to exist in many taxa. These mechanisms are expected to provide fitness benefits by enabling organisms to anticipate environmental changes and adapt accordingly. However, very few studies have addressed the adaptive value of proper timing. The objective of this PhD-project was to investigate mechanisms and fitness consequences of timing decisions concerning colony phenology and foraging activity in the honey bee (Apis mellifera), a social insect species with a high degree of social organization and one of the most important pollinators of wild plants and crops. In chapter II, a study is presented that aimed to identify the consequences of disrupted synchrony between colony phenology and the local environment by manipulating the timing of brood onset after hibernation. In a follow-up experiment, the importance of environmental factors for the timing of brood onset was investigated to assess the potential of climate change to disrupt synchronization of colony phenology (Chapter III). Chapter IV aimed to prove for the first time that honey bees can use interval time-place learning to improve foraging activity in a variable environment. Chapter V investigates the fitness benefits of information exchange between nest mates via waggle dance communication about a resource environment that is heterogeneous in space and time.
II. In the study presented in chapter II, the importance of the timing of brood onset after hibernation as critical point in honey bee colony phenology in temperate zones was investigated. Honey bee colonies were overwintered at two climatically different sites. By translocating colonies from each site to the other in late winter, timing of brood onset was manipulated and consequently colony phenology was desynchronized with the local environment. Delaying colony phenology in respect to the local environment decreased the capability of colonies to exploit the abundant spring bloom. Early brood onset, on the other hand, increased the loads of the brood parasite Varroa destructor later in the season with negative impact on colony worker population size. This indicates a timing related trade-off and illustrates the importance of investigating effects of climate change on complex multi-trophic systems. It can be concluded that timing of brood onset in honey bees is an important fitness relevant step for colony phenology that is highly sensitive to climatic conditions in late winter. Further, phenology shifts and mismatches driven by climate change can have severe fitness consequences.
III. In chapter III, I assess the importance of the environmental factors ambient temperature and photoperiod as well as elapsed time on the timing of brood onset. Twenty-four hibernating honey bee colonies were placed into environmental chambers and allocated to different combinations of two temperature regimes and three different light regimes. Brood onset was identified non-invasively by tracking comb temperature within the winter cluster. The experiment revealed that ambient temperature plays a major role in the timing of brood onset, but the response of honey bee colonies to temperature increases is modified by photoperiod. Further, the data indicate the involvement of an internal clock. I conclude that the timing of brood onset is complex but probably highly susceptible to climate change and especially spells of warm weather in winter.
IV. In chapter IV, it was examined if honey bees are capable of interval time-place learning and if this ability improves foraging efficiency in a dynamic resource environment. In a field experiment with artificial feeders, foragers were able to learn time intervals and use this ability to anticipate time periods during which feeders were active. Further, interval time-place learning enabled foragers to increase nectar uptake rates. It was concluded that interval time-place learning can help honey bee foragers to adapt to the complex and variable temporal patterns of floral resource environments.
V. The study presented in chapter V identified the importance of the honey bee waggle dance communication for the spatiotemporal coordination of honey bee foraging activity in resource environments that can vary from day to day. Consequences of disrupting the instructional component of honey bee dance communication were investigated in eight temperate zone landscapes with different levels of spatiotemporal complexity. While nectar uptake of colonies was not affected, waggle dance communication significantly benefitted pollen harvest irrespective of landscape complexity. I suggest that this is explained by the fact that honey bees prefer to forage pollen in semi-natural habitats, which provide diverse resource species but are sparse and presumably hard to find in intensively managed agricultural landscapes. I conclude that waggle dance communication helps to ensure a sufficient and diverse pollen diet which is crucial for honey bee colony health.
