Synthesis and evaluation of 18-F-radiopharmaceuticals within the serotonergic receptor system for molecular imaging

Molecular imaging technologies as Positron Emission Tomography (PET) are playing a key role in drug discovery, development and delivery due to the possibility to quantify e.g. the binding potential in vivo, non-invasively and repetitively. In this context, it provides a significant advance in the understanding of many CNS disorders and conditions. The serotonergic receptor system is involved in a number of important physiological processes and diseases such as depression, schizophrenia, Alzheimer’s disease, sleep or sexual behaviour. Especially, the 5-HT2A and the 5-HT1A receptor subtypes are in the focus of fundamental and clinical research due to the fact that many psychotic drugs interact with these neuronal transmembrane receptors. This work describes the successful development, as well as in vitro and in vivo evaluation of 5-HT2A and 5-HT1A selective antagonistic PET-radiotracers. The major achievements obtained in this thesis are: 1. the development and in vitro evaluation of several 5-HT2A antagonistic compounds, namely MH.MZ (Ki = 9.0 nM), (R)-MH.MZ (Ki = 0.72 nM) and MA-1 (Ki = 3.0 nM). 2. the 18F-labeling procedure of these compounds and their optimization, whereby radiochemical yields > 35 % in high specific activities (> 15 GBq/µmol) could be observed. Synthesis time inclusive secondary synthon synthesis, the radioactive labeling procedure, separation and final formulation took no longer than 120 min and provided the tracer in high radiochemical purity. 3. the in vivo µPET evaluation of [18F]MH.MZ and (R)-[18F]MH.MZ resulting in promising imaging agents of the 5-HT2A receptor status; from which (R)-[18F]MH.MZ seems to be the most promising ligand. 4. the determination of the influence of P-gp on the brain biodistribution of [18F]MH.MZ showing a strong P-gp dependency but no regional alteration. 5. the four-step radiosynthesis and evaluation of [18F]MDL 100907 resulting in another high affine tracer, which is, however, limited due to its low radiochemical yield. 6. the development and evaluation of 3 novel possible 5-HT2A imaging agents combining structural elements of altanserin, MDL 100907 and SR 46349B demonstrating different binding modes of these compounds. 7. the development, the labeling and in vitro evaluation of the novel 5-HT1A antagonistic tracer [18F]AH1.MZ (Ki = 4.2 nM).

Flagellin:allergen fusion proteins as novel vaccines for the treatment of severe type I allergies

Over the last decades the prevalence of food allergies has continually increased on a world wide scale. While there are effective treatments available for bee and wasp venom allergic patients, there is currently no established therapy for the treatment of severe food allergies. Aim of the project was to genetically fuse different food allergens with the immune modulating Toll-like receptor 5 (TLR5)-ligand flagellin and to test these constructs for their immune modulatory capacities both in vitro and in vivo. Chicken ovalbumin (Ova) as model antigen, Pru p 3, and Ara h 2 the respective major allergens from peach and peanut were used as allergens. The potential vaccine candidates were characterized by protein biochemical methods (purity, folding, endotoxin contaminations). Moreover, their immune modulating effects on cell culture lines (TLR5-receptor activation) and primary mouse immune cells (myeloid and plasmacytoid dendritic cells) were investigated. Additionally, the prophylactic and therapeutic use of the flagellin Ova fusion protein (rflaA:Ova) were investigated in a mouse model of intestinal allergy. In myeloid dendritic cells (mDC) stimulation with the fusion proteins led to a strong cell activation and cytokine secretion. Here, the fusion proteins proved to be a much stronger stimulus than the equimolar amount of both proteins provided alone or as a mixture. Noteworthy, stimulation with rflaA:Ova induced the secretion of the anti-inflammatory cytokine IL-10 from mDC. In co-culture experiments this IL-10 secretion suppressed the Ova-induced secretion of Th1 and Th2 cytokines from Ova-specific CD4 T cells. Using MyD88-deficient mDC this repression of cytokine secretion was shown to be TLR-dependent. Finally, the potency of the rflaA:Ova construct was investigated in a mouse model of Ova-induced intestinal allergy. In a prophylactic vaccination approach rflaA:Ova was shown to prevent the establishment of the intestinal allergy and all associated symptoms (weight loss, temperature drop, soft faeces). This fusion protein-mediated protection was accompanied by a reduced T cell activation, and reduced Th2 cytokines in intestinal homogenates. These effects were paralleled by a strong induction of Ova-specific IgG2a antibodies in rflaA:Ova-vaccinated sera, while Ova-specific IgE antibody production was significantly reduced. Therapeutic vaccination with rflaA:Ova reduced allergic symptoms and T cell activation but did not influence weight loss and antibody production. In all in vivo experiments vaccination with both proteins either provided alone or as a mixture did not have comparable effects. Future experiments aim at elucidating the mechanism and further optimization of the therapeutic vaccination approach. The results presented in this thesis demonstrate, that fusion proteins containing flagellin have strong immune modulatory capacities both in vitro and in vivo. Therefore, such constructs are promising vaccine candidates for the therapy of type I allergies.

Characterization and optimization of an x-ray laser for the spectroscopy of Li-like heavy-ions

Recent developments in the theory of plasma-based collisionally excited x-ray lasers (XRL) have shown an optimization potential based on the dependence of the absorption region of the pumping laser on its angle of incidence on the plasma. For the experimental proof of this idea, a number of diagnostic schemes were developed, tested, qualified and applied. A high-resolution imaging system, yielding the keV emission profile perpendicular to the target surface, provided positions of the hottest plasma regions, interesting for the benchmarking of plasma simulation codes. The implementation of a highly efficient spectrometer for the plasma emission made it possible to gain information about the abundance of the ionization states necessary for the laser action in the plasma. The intensity distribution and deflection angle of the pump laser beam could be imaged for single XRL shots, giving access to its refraction process within the plasma. During a European collaboration campaign at the Lund Laser Center, Sweden, the optimization of the pumping laser incidence angle resulted in a reduction of the required pumping energy for a Ni-like Mo XRL, which enabled the operation at a repetition rate of 10 Hz. Using the experiences gained there, the XRL performance at the PHELIX facility, GSI Darmstadt with respect to achievable repetition rate and at wavelengths below 20 nm was significantly improved, and also important information for the development towards multi-100 eV plasma XRLs was acquired. Due to the setup improvements achieved during the work for this thesis, the PHELIX XRL system now has reached a degree of reproducibility and versatility which is sufficient for demanding applications like the XRL spectroscopy of heavy ions. In addition, a European research campaign, aiming towards plasma XRLs approaching the water-window (wavelengths below 5 nm) was initiated.

Thermoelectric performance of p-type TiCoSb half-Heusler compounds - Intrinsic phase separation and charge carrier concentration optimization as key to high efficiency

The world's rising demand of energy turns the development of sustainable and more efficient technologies for energy production and storage into an inevitable task. Thermoelectric generators, composed of pairs of n-type and p-type semiconducting materials, di¬rectly transform waste heat into useful electricity. The efficiency of a thermoelectric mate¬rial depends on its electronic and lattice properties, summarized in its figure of merit ZT. Desirable are high electrical conductivity and Seebeck coefficients, and low thermal con¬ductivity. Half-Heusler materials are very promising candidates for thermoelectric applications in the medium¬ temperature range such as in industrial and automotive waste heat recovery. The advantage of Heusler compounds are excellent electronic properties and high thermal and mechanical stability, as well as their low toxicity and elemental abundance. Thus, the main obstacle to further enhance their thermoelectric performance is their relatively high thermal conductivity.rn rnIn this work, the thermoelectric properties of the p-type material (Ti/Zr/Hf)CoSb1-xSnx were optimized in a multistep process. The concept of an intrinsic phase separation has recently become a focus of research in the compatible n-type (Ti/Zr/Hf)NiSn system to achieve low thermal conductivities and boost the TE performance. This concept is successfully transferred to the TiCoSb system. The phase separation approach can form a significant alternative to the previous nanostructuring approach via ball milling and hot pressing, saving pro¬cessing time, energy consumption and increasing the thermoelectric efficiency. A fundamental concept to tune the performance of thermoelectric materials is charge carrier concentration optimization. The optimum carrier concentration is reached with a substitution level for Sn of x = 0.15, enhancing the ZT about 40% compared to previous state-of-the-art samples with x = 0.2. The TE performance can be enhanced further by a fine-tuning of the Ti-to-Hf ratio. A correlation of the microstructure and the thermoelectric properties is observed and a record figure of merit ZT = 1.2 at 710°C was reached with the composition Ti0.25Hf0.75CoSb0.85Sn0.15.rnTowards application, the long term stability of the material under actual conditions of operation are an important issue. The impact of such a heat treatment on the structural and thermoelectric properties is investigated. Particularly, the best and most reliable performance is achieved in Ti0.5Hf0.5CoSb0.85Sn0.15, which reached a maximum ZT of 1.1 at 700°C. The intrinsic phase separation and resulting microstructure is stable even after 500 heating and cooling cycles.