Characterization of primary biogenic aerosol particles by DNA analysis: Diversity of airborne Ascomycota and Basidiomycota

Primary biogenic aerosol (PBA) particles account for large proportions of air particulate matter, and they can influence the hydrological cycle and climate as nuclei for water droplets and ice crystals in clouds, fog, and precipitation. Moreover, they can cause or enhance human, animal, and plant diseases. The actual abundance and properties of PBA particles and components in the atmosphere are, however, still poorly understood and quantified. rnIn this study, the identity, diversity, and frequency of occurrence of PBA particles were investigated by DNA analysis. Methods for the extraction, amplification, and analysis of DNA from aerosol filter samples were developed and optimized for different types of organisms, including fungi, bacteria, and plants. The investigations were focused on fungal DNA, and over 2500 sequences were obtained from air samples collected at different locations and climatic zones around the world (tropical, mid-latitude, sub-polar; continental, marine). rnNearly all fungal DNA sequences could be attributed to the phyla of Ascomycota and Basidiomycota. With regard to species richness, the ratio of Basidiomycota to Ascomycota was much higher in continental air samples (~60:40) than in marine air samples (~30:70). Pronounced differences in the relative abundance and seasonal cycles of various groups of fungi were detected in coarse and fine particulate matter from continental air, with more plant pathogens in the coarse and more human pathogens and allergens in the respirable fine particle fraction (<3 µm). The results of this study provide new information and insights into the sources of PBA particles and the interactions of the biosphere with the atmosphere, climate, and public health. rn

Blood-CNS barriers in neurodegenerative diseases

The blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) separate the brain and the spinal cord from the circulating blood and are important for the maintenance of the CNS homeostasis. They build a physical barrier thereby protecting the CNS from pathogens and toxic agents, and their disruption plays a crucial role in the pathogenesis of several CNS disorders. In this thesis, the blood-CNS-barriers were studied via in vitro models in two case studies for neurodegenerative disorders, in particular Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). The first model evaluates treatment possibilities of AD using nanotechnology-based strategies. Since the toxic amyloid-β42 (Aβ42) peptide plays a crucial role in the pathogenesis of AD, reduced generation or enhanced clearance of Aβ42 peptides are expected to modify the disease course in AD. Therefore, several Aβ42-lowering drugs like flurbiprofen had been tested in clinical trials, but most of them failed due to their low brain penetration. Here, flurbiprofen was embedded in polylactide (PLA) nanoparticles and its transport was examined in an in vitro BBB model. The embedding of flurbiprofen into the nanoparticles disguised its cytotoxic potential and enabled the administration of higher drug concentrations which resulted in a sufficient transport of the drug across an endothelial cell monolayer. These results demonstrate that non-permeable drugs can be transported efficiently via nanoparticles and that these nanotechnology-based strategies are a promising tool to generate novel therapeutic options for AD and other CNS diseases. rnThe focus of the second project was to investigate the impaired integrity of the BSCB in a mouse model for ALS. About 20% of all familial ALS cases are associated with missense mutations or small deletions in the gene that encodes Cu/Zn-superoxide dismutase 1 (SOD1). To date, the molecular mechanisms resulting in ALS are still unknown, but there is evidence that the disruption of the BSCB is one of the primary pathological events. In both familial and sporadic ALS patients, loss of endothelial integrity and endothelial cell damage was observed, and studies with SOD1 transgenic mice demonstrated that the BSCB disruption was found prior to motor neuron degeneration and neurovascular inflammation. Thus, an in vitro model for ALS endothelial cells was generated which exhibited comparable integrity characteristics and tight junction (TJ) protein expression profiles as isolated primary endothelial cells of the BSCB of SOD1 transgenic mice. In this, an alteration of the βcat/AKT/FoxO1 pathway, which regulates the expression of the TJ protein claudin-5, could be observed. These data furthermore indicate that ALS is a neurovascular disease, and understanding of the primary events in ALS pathogenesis will hopefully provide ideas for the development of new therapeutic strategies. rn