VI. In my PhD-project, I could show that honey bee colonies are able to adapt their activities to a seasonally and daily changing environment, which affects resource uptake, colony development, colony health and ultimately colony fitness. Ongoing global change, however, puts timing in honey bee colonies at risk. Climate change has the potential to cause mismatches with the local resource environment. Intensivation of agricultural management with decreased resource diversity and short resource peaks in spring followed by distinctive gaps increases the probability of mismatches. Even the highly efficient foraging system of honey bees might not ensure a sufficiently diverse and healthy diet in such an environment. The global introduction of the parasitic mite V. destructor and the increased exposure to pesticides in intensively managed landscapes further degrades honey bee colony health. This might lead to reduced cognitive capabilities in workers and impact the communication and social organization in colonies, thereby undermining the ability of honey bee colonies to adapt to their environment.
For cellular viability, transcription is a fundamental process. Hereby, the DNA plays the most elemental and highly versatile role. It has long been known that promoters contain conserved and often well-defined motifs, which dictate the site of transcription initiation by providing binding sites for regulatory proteins. However, research within the last decade revealed that it is promoters lacking conserved promoter motifs and transcribing constitutively expressed genes that constitute the majority of promoters in eukaryotes. While the process of transcription initiation is well studied, whether defined DNA sequence motifs are required for the transcription of constitutively expressed genes in eukaryotes remains unknown. In the highly divergent protozoan parasite Trypanosoma brucei, most of the proteincoding genes are organized in large polycistronic transcription units. The genes within one polycistronic transcription unit are generally unrelated and transcribed by a common transcription start site for which no RNA polymerase II promoter motifs have been identified so far. Thus, it is assumed that transcription initiation is not regulated but how transcription is initiated in T. brucei is not known. This study aimed to investigate the requirement of DNA sequence motifs and chromatin structures for transcription initiation in an organism lacking transcriptional regulation. To this end, I performed a systematic analysis to investigate the dependence of transcription initiation on the DNA sequence. I was able to identify GT-rich promoter elements required for directional transcription initiation and targeted deposition of the histone variant H2A.Z, a conserved component during transcription initiation. Furthermore, nucleosome positioning data in this work provide evidence that sites of transcription initiation are rather characterized by broad regions of open and more accessible chromatin than narrow nucleosome depleted regions as it is the case in other eukaryotes. These findings highlight the importance of chromatin during transcription initiation. Polycistronic RNA in T. brucei is separated by adding an independently transcribed miniexon during trans-splicing. The data in this work suggest that nucleosome occupancy plays an important role during RNA maturation by slowing down the progressing polymerase and thereby facilitating the choice of the proper splice site during trans-splicing. Overall, this work investigated the role of the DNA sequence during transcription initiation and nucleosome positioning in a highly divergent eukaryote. Furthermore, the findings shed light on the conservation of the requirement of DNA motifs during transcription initiation and the regulatory potential of chromatin during RNA maturation. The findings improve the understanding of gene expression regulation in T. brucei, a eukaryotic parasite lacking transcriptional Regulation.
Cadherin-13 (CDH13) is an atypical member of the cadherin superfamily, a group of membrane proteins mediating calcium-dependent cellular adhesion. Although CDH13 shows the classical extracellular cadherin structure, the typical transmembrane and cytoplasmic domains are absent. Instead, CDH13 is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. These findings and many studies from different fields suggest that CDH13 also plays a role as a cellular receptor. Interestingly, many genome-wide association studies (GWAS) have found CDH13 as a risk gene for attention-deficit/hyperactivity disorder (ADHD) and other neurodevelopmental disorders. In previous work from our research group, strong expression of Cdh13 mRNA in interneurons of the hippocampal stratum oriens (SO) was detected. Therefore, double-immunofluorescence studies were used to evaluate the degree of co-expression of CDH13 with seven markers of GABAergic interneuron subtypes. For this purpose, murine brains were double stained against CDH13 and the respective marker and the degree of colocalization in the SO of the hippocampus was assessed. Based on the result of this immunofluorescence study, quantitative differences in interneuron subtypes of the SO between Cdh13 knockout (ko), heterozygote (het) and wildtype (wt) mice were investigated in this dissertation using stereological methods. In addition, genotype- dependent differences in the expression of genes involved in GABAergic and glutamatergic neurotransmission were analyzed by quantitative real-time PCR (qRT-PCR). Primers targeting different GABA receptor subunits, vesicular GABA and glutamate transporter, GABA synthesizing enzymes and their interaction partners were used for this purpose.
The results of the stereological quantification of the interneuron subtypes show no significant differences in cell number, cell density or volume of the SO between Cdh13 ko, het and wt mice. On the other hand, qRT-PCR results indicate significant differences in the expression of tropomyosin-related kinase B gene (TrkB), which encodes the receptor of brain-derived neurotrophic factor (BDNF), a regulator of GABAergic neurons. This finding supports a role for CDH13 in the regulation of BDNF signaling in the hippocampus.
The aim of this thesis was the development of a multifunctional coating system for AuNPs based on thioether polymers, providing both excellent colloidal stability and a variable possibility to introduce functionalities for biological applications.
First, two thioether-polymer systems were synthesised as a systematic investigation into colloidal stabilisation efficacy. Besides commonly used monovalent poly(ethylene glycol) (PEG-SR), its structural analogue linear poly(glycidol) (PG-SR) bearing multiple statistically distributed thioether moieties along the backbone was synthesised. Additionally, respective thiol analogues (PEG-SH and PG-SH) were produced and applied as reference.
Successive modification of varyingly large AuNPs with aforementioned thiol- and thioether-polymers was performed via ligand exchange reaction on citrate stabilised AuNPs. An increased stabilisation efficacy of both thioether-polymers against biological and physiological conditions, as well as against freeze-drying compared to thiol analogues was determined.
Based on the excellent colloidal stabilisation efficacy and multi-functionalisability of thioether-PG, a plethora of functional groups, such as charged groups, hydrophilic/hydrophobic chains, as well as bio-active moieties namely diazirine and biotin was introduced to the AuNP surface. Moreover, the generic and covalent binding of diazirine-modified PG-SR with biomolecules including peptides and proteins was thoroughly demonstrated.
Lastly, diverse applicability and bioactivity of aforementioned modified particles in various studies was displayed, once more verifying the introduction of functionalities. On the one hand the electrostatic interaction of charged AuNPs with hydrogels based on hyaluronic acid was applied to tune the release kinetics of particles from three-dimensional scaffolds. On the other hand the strong complexation of siRNA onto two positively charged AuNPs was proven. The amount of siRNA payload was tuneable by varying the surface charge, ionic strength of the surrounding medium and the N/P ratio. Moreover, the biological activity and selectivity of the biotin-streptavidin conjugation was verified with respectively functionalised particles in controlled agglomeration test and in laser-triggered cell elimination experiments. In the latter, streptavidin-functionalised AuNPs resulted in excellent depletion of biotinylated cells whereas unfunctionalised control particles failed, excluding unspecific binding of these particles to the cell surface.
The noninvasive magnetic resonance imaging technique allows for the investigation of functional processes in the living plant. For this purpose during this work, different NMR imaging methods were further developed and applied.
For the localisation of the intrusion of water into the germinating rape seed with the simultaneous depiction of the lipid-rich tissue via a 3D rendering, in Chap. 5 the technique of interleaved chemical selective acquisition of water and lipid was used in the germinating seed. The utilization of high-resolution MR images of germinated seeds enabled the localization of a predetermined water gap in the lipid-rich aleurone layer, which resides directly under the seed coat. The for a long time in biology prevalent discussion, whether such a gap exists or the seed soaks up the water from all sides, rather like a sponge, could hereby, at least for the rapeseed seed, be answered clearly. Furthermore, the segmentation and 3D visualization of the vascular tissue in the rapeseed seeds was enabled by the high-resolution datasets, a multiply branched structure preconstructed in the seed could be shown. The water is directed by the vascular tissue and thus awakens the seed gradually to life. This re-awakening could as well be tracked by means of invasive imaging via an oxygen sensor. In the re-awakened seeds, the lipid degradation starts, other than expected, not in the lipid-rich cotyledons but in the residual endosperm remaining from seed development and in the aleurone layer which previously protected the embryo. Within this layer, the degradation could be verified in the high-resolution MR datasets.
The method presented in Chap. 6 provides a further characteristic trait for phenotyping of seeds and lipid containing plants in general. The visualization of the compounds of fatty acids in plant seeds and fruits could be achieved by the distinct utilization of chemical shift-selective imaging techniques. Via the application of a CSI sequence the fatty acid compounds in an olive were localized in a 2D slice. In conjunction with an individually adjusted CHESS presaturation module Haa85 the high-resolution 3D visualization of saturated and unsaturated fatty acid compounds in different seeds was achieved. The ratio maps calculated from these datasets allow to draw conclusions from the developmental stage or the type of seed. Furthermore, it could be shown that the storage condition of two soybean seeds with different storage time durations lead to no degradation of the fatty acid content.
Additional structural information from inside of dry seeds are now accessible via MRI. In this work the imaging of cereal seeds could be significantly improved by the application of the UTE sequence. The hitherto existing depictions of the lipid distribution, acquired with the spin echo sequence, were always sufficient for examinations of the lipid content, yet defects in the starchy endosperm or differences in the starch concentration within the seed remained constantly unseen with this technique. In a direct comparison of the datasets acquired with the previous imaging technique (spin echo) and with UTE imaging, the advantage of data acquisition with UTE could be shown. By investigating the potential seed compounds (starch, proteins, sugar) in pure form, the constituent parts contributing to the signal could be identified as bound water (residual moisture) and starch. The application of a bi-exponential fit on the datasets of the barley seed enabled the separate mapping of magnetization and of relaxation time of two components contributing to the NMR signal. The direct comparison with histological stainings verified the previous results, thus this technique can be used for the selective imaging of starch in dry seeds.
Conclusions on the translocation characteristics in plants can be drawn by the technique proposed in Chap. 8. The associated translocation velocities can now, even in the range of several um/h, be determined in the living plant. Based on calculated concentrations of an MR contrast agent, which was taken up by the plant, these translocation velocities were estimated both in longitudinal direction, thus along the vascular bundle, and in horizontal direction, thus out of the bundle. The latter velocity is located below the contrast agent's velocity value of free diffusion. By adjusting a dynamic contrast-enhancing imaging technique (DCE-Imaging, Tof91) the acquisition duration of a T1-map was significantly reduced. By means of these maps, local concentrations of the contrast agent in plant stems and the siliques of the rapeseed plant could be determined.
Numerous questions in plant science can only be answered by non-invasive techniques such as MRI. For this reason, besides the experimental results achieved in this work, further NMR methods were tested and provided for the investigation of plants.
As an example, the study on the imaging of magnetic exchange processes are mentioned, which provided the groundwork for a possible transfer of CEST experiments (Chemical Exchange Saturation Transfer) to the plant. The results are presented in the bachelor thesis of A. Jäger Jae17, which was performed under my supervision, they find great interest under biologists.
The development of new technologies, which extend the possibilities for the investigation of living organisms, is of great importance. For this reason, I have contributed to the development of the currently unpublished method RACETE (Refocused Acquisition of Chemical Exchange Transferred Excitations [Jak17, Reu17, Gut18a]). By rephasing the transferred magnetization the utilization of properties which have not been available in chemical "`exchange"' experiments is enabled. With this method a positive contrast is generated, thus a reference experiment is not mandatory. Furthermore, the image phase, which in classical experiments contains no information about the exchanged protons, can be used for the distinct identification of multiple substances which have been excited simultaneously.
This recently at the Department of Experimental Physics V developed method can be used in particular for the identification of lipids and for the localization of sugars and amino acids, thus it can serve the enhancement and improvement of non-invasive analytical methods.
Improved treatment options for the degenerative joint disease osteoarthritis (OA) are of major interest, since OA is one of the main sources of disability, pain, and socioeconomic burden worldwide [202]. According to epidemiological data, already 27 million people suffer from OA in the US [23]. Moreover, the WHO expects OA to be the fourth most common cause of disability in 2020 [203], illustrating the need for effective and long-lasting therapy options of severe cartilage defects. Despite numerous clinically available products for the treatment of cartilage defects [62], the development of more cartilage-specific materials is still at the beginning.
Hyaluronic acid (HA) is a major component of the cartilaginous extracellular matrix (ECM) and inherently creates a cell-friendly niche by providing cell attachment and migration sites. Furthermore, it is known that the functional groups of HA are well suited for chemical modification. These characteristics render HA an attractive material for hydrogel-based tissue engineering approaches. Poly(glycidol) (PG) as chemical crosslinker basically features similar chemical characteristics as the widely used poly(ethylene glycol) (PEG), but provides additional side groups at each repeating unit that can be further chemically functionalized. With the introduction of PG as multifunctional crosslinker for HA gels, a higher cross-linking density and, accordingly, a greater potential for biomimetic functionalization may be achieved. However, despite the mentioned potential benefits, PG has not been used for cartilage regeneration approaches so far.
The initial aim of the study was to set up and optimize a HA-based hydrogel for the chondrogenic differentiation of mesenchymal stromal cells (MSCs), using different amounts and variations of cross-linkers. Therefore, the hydrogel composition was optimized by the utilization of different PEG diacrylate (PEGDA) concentrations to cross-link thiol-modified HA (Glycosil, HA-SH) via Michael addition. We aimed to generate volumestable scaffolds that simultaneously enable a maximum of ECM deposition. Histological and biochemical analysis showed 0.4% PEGDA as the most suitable concentration for these requirements (Section 5.1.2).
In order to evaluate the impact of a differently designed cross-linker on MSC chondrogenesis, HA-SH was cross-linked with PEGTA (0.6%) and compared to PEGDA (0.4%) in a next step. Following this, acrylated PG (PG-Acr) as multifunctional cross-linker alternative to acrylated PEG was evaluated. It provides around five times more functional groups when utilized in PG-Acr (0.6%) HA-SH hydrogels compared to PEGTA (0.6%) HA-SH hydrogels, thus enabling higher degrees of biomimetic functionalization. Determination of cartilage-specific ECM components showed no substantial differences between both cross-linkers while the deposition of cartilaginous matrix appeared more homogeneous in HA-SH PG-Acr gels. Taken together, we were able to successfully increase the possibilities for biomimetic functionalization in the developed HA-SH hydrogel system by the introduction of PG-Acr as cross-linker without negatively affecting MSC chondrogenesis (Section 5.1.3).
The next part of this thesis focused extensively on the biomimetic functionalization of PG-Acr (0.6%) cross-linked HA-SH hydrogels. Here, either biomimetic peptides or a chondrogenic growth factor were covalently bound into the hydrogels.
Interestingly, the incorporation of a N-cadherin mimetic (HAV), a collagen type II binding (KLER), or a cell adhesion-mediating peptide (RGD) yielded no improvement of MSC chondrogenesis. For instance, the covalent binding of 2.5mM HAV changed morphology of cell nuclei and reduced GAG production while the incorporation of 1.0mM RGD impaired collagen production. These findings may be attributed to the already supportive conditions of the employed HA-based hydrogels for chondrogenic differentiation. Most of the previous studies reporting positive peptide effects on chondrogenesis have been carried out in less supportive PEG hydrogels or in significantly stiffer MeHA-based hydrogels [99, 101, 160]. Thus, the incorporation of peptides may be more important under unfavorable conditions while inert gel systems may be useful for studying single peptide effects (Section 5.2.1).
The chondrogenic factor transforming growth factor beta 1 (TGF-b1) served as an example for growth factor binding to PG-Acr. The utilization of covalently bound TGF-b1 may thereby help overcome the need for repeated administration of TGF-b1 in in vivo applications, which may be an advantage for potential clinical application. Thus, the effect of covalently incorporated TGF-b1 was compared to the effect of the same amount of TGF-b1 without covalent binding (100nM TGF-b1) on MSC chondrogenesis. It was successfully demonstrated that covalent incorporation of TGF-b1 had a significant positive effect in a dose-dependent manner. Chondrogenesis of MSCs in hydrogels with covalently bound TGF-b1 showed enhanced levels of chondrogenesis compared to hydrogels into which TGF-b1 was merely mixed, as shown by stronger staining for GAGs, total collagen, aggrecan and collagen type II. Biochemical evaluation of GAG and collagen amounts, as well as Western blot analysis confirmed the histological results. Furthermore, the positive effect of covalently bound TGF-b1 was shown by increased expression of chondrogenic marker genes COL2A1, ACAN and SOX9. In summary, covalent growth factor incorporation utilizing PG-Acr as cross-linker demonstrated significant positive effects on chondrogenic differentiation of MSCs (Section 5.2.2).
In general, PG-Acr cross-linked HA hydrogels generated by Michael addition represent a versatile hydrogel platform due to their high degree of acrylate functionality. These hydrogels may further offer the opportunity to combine several biological modifications, such as the incorporation of biomimetic peptides together with growth factors, within one cell carrier.
A proof-of-principle experiment demonstrated the suitability of pure PG gels for studying single peptide effects. Here, the hydrogels were generated by the utilization of thiol-ene-click reaction. In this setting, without the supportive background of hyaluronic acid, MSCs showed enhanced chondrogenic differentiation in response to the incorporation of 1.0mM HAV. This was demonstrated by staining for GAGs, the cartilage-specific ECM molecules aggrecan and type II collagen, and by increased GAG and total collagen amounts shown by biochemical analysis. Thus, pure PG gels exhibit the potential to study the effects and interplay of peptides and growth factors in a highly modifiable, bioinert hydrogel environment.
The last section of the thesis was carried out as part of the EU project HydroZONES that aims to develop and generate zonal constructs. The importance of zonal organization has attracted increased attention in the last years [127, 128], however, it is still underrepresented in tissue engineering approaches so far. Thus, the feasibility of zonal distribution of cells in a scaffold combining two differently composed hydrogels was investigated. A HA-SH(FMZ) containing bottom layer was generated and a pure PG top layer was subsequently cast on top of it, utilizing both times thiol-ene-click reaction. Indeed, stable, hierarchical constructs were generated that allowed encapsulated MSCs to differentiate chondrogenically in both zones as shown by staining for GAGs and collagen type II, and by quantification of GAG amount. Thus, the feasibility of differently composed zonal hydrogels utilizing PG as a main component was successfully demonstrated (Section 5.4).
With the first-time utilization and evaluation of PG-Acr as versatile multifunctional cross-linker for the preparation of Michael addition-generated HA-SH hydrogels in the context of cartilage tissue engineering, a highly modifiable HA-based hydrogel system was introduced. It may be used in future studies as an easily applicable and versatile toolbox for the generation of biomimetically functionalized hydrogels for cell-based cartilage regeneration. The introduction of reinforcement structures to enhance mechanical resistance may thereby further increase the potential of this system for clinical applications.
Additionally, it was also demonstrated that thiol-ene clickable hydrogels can be used for the generation of cell-laden, pure PG gels or for the generation of more complex, coherent zonal constructs. Furthermore, thiol-ene clickable PG hydrogels have already been further modified and successfully been used in 3D bioprinting experiments [204]. 3D bioprinting, as part of the evolving biofabrication field [205], offers the possibilities to generate complex and hierarchical structures, and to exactly position defined layers, yet at the same time alters the requirements for the utilized hydrogels [159, 206–209]. Since a robust chondrogenesis of MSCs was demonstrated in the thiol-ene clickable hydrogel systems, they may serve as a basis for the development of hydrogels as so called bioinks which may be utilized in more sophisticated biofabrication processes.
Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Here we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N\(^6\)-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. Using in vitro selection from random DNA, we developed deoxyribozymes that are sensitive to the presence of N\(^6\)-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m\(^6\)A as a regulatory element that influences RNA folding and protein binding.