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Small-angle X-ray scattering (SAXS) is a universal low-resolution method to study proteins in solution and to analyze structural changes in response to variations of conditions (pH, temperature, ionic strength etc). SAXS is hardly limited by the particle size, being applicable to the smallest proteins and to huge macromolecular machines like ribosomes and viruses. SAXS experiments are usually fast and require a moderate amount of purified material. Traditionally, SAXS is employed to study the size and shape of globular proteins, but recent developments have made it possible to quantitatively characterize the structure and structural transitions of metastable systems, e.g. partially or completely unfolded proteins. In the absence of complementary information, low-resolution macromolecular shapes can be reconstructed ab initio and overall characteristics of the systems can be extracted. If a high or low-resolution structure or a predicted model is available, it can be validated against the experimental SAXS data. If the measured sample is polydisperse, the oligomeric state and/or oligomeric composition in solution can be determined. One of the most important approaches for macromolecular complexes is a combined ab initio/rigid body modeling, when the structures (either complete or partial) of individual subunits are available and SAXS data is employed to build the entire complex. Moreover, this method can be effectively combined with information from other structural, computational and biochemical methods. All the above approaches are covered in a comprehensive program suite ATSAS for SAXS data analysis, which has been developed at the EMBL-Hamburg. In order to meet the growing demands of the structural biology community, methods for SAXS data analysis must be further developed. This thesis describes the development of two new modules, RANLOGS and EM2DAM, which became part of ATSAS suite. The former program can be employed for constructing libraries of linkers and loops de novo and became a part of a combined ab initio/rigid body modeling program CORAL. EM2DAM can be employed to convert electron microscopy maps to bead models, which can be used for modeling or structure validation. Moreover, the programs CRYSOL and CRYSON, for computing X-ray and neutron scattering patterns from atomic models, respectively, were refurbished to work faster and new options were added to them. Two programs, to be contributed to future releases of the ATSAS package, were also developed. The first program generates a large pool of possible models using rigid body modeling program SASREF, selects and refines models with lowest discrepancy to experimental SAXS data using a docking program HADDOCK. The second program refines binary protein-protein complexes using the SAXS data and the high-resolution models of unbound subunits. Some results and conclusions from this work are presented here. The developed approaches detailed in this thesis, together with existing ATSAS modules were additionally employed in a number of collaborative projects. New insights into the “structural memory” of natively unfolded tau protein were gained and supramodular structure of RhoA-specific guanidine nucleotide exchange factor was reconstructed. Moreover, high resolution structures of several hematopoietic cytokine-receptor complexes were validated and re-modeled using the SAXS data. Important information about the oligomeric state of yeast frataxin in solution was derived from the scattering patterns recorded under different conditions and its flexibility was quantitatively characterized using the Ensemble Optimization Method (EOM).
Monoamine oxidase inhibitors (MAO-I) belong to the earliest drugs tried in Parkinson's disease (PD). They have been used with or without levodopa (L-DOPA). Non-selective MAO-I due to their side-effect/adverse reaction profile, like tranylcypromine have limited use in the treatment of depression in PD, while selective, reversible MAO-A inhibitors are recommended due to their easier clinical handling. For the treatment of akinesia and motor fluctuations selective irreversible MAO-B inhibitors selegiline and rasagiline are recommended. They are safe and well tolerated at the recommended daily doses. Their main differences are related to (1) metabolism, (2) interaction with CYP-enzymes and (3) quantitative properties at the molecular biological/genetic level. Rasagiline is more potent in clinical practise and has a hypothesis driven more favourable side effect/adverse reaction profile due to its metabolism to aminoindan. Both selegiline and rasagiline have a neuroprotective and neurorestaurative potential. A head-to head clinical trial would be of utmost interest from both the clinical outcome and a hypothesis-driven point of view. Selegiline is available as tablet and melting tablet for PD and as transdermal selegiline for depression, while rasagiline is marketed as tablet for PD. In general, the clinical use of MAO-I nowadays is underestimated. There should be more efforts to evaluate their clinical potency as antidepressants and antidementive drugs in addition to the final proof of their disease-modifying potential. In line with this are recent innovative developments of MAO-I plus inhibition of acetylcholine esterase for Alzheimer's disease as well as combined MAO-I and iron chelation for PD.
HINTERGRUND: Der brain-derived neurotrophic factor (BDNF) reguliert die synaptische Plastizität und spielt somit eine wichtige Rolle in der Gedächtnisbildung und -erhaltung. Deswegen gibt es eingehende Untersuchungen dieses neurotrophischen Faktors in Bezug auf Demenzerkrankungen, vor allem der Alzheimer Demenz. In dieser Studie wurde nach einem Zusammenhang zwischen BDNF Blutplasmawerten und der Alzheimer Demenz in einer longitudinalen Kohortenstudie, der Vienna-Transdanube-Aging(VITA)-Studie gesucht. METHODEN: Die VITA-Studie ist eine kommunale Kohortenstudie aller 75jährigen Einwohner einer geographischen Region Wiens. Es wurden die BDNF Plasmawerte der Basisuntersuchung und der ersten Folgeuntersuchung 30 Monate später als mögliche Biomarker für die Alzheimer Demenz untersucht. Assoziationen zwischen BDNF Plasmawerten und anderen epidemiologischen Eckdaten wurden ebenfalls analysiert. ERGEBNISSE: Wir konnten keine Assoziation zwischen BDNF Plasmawerten und der Entwicklung oder einer bereits bestehenden Alzheimer Demenz finden. Geschlecht, Body-Maß-Index und Depression stellten sich als Komorbiditäts-Faktoren von Demenz-erkrankungen dar. SCHLUSSFOLGERUNG: BDNF Plasmawerte sind diesen Ergebnissen nach kein so viel versprechender molekularer Marker für Alzheimer Demenz wie erhofft. BDNF wird jedoch weiterhin in vielen interessanten Studienprotokollen untersucht, da es sowohl im Blutserum als auch im Hirngewebe nachgewiesen werden kann und somit viele diagnostische und therapeutische Ansätze inspiriert.