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Oils and fats from natural origin are sustainable sources for a broad range of economically relevant products in food, feed, fuel, oleochemical, and cosmetic industries. Thereby, a huge variety of lipids or lipid-derived products exist which distinguish themselves by their unique physical properties making them suitable for their individual applications. To obtain such functional lipids in an environmentally friendly manner, enzymes can be employed. In that context, lipases have been proven to be valuable biocatalysts in lipid modification, which are broadly applied in industry. Even though they have been implemented successfully in the dairy, baking, and detergent industries, there is an increasing demand for the expansion of their utilization. New technologies like protein engineering and the implementation of process development are employed in solving this task. Within the enzymes in lipid modification, lipases are the most applied catalysts and in this thesis their utilization was expanded successfully to the implementation of novel separation processes and the production of improved drug delivery matrices.
Multiproxy investigations of lacustrine sediments from Laguna Azul (52 °S) document multi-millennial Holocene influences of Southern Hemispheric Westerlies (SHW) on the hydroclimatic variability of south-eastern Patagonia. During the last 4000 years, this hydroclimatic variability is overprinted by centennial warm/dry periods. A cool/wet period from 11,600 to 10,100 cal. BP is succeeded by an early Holocene dry period (10,100–8300 cal. BP) with a shallow lake, strong anoxia, methanogenesis and high salinity. Between 8300 and 4000 cal. BP the influence of SHW weakened, resulting in a freshwater lake considered to be related to less arid conditions. Since 4000 cal. BP, regional temperature decreased accompanied by re-intensification of SHW reaching full strength since 3000 cal. BP. Centred around 2200, 1000 cal. BP and in the 20th century, Laguna Azul experienced century-long warm/dry spells. Between these dry periods, two pronounced moist periods are suggested to be contemporaneous to the ‘Dark Age Cold Period’ and the ‘Little Ice Age’. Different from millennial SHW variations, centennial fluctuations appear to be synchronous for South America and the Northern Hemisphere. Changes in solar activity, large volcanic eruptions and/or modulations of ocean circulation are potential triggers for this synchronicity.
Microalgae are aquatic, unicellular, eukaryotic organisms, which perform photosynthesis. They have gained interest within the last decades not only for biofuel production due to their high amount of lipids, but also for pharmaceutical and for nutraceutical purposes. Interesting compounds are proteins, carbohydrates, or pigments, such as carotenoids. However, microalgae possess strong and rigid cell walls, which hinder a sufficient and yet, gentle extraction of those valuable compounds. Although standard extraction techniques are available, several shortcomings occur, e.g. high energy demand, use of environmentally harmful solvents or alteration of compounds due to heat or chemicals. Therefore, an alternative method is needed, which is able to address these disadvantages. Physical plasmas were thus studied to answer the question whether they are able to disintegrate the cell walls of microalgae effectively and yet, without degradation of the extractives.
First step of the thesis was to find a suitable plasma source that has an effect on the cell walls because plasma effects, such as electric fields, shockwaves, UV light emission, and the generation of reactive species can be tailored with the respective setup. It was found that spark discharges are most effective for the extraction of Chlorella vulgaris, which was chosen as model organism. All extraction yields were compared to reference methods, whereat microwave radiation was found to be the most effective reference method and were hence, applied for comparative studies.
For the next step, proteins were selected as targets to answer the question, which differences can be determined between plasms-treated and microwave-radiated proteins are observable although the extraction yields were equal. Furthermore, plasma effects, especially the effects of reactive species on the extracted proteins had to be studied. Findings indicate that heat sensitive proteins, such as photosystem-related proteins, or histones are better extractable with spark discharges than with microwave exposure and the effect of reactive species is only minor.
The last step was to determine, which plasma effect is responsible for the observed cell wall disintegration. Therefore, the tensile strength of Chlorella vulgaris was determined and compared to the shockwave pressure, which is generated from the spark channel. It was proven that the shockwave pressure exceeds by far the tensile strength of the microalgae an can be thus held responsible for mechanism for cell wall rupture.
In this thesis, it was found that spark discharges are a promising alternative for the extraction of valuable compounds from microalgae. The discharges are not only effective, but also gentle enough for sensitive compounds, such as proteins or pigments.
Staphylococcus aureus has acquired resistance to antibiotics since their first use. The S. aureus protein NorA, an efflux pump belonging to the major facilitator superfamily (MFS), contributes to resistance to fluoroquinolones (e.g., ciprofloxacin), biocides, dyes, quaternary ammonium compounds, and antiseptics. Different compounds have been identified as potential efflux pump inhibitors (EPIs) of NorA that result in increased intracellular concentration of antibiotics, restoring their antibacterial activity and cell susceptibility. However, none of the currently known EPIs have been approved for clinical use, probably due to their toxicity profiles. In the present study, we screened approved drugs for possible efflux pump inhibition. By screening a compound library of approximately 1200 different drugs, we identified nilotinib, a tyrosine kinase inhibitor, as showing the best efflux pump inhibitory activity, with a fractional inhibitory concentration index of 0.1875, indicating synergism with ciprofloxacin, and a minimum effective concentration as low as 0.195 μM. Moreover, at 0.39 μM, nilotinib, in combination with 8 μg/mL of ciprofloxacin, led to a significant reduction in biofilm formation and preformed mature biofilms. This is the first description of an approved drug that can be used as an efflux pump inhibitor and to reduce biofilms formation at clinically achievable concentrations.
Bats (Chiroptera) form the second largest order of mammals and with over 1,250 species, they represent about 20% of all mammalian species worldwide. They are the only mammals with true and sustained flight and distributed all over the world except the arctic regions. Moreover, bats entered specific ecological niches and with their food spectra, they reduce different arthropod populations as well as disperse seeds and pollen of plant species in various regions and habitats.
Bats also have a crucial role in spreading high-pathogenic and zoonotic viruses, harbor in general more viruses (zoonotic and non-zoonotic), and, related to the species, number even more than rodents. However, clinical symptoms of viral diseases are rarely reported in bat communities. Also seroconversions after infection were not reported for a variety of viruses found in bats. Since the incidence of virus-positive bats estimated in passive surveillance studies is usually very low, it is a question how such viruses can use bats as reservoir hosts. There is obviously a special evolutionary relationship between the pathogens and bats as hosts, which are based on possibly physiologic adaptations also in resistance and immunity.
In this thesis, the two lyssaviruses, European Bat Lyssavirus 1 and 2 (EBLV-1 and -2) were chosen as a model to investigate the immune response of European bats against viral infection in vitro. Lyssaviruses are the causative agents of rabies, a fatal zoonotic disease with neurotropic characteristics.
One main question to investigate was in which way bats act as reservoir host and developed a high disease resistance. The present thesis is based on three hypotheses about innate immune response against lyssavirus infection:
A) In bats specific peripheral resistance mechanisms evolved which reduce the risk of systemic viral infection after a hypothesized airborne transmission and infection via nasal epithelium supported by the social structure of and communication within bat communities.
B) The co-evolution of EBLV and the innate resistance of bats resulted in a very effective type I interferon response to inhibit a systemic lyssavirus infection.
C) The specific physiology of body temperature of bats with daily torpor depresses the viral replication but favours the type I interferon response.
To analyze the interferon-based resistance mechanisms, the type I interferon (IFN) genes of two European bats species (Eptesicus serotinus and Myotis myotis) were cloned and sequenced. Using established cell lines from the respiratory nasal epithelium (MmNep), olfactory nasal epithelium (MmNol), and Bulbus olfactorius brain (MmBr), the type I IFN response along a possible airborne infection route was investigated. The anti-viral effects and induction of IFNs/interferon stimulated genes (ISGs) in each cell line were also investigated in detail after infection in vitro. Finally, the influence of different temperatures on lyssavirus replication was analyzed in cell culture experiments.
The results indicated that (a) along the hypothesized airborne infection route the susceptibility for lyssavirus infections is decreased, (b) the type I IFN activity in contrast is increased contributing to a limitation of lyssavirus replication and (c) an obvious influences of varying cultivation temperatures on the resistance against lyssavirus infections, which favor the IFN response and repressing lyssavirus replication.
The result from these in vitro studies supports the hypothesis of a special co-evolution between lyssaviruses and bats. However, in vivo studies on the relevance in infected animals are missing so far. This model could also explain the generally limited pathogenicity of bat-associated viruses.
Global climate change is occurring all over the world, but in the Arctic the climate is changing more rapidly and drastically than in many other parts of our planet. Many species that are already at their climatic limit need to adapt to recent climate conditions or migrate in order to not go extinct. The possibilities of adaption include phenotypic plasticity and adaptation to various extents. This is also the case for white spruce P. glauca, which belongs to the conifers and thus in the largest group of gymnosperms still living today. Among the approx. 600 extant conifer species white spruce is one of the most widespread trees in North American boreal forests. Its range extends from 69° N in the Canadian Northwest Territories to the Great Lakes at about 44° N, where it occurs from sea level to an altitude of about 1520 m (Burns and Honkala, 1990). Site related, climate-dependent differences in white spruce reproduction can be seen as a strategy to survive under the harsh climatic conditions at Alaska's treelines: Besides sexual reproduction, the vegetative propagation occurs in the white spruce as an additional reproductive mechanism. This can be realized by "layering" when the lower branches of the tree crown touch the ground and develop roots to later grow as a separate individual with or without a connection to the mother tree. Known as other mechanisms of vegetative propagation are also the rooting of fallen trees which were not completely uprooted, and the "root suckering", in which new shoots sprout from the roots of the tree. However, the latter was not yet observed in the genus Picea. With the help of short, repetitive, non-coding sequences in the genome, which are therefore not subject to selection and are called microsatellites, these clones can be determined by genotyping.
For this purpose, using different polymorphic microsatellites, an individual multilocus genotype is created for each tree, by means of which it can be compared with all other trees of the same species.
In the first part of this work (article I), the occurrence of clones in three study areas at Alaskan treelines are examined and the reasons for their appearance in variable numbers are discussed. For this purpose, 2571 white spruces (P. glauca) were genotyped and their position was determined via differential GPS in the field. The percentage of clonal trees is higher in areas with harsh climatic conditions and correlates with the height of the lowest branches of the tree crown. This suggests that the vegetative propagation of white spruce is a backup strategy for times when climatic conditions hamper sexual reproduction. The correlation between clone numbers and tree crown height suggests "layering" as the main mechanism for cloning whereas selection for vegetative reproduction seems to be very unlikely shown by the results for genetic differentiation between the clonal and the singleton trees in this study.
In the second part of this work (articles II and III), the influence of environmental factors and phenotypic traits on the mycobiome of the needles (including all fungi living on (epiphytic) and in (endophytic) the needles) in our study areas in Alaska was investigated. The mycobiome of the white spruce needles was chosen as a proxy for the parasite infection rate by fungi and thus serves as a fitness parameter. For this purpose, all epiphytic and endophytic fungal species were analyzed by a metabarcoding analysis.
In article II, 48 trees of one study area at Alaska’s northern treeline (Brooks Range) were examined for differences in mycobiome due to genetic differentiation, phenotypic characteristics and / or habitat characteristics. The trees used for this study were sampled from two adjacent plots on a south-facing mountain slope with an elevation gradient from 875 to 950 meters above sea level. It could be shown that, in contrast to the trees genotype, the height above sea level, the mountain slope, as well as the height and age of the trees have a significant impact on the mycobiome. The genetic differentiation between the tree individuals, however, showed no significant effect.
Based on article II we examined the mycobiome composition of a total of 96 trees in 2 plots (16 trees each) at three sites in Alaska over a distance of 500 kilometers. Additionally, we sampled needles of two different ages for each tree (current year and three years old needles) summing up to 192 samples in total. The incentive of this study (article III) was to investigate the influence of origin and age of spruce needles on their mycobiome and if there is a genetic predisposition that is related to the fungal species community. In addition, the sampling design was improved by collecting needles from all four orientations (North, South, East and West) and sampling trees at a standardized distance to each other to avoid systematic errors. Comparable to article II the influence of the trees genetics on the species community of the epiphytic and endophytic fungi of the white spruce needles seems to be very unlikely. In contrast, a significant influence of the geographic origin and the needle age on the species structure of the needle inhabiting fungal species was found. The phenotypic tree traits height and dbh (diameter at breast height) had only minor influence and did in fact explain less than 2% of the mycobiome variance. Using Illumina sequencing, 10.2 million reads from the nucleotide sequence between the internal transcribed spacer (ITS) genes could be obtained, which yielded in 1575 ribotypes (called operational taxonomic unit, OTU) for the fungi. These were compared with a reference database to compare and assign them to known fungal species. For example, 942 OTUs with >95% similarity could be identified as known species, with 1975 samples identified on genus level and 2683 when determined to family level. The most pronounced difference between the two studies (article II and III) were due to the fungal species of the class of Pucciniomycetes, more specifically the genus Chrysomyxa which belongs to the rust fungi and is plant pathogenic. In the study of article II (sampling in 2012), Pucciniomycetes accounted for only a minor portion of the assigned DNA sequences. In the second study (article III, sampling in 2015) they accounted for more than half of all basidiomycetes found, which in turn contain 20.0% of all DNA sequences, the second largest phylum found beside Ascomycetes (51.4%).
Die linksventrikuläre Herzhypertrophie ist ein zentraler Vorhersageparameter für das Auftreten kardiovaskulärer Ereignisse. Durch eine permanent gesteigerte Belastung des Herzens kommt es dabei auf zellulärer Ebene zu Umbauprozessen, die mit einer Hypertrophie der einzelnen Kardiomyozyten einhergehen. An der prohypertrophen Signaltransduktion sind zahlreiche Signalwege beteiligt, deren jeweilige Bedeutung und Interaktion untereinander noch nicht ausreichend untersucht ist. Seit einiger Zeit ist bekannt, dass auch das Wnt-Signalling im adulten Herzen bei Schädigung reaktiviert werden kann. Auch wenn die Beteiligung vieler Mediatoren des Wnt-Signallings für die Hypertrophieentstehung bereits gezeigt werden konnte, ist es aufgrund der Interaktionen der verschiedenen Signalwege notwendig, den Effekt der Wnt-Proteine auf die Kardiomyozyten direkt nachzuweisen. Da sowohl eigene Voruntersuchungen als auch viele In-vivo-Studien an Mäusen des Stammes C57/BL6 durchgeführt wurden, sollten in dieser Arbeit die Wirkungen verschiedener Wnt-Proteine hinsichtlich Hypertrophie und Zellsignalling auf isolierte murine neonatale Kardiomyozyten untersucht werden. Dazu wurden zunächst der Isolationsprozess dieser Primärzellen etabliert und optimale Kultur- und Stimulationsprotokolle bestimmt. Anschließend wurde das Ausmaß der zellulären Hypertrophie immunfluoreszenzmikroskopisch mittels myozytenspezifischer Anfärbung von α-Actinin untersucht. Es konnte gezeigt werden, dass eine Isolation und Kultivierung der neonatalen Kardiomyozyten im Gewebeverband über eine längere Versuchsdauer von bis zu sieben Tagen möglich ist. Die Zellen waren reproduzierbar vital und wiesen stabil kontraktile Eigenschaften auf. Zusätzlich wurde nachgewiesen, dass diese Zellen bei Stimulation mit dem Wnt-Protein Wnt3A im Mangelmedium Panserin mit einer konzentrationsabhängigen Hypertrophie antworten, die dem bekannten prohypertrophen Stimulus Endothelin-1 sogar überlegen ist.
Ein zweiter Teil der Arbeit beschäftigte sich mit spezifischen Signalwegen, die von verschiedenen Wnt-Proteinen angesprochen werden können. Dabei wurden sowohl Veränderungen von Kinaseaktivitäten wie der AKT und der GSK-3β als auch Proteinakkumulationen wie die von β-Catenin untersucht. Auch Kerntranslokationen der Transkriptionsfaktoren β-Catenin und NFAT c1 – c4 wurden überprüft. Leider konnten an den neonatalen Mauskardiomyozyten keine Wnt-induzierten Veränderungen von Enzymaktivitäten bzw. veränderte Lokalisierungen einzelner Transkriptionsfaktoren nachgewiesen werden. Einzig ein detektierbarer Anstieg der intrazellulären Kalziumkonzentrationen, der aber gegenüber unstimulierten Zellen keine Signifikanz erreichte, könnte auf eine mögliche Beteiligung des Wnt/Ca2+-Weges hindeuten.
Menschen mit Demenz (MmD) sind oft multimorbid und haben komplexe Pharmakotherapie, die mit einer zunehmenden Anzahl von arzneimittelbezogenen Problemen (ABP) einhergeht. Dies kann zu einer Verringerung der Lebensqualität und zu erhöhten Krankenhausaufenthaltsraten für MmD sowie zu hohen Kosten für das Gesundheitssystem führen. Daher ist es wichtig, die Prävention von ABP bei der Planung von Versorgungsprogrammen für MmD zu berücksichtigen. Frühere Studien zur Arzneimitteltherapiesicherheit konzentrierten sich hauptsächlich auf institutionalisierte MmD. Über die Situation in der ambulanter Versorgung ist wenig bekannt. Das Ziel dieser Arbeit ist es, Folgendes zu bestimmen: (a) Häufigkeit und Art der Behandlung mit Antidementiva und damit assoziierte soziodemografische und klinische Prädiktoren (b) Prävalenz von potenziell inadäquater Medikation (PIM) gemäß der Priscus-Liste und damit verbundene soziodemographische und klinische Prädiktoren (c) Häufigkeit und Art der ABP und damit verbundene soziodemografische und klinische Prädiktoren bei MmD in primärärztlicher Versorgung, die positiv auf Demenz gescreent wurden. Die Analysen basieren auf den Daten der DelpHi-MV-Studie, einer clusterrandomisierten, kontrollierten Interventionsstudie zur Evaluation eines innovativen Konzepts des Dementia Care Managements (DCM) in Deutschland. Die Daten von N = 448 Studienteilnehmern (≥70 Jahre, zu Hause lebend), die positiv auf Demenz (DemTect <9) getestet wurden, wurden in die Analyse einbezogen. Für jeden Studienteilnehmer wurde eine Medikationsanalyse auf der Grundlage der häuslichen Medikationsanamnese durchgeführt. Die Ergebnisse zeigten, dass 30% der MmD Antidementiva erhielten, von denen 63% hatten bereits eine formale Demenzdiagnose. In der Untergruppe der Studienteilnehmer mit einer formalen Demenzdiagnose erhielten 46% die Antidementiva. Die Verschreibung von Antidementiva war signifikant mit dem Schweregrad der kognitiven Beeinträchtigung und einer formalen Demenzdiagnose assoziiert. 38% der Studienteilnehmer ohne Demenzdiagnose erhalten Antidementiva. 22% der Studienteilnehmer erhielten mindestens ein PIM gemäß der Priscus-Liste. Die kognitive Beeinträchtigung war nicht mit der Verwendung von PIM assoziiert. Die Verschreibung eines PIM war signifikant mit der Gesamtzahl der eingenommenen Medikamente sowie mit der Diagnose von psychischen und Verhaltenssymptomen assoziiert. Polypharmazie, definiert als die dauerhafte Anwendung von fünf oder mehr Medikamenten, wurde bei 67% der Studienteilnehmer identifiziert, 93% hatten mindestens ein ABP, das von einem Apotheker oder einer Krankenschwester während eines Hausbesuchs entdeckt wurde. Insgesamt wurden 1077 ABP registriert. Probleme im Zusammenhang mit Anwendung und Compliance waren die häufigste Gruppe von ABP, gefolgt von Problemen mit Arzneimittelwechselwirkungen, Problemen mit unangemessener Arzneimittelwahl, Problemen mit der Dosierung und Problemen mit unerwünschten Arzneimittelereignissen. Die kognitive Beeinträchtigung war nicht mit der Gesamtzahl der ABP assoziiert. Die Gesamtzahl der eingenommenen Medikamente und das Vorhandensein einer Diagnose von psychischen und Verhaltenssymptomen waren mit der Gesamtzahl der ABP assoziiert.
Die Dissertation gibt einen Überblick über die Gesamtmedikation und ABP von MmD in der Primärversorgung. Unter anderem wurden die Probleme der Behandlung mit Antidementiva und der Einnahme von PIM diskutiert. Die These legt nahe, dass das Medikationsanalyse auf der Grundlage der häuslichen Medikationsanamnese für MmD wichtig ist, da eine solche Überprüfung die Verfügbarkeit umfassender Informationen über die Gesamtmedikation, Ernährungsfaktoren und die tatsächliche Anwendung von Medikamenten gewährleistet. Die Dissertation bietet somit eine Basis für die Verbesserung der Arzneimitteltherapiesicherheit und die Entwicklung innovativer Versorgungskonzepte für MmD. Die hohe Prävalenz von ABP in dieser Population unterstreicht die Wichtigkeit der Einbeziehung eines Medikationsmanagements in die Versorgungsprogramme für MmD. Identifizierte Risikofaktoren für ABP können dazu beitragen, das Medikationsmanagement für die Patientengruppen anzubieten, die das höchste Risiko aufweisen.