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Für die Lösung der quantenmechanischen Bewegungsgleichungen, die komplexe, molekulare Systeme beschreiben, sind effiziente und verlässliche Näherungsverfahren erforderlich. Die Dichtefunktionaltheorie (DFT) stellt für die Behandlung der Elektronenwechselwirkung in vielen Fällen den besten Kompromiss zwischen Effizienz und Genauigkeit dar. Im Rahmen der DFT wird die gesamte nicht-klassische Elektron-Elektron-Wechselwirkung im so genannten Austausch-Korrelationsfunktional angenähert. Viele solcher Näherungen sind semi-empirischer Natur, andere wurden ausschließlich von physikalischen Überlegungen abgeleitet. In globalen Hybridfunktionale wird ein konstanter Anteil der integrierten DFT-Austauschenergiedichte durch exakten Austausch aus der Hartree-Fock Näherung ersetzt. Das populärste Funktional B3LYP enthält 20 % exakten Austausch und mehrere empirische Parameter. Der optimale Prozentsatz hängt allerdings sehr stark von den zu berechnenden Systemen und molekularen Eigenschaften ab. Eine Lösung dieses Problems sollten lokale Hybridfunktionale liefern, in denen die Beimischung der exakten Austauschenergiedichte über eine lokale Mischfunktion (LMF) gesteuert wird und daher positions- und molekülabhängig ist. In dieser Arbeit wird ein semi-empirischer Ansatz für die Entwicklung neuer lokaler Hybridfunktionale verfolgt: während die Energiedichten unverändert aus etablierten Näherungen zum Austauschkorrelationsfunktional übernommen werden, stehen parametrisierte LMFs im Zentrum der Untersuchungen. Die verschiedenen LMFs beinhalten neben mindestens einem empirischen Parameter eine Variable die vom Quotienten der von-Weizsäcker kinetischen Energiedichte und der korrelierten kinetischen Energiedichte (sogenannte t-LMFs) bzw. dem reduzierten Dichtegradienten (bezeichnet als t-LMFs) abhängt. Weitere LMFs werden durch zusätzliche Berücksichtigung der Spinpolarisation erhalten. Alle Parameter werden an Atomisierungsenergien bzw. Reaktionsbarrieren bekannter molekularer Testsätze gefittet. Durch Visualisierung der LMFs können zusätzlich Einblicke in den physikalischen Hintergrund und in Möglichkeiten der Weiterentwicklung gewonnen werden. Es wurde beispielsweise beobachtet, dass entlang einer gedehnten Bindung höhere Werte der LMF und damit größere Beimischungen exakter Austauschenergie in Übergangszuständen einhergehen. Dieser Effekt ist für t-LMFs am ausgeprägtesten und korreliert mit besseren Ergebnissen für Reaktionsbarrieren mit lokalen Hybridfunktionalen, die auf einer t-LMF basieren. Bis auf wenige Ausnahmen leiten sich die lokalen Hybridfunktionale in dieser Arbeit aus dem Austausch- und Korrelationsfunktional der lokalen Dichtenäherung (LSDA) ab und enthalten keine Gradientenkorrektur im Sinne der GGA (generalized gradient approximation). Die neuen Funktionale wurden zunächst nicht-selbstkonsistent in eine Entwicklerversion des quantenchemischen Programmpaketes Turbomole implementiert. Das bedeutet, für gegebene Molekülorbitale bzw. eine gegeben Elektronendichte kann lediglich die Gesamtenergie berechnet werden. Dies ist eine anerkannte Näherung, die vor allem für die Optimierung der Parameter eine große Zeitersparnis darstellt. Um letztlich orbitalabhängige, molekulare Eigenschaften berechnen zu können wird neben der Gesamtenergie auch noch das zugehörige lokale Hybridpotential benötigt. Für die selbstkonsistente Implementierung wird die funktionale Ableitung der Austauschkorrelationsenergie nach den Orbitalen bestimmt. Daraus resultierend müssen neben den üblichen lokalen Austauschkorrelationspotentialtermen auch Integrale berechnet werden, die das mit der LMF gewichtete nicht-lokale exakte Austauschpotential enthalten. Die entsprechenden Terme kann man, genauso wie die exakte Austauschenergiedichte an sich, nicht analytisch berechnen. Früheren Ansätzen folgend wurden sie in der vorliegenden Arbeit in einer Basissatzentwicklung angenähert, wobei der Einfachheit halber die atomaren Basisfunktionen verwendet wurden. Um die Genauigkeit dieser sogenannten RI (resolution of the identity)-Näherung validieren zu können und auch schon im Hinblick auf die Anpassung einer Hilfsbasis, wurde darüber hinaus die numerische Berechnung aller Integrale, die das exakte Austauschpotential und die entsprechende Energiedichte enthalten, implementiert. Unter Verwendung der RI-Näherung ist der Rechenaufwand lokaler Hybride vergleichbar mit dem globaler Hybridfunktionale: Während die formale Skalierung in Abhängigkeit der Systemgröße gleich ist, ergab sich ein etwas höherer Vorfaktor für die lokalen Hybride. Verschiedene Literaturbekannte Testsätze mit Atomisierungsenergien, Reaktionsbarrieren, Dissoziationsenergien oder Gleichgewichtsabständen, die teilweise einige Schwächen bisheriger Dichtefunktionalnäherungen aufdecken, wurden berücksichtigt. Für die 223 Atomisierungsenergien des G3 Testsatzes stellen alle unsere Funktionale eine signifikante Verbesserung gegenüber B3LYP dar. Atomisierungsenergien sind insofern ein sensibler Test, da alle Bindungen gebrochen werden und Fehlerkompensation eine untergeordnete Rolle spielt. Vor allem lokale Hybridfunktionale, deren LMFs neben der kinetischen Energiedichte explizit von der Spinpolarisation abhängen, lieferten hervorragende Resultate. Obwohl im Vergleich zu Atomisierungsenergien für die korrekte Berechnung von Reaktionsbarrieren im Allgemeinen mehr exakter Austausch benötigt wird, sind unsere Funktionale auch für zwei Testsätze mit jeweils 38 Reaktionsbarrieren besser als B3LYP. Zwar kann mit einem globalen Hybrid mit 50 % exaktem Austausch eine geringere Abweichung von den Richtwerten erzielt werden, aber ein solches Funktional ist für thermochemische Daten unzureichend. Hier wurde erstmals gezeigt, dass lokale Hybridfunktionale ohne Gradientenkorrektur sowohl für Thermochemie als auch für Kinetik zufrieden stellende Ergebnisse liefern können. Das Dissoziationsverhalten symmetrischer Radikalkationen stellt für die hier diskutierten Dichtefunktionale nach wie vor eine Herausforderung dar: Die Dissoziationsenergien von sieben Modellsystemen werden mit unseren Funktionalen stark überschätzt und Gleichgewichtsabstände unterschätzt. Insgesamt sind die Werte nur marginal besser als mit B3LYP. Neben Eigenschaften von Hauptgruppenverbindungen wurden zudem Übergangsmetalldimere und -monohydride untersucht. Für erstere ist eine gute Beschreibung dynamischer sowie statischer Elektronenkorrelation ausschlaggebend. In den Hydriden andererseits dominiert mit gängigen Dichtefunktionalen die unphysikalische Selbstwechselwirkung eines Elektrons mit sich selbst. Für die 3d-Übergangsmetalldimere sind die getesteten Funktionale genauso gut wie B3LYP und für die Hydride etwas besser. Atomare s-d Transferenergien von 3d Übergangsmetallen verbleiben auch für unsere lokalen Hybridfunktionale, die insgesamt schlechtere Ergebnisse erzielen als B3LYP, noch problematisch. Das hierfür geeignetste lokale Hybridfunktional basiert auf einer s-LMF und beinhaltet LYP Korrelation. Für die isotropen Hyperfeinkopplungskonstanten (HFCCs) kleiner Hauptgruppenverbindungen wurden zufriedenstellende Ergebnisse (ähnlich wie B3LYP) mit einem t-LMF basierten lokalen Hybrid erzielt. Die RI Näherung zum lokalen Hybridpotential wurde dem numerisch exakten Potential für die Berechnung von Gesamtenergien, isotrope HFCCs und Orbitalenergien für verschiedene Basissätze gegenübergestellt. Wie erwartet ist der Fehler für Gesamtenergien mit der RI-Näherungen vergleichsweise gering, vor allem relativ zu den verbleibenden Abweichungen von experimentellen Energien. Der Vergleich der mittleren absoluten Abweichung von experimentellen Werten für 26 isotrope HFCCs zeigt sogar für mittelgroße und kontrahierte IGLO Basissätze nur geringe Unterschiede zwischen dem RI-Potential und dem numerisch exakten lokalen Hybridpotential. Die Analyse der HFCCs einzelner Moleküle und der Orbitalenergien des CN Moleküls offenbart allerdings, dass Ungenauigkeiten aufgrund der RI-Näherung hier eine größere Rolle spielen, vor allem wenn zu kleine atomare Basissätze verwendet werden. Von den untersuchten lokalen Hybriden stellen sich einige als hervorragende Kandidaten für die Berechnung thermochemischer und kinetischer Eigenschaften heraus. Jeweils unterschiedliche Funktionale erzielen darüber hinaus mit den besten bekannten Funktionalen vergleichbare Ergebnisse für isotrope Hyperfeinkopplungskonstanten und ausgewählte Eigenschaften kleiner Übergangsmetallverbindungen. Die in dieser Arbeit präsentierten lokalen Hybridfunktionale stellen daher einen wichtigen Schritt in der Entwicklung universeller Näherungen zum Austauschkorrelationsfunktional dar. Zur akkuraten Beschreibung molekularer Eigenschaften von Übergangsmetallkomplexen und dem Dissoziationsverhalten von Radikal-Kation-Dimeren neben Thermochemie und Kinetik, werden in Zukunft wohl komplexere LMFs benötigt. Um konkurrenzfähige lokale Hybride mit gradientenkorrigierter Austausch- und Korrelationsenergiedichte zu entwickeln, müssen darüber hinaus weitere Studien zum Einfluss des abweichenden Eichursprungs der miteinander kombinierten Austauschenergiedichten durchgeführt werden. Eine andere Möglichkeit ist die Entwicklung speziell abgestimmter Korrelationsfunktionale für lokale Hybride. Außerdem sollte die Qualität der RI-Näherung zum lokalen Hybridpotential detaillierter untersucht werden. Hierfür könnten zum Beispiel Ionisierungsenergien und Elektronenaffinitäten herangezogen werden. Um zusätzliche Abweichungen oder sogar fälschlicherweise "zu gute" Ergebnisse bei Validierungsrechnungen zu vermeiden, sollten Hilfsbasen für die Entwicklung des nicht-lokalen exakten Austauschpotentials implementiert und optimiert werden. Einer der nächsten Implementierungsschritte sollte auch Gradienten bezüglich der Kernkoordinaten beinhalten, um die Validierung der neuen lokalen Hybridfunktionale auf Strukturoptimierungen auszuweiten.
In this work, we take a look at the connection of gamma-ray bursts (GRBs) and ultra-high-energy cosmic rays (UHECR) as well as the possibilities how to verify this connection. The currently most promising approach is based on the detection of high-energy neutrinos, which are associated with the acceleration of cosmic rays. We detail how the prompt gamma-ray emission is connected to the prediction of a neutrino signal. We focus on the interactions of photons and protons in this regard. At the example of the current ANTARES GRB neutrino analysis, we show the differences between numerical predictions and older analytical methods. Moreover, we discuss the possibilities how cosmic ray particles can escape from GRBs, assuming that UHECR are entirely made up of protons. For this, we compare the commonly assumed neutron escape model with a new component of direct proton escape. Additionally, we will show that the different components, which contribute to the cosmic ray flux, strongly depend on the burst parameters, and test the applicability on some chosen GRBs. In a further step, we continue with the considerations regarding the connection of GRBs and UHECR by connecting the GRB source model with the cosmic
ray observations using a simple cosmic ray propagation code. We test if it is possible to achieve the observed cosmic ray energy densities with our simple model and what the consequences are regarding the prompt GRB neutrino flux predictions as well as the cosmogenic neutrinos. Furthermore, we consider the question of neutrino lifetime and how it affects the prompt GRB neutrino flux predictions. In a final chapter, we show that it is possible to apply the basic source model with photohadronic interactions to other types of sources, using the example of the microquasar Cygnus X-3.
Parkinson’s disease (PD) is the second most common neurodegenerative disease with still no cure available. The prominent feature of PD is the loss of dopaminergic neurons at the Substantia nigra (SN). Genetic and environmental insults affecting the SNCA gene encoding the alpha-Synuclein (alpha-Syn) protein result into an aberrant form of the protein with higher propensity towards oligomerization becoming part of insoluble inclusions called Lewy Bodies (LB). LB impart cytotoxicity leading to neurodegeneration, activate resident microglia and escape to the periphery where they get captured by dendritic cells and presented to naïve T cells. Proliferating effector T lymphocytes invade the brain releasing proinflammatory cytokines and performing a cytotoxic effect on neurons.
In this study, we examine the hypothesis that the expansion of regulatory T cells (Treg) could exert an anti-inflammatory effect that averts neurodegeneration in the AAV1/2-A53T-alpha-Syn mouse model for PD.
Mice brains were transfected by a unilateral stereotaxic injection at the SN region with a chimeric Adeno-Associated Viral vector of serotypes 1 and 2 (AAV1/2) carrying the A53T-mutated human SNCA gene encoding the readily aggregating aberrant alpha-Syn (AAV1/2-A53T-alpha-Syn). One week after injection, mice were treated with the CD28 superagonistic antibody (CD28SA), known to significantly expand the Treg population. Mice were then analyzed by behavioral analysis using the Rotarod performance test and the Cylinder test. The impact of CD28SA on the immune system was examined by flow cytometry. The integrity of the nigrostriatal system was assessed by stereological quantification of Tyrosine hydroxylase (TH)-stained dopaminergic neurons in SN and optical density measurements of TH-stained striatum. The mechanism of action of CD28SA was analyzed by treating PD mice alternatively with a Treg adoptive transfer, while CD28SA effect on levels of neurotrophic factors was quantified by ELISA.
We observed an expansion of Treg by FACS analyses three days after CD28SA treatment, demonstrating target engagement. CD28SA treatment of AAV1/2-A53T-alpha-Syn mice provided neuroprotection evident through elevated numbers of dopaminergic neurons in the SN and higher optical density of TH-staining in the striatum, in CD28SA-treated mice compared to PBS-treated control mice, and that was reflected in an enhanced performance in behavioral studies. Additionally, brain infiltration of proinflammatory activated T lymphocytes (CD4+CD69+ and CD8+CD69+ cells), that were obvious in PBS-treated AAV1/2-A53T-alpha-Syn control mice, was augmented in PD mice receiving CD28SA. The alternative treatment with Treg adoptive transfer did replicate the beneficial effects of CD28SA indicating that Treg expansion is the main effector mechanism by which it exerts its neuroprotective effect. CD28SA treatment of PD mice led to an increase of GDNF and BDNF in some brain structures that was not observed in untreated mice.
We conclude that in the AAV1/2-A53T-alpha-Syn PD mouse model, CD28SA suppresses proinflammation, reverses behavioral deficits and is neuroprotective on SN dopaminergic cells.
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.
Clostridial neurotoxins (botulinum toxins and tetanus toxin) disrupt neurotransmitter release by cleaving neuronal SNARE proteins. We generated transgenic flies allowing for conditional expression of different botulinum toxins and evaluated their potential as tools for the analysis of synaptic and neuronal network function in Drosophila melanogaster by applying biochemical assays and behavioral analysis. On the biochemical level, cleavage assays in cultured Drosophila S2 cells were performed and the cleavage efficiency was assessed via western blot analysis. We found that each botulinum toxin cleaves its Drosophila SNARE substrate but with variable efficiency. To investigate the cleavage efficiency in vivo, we examined lethality, larval peristaltic movements and vision dependent motion behavior of adult Drosophila after tissue-specific conditional botulinum toxin expression. Our results show that botulinum toxin type B and botulinum toxin type C represent effective alternatives to established transgenic effectors, i.e. tetanus toxin, interfering with neuronal and non-neuronal cell function in Drosophila and constitute valuable tools for the analysis of synaptic and network function.
Magnetic resonance imaging is derogated by the presence of metal implants and image quality is impaired. Artifacts are categorized according to their sources, the differences in susceptibility between metal and tissue and the modulation of the magnetic radiofrequency (RF) transmit field. Generally, these artifacts are intensified at higher field strength. The purpose of this work is to analyze the efficiency of current methods used for metal artifact reduction at 3T and to investigate improvements. The impact of high-bandwidth RF pulses on susceptibility-induced artifacts is tested. In addition, the benefit of a two-channel transmit system with respect to shading close to total hip replacements and other elongated metal structures in parallel to the magnetic field is analyzed.
Local transmit/receive coils feature a higher peak B1 amplitude than conventional body coils and thus enable high-bandwidth RF pulses. Susceptibility-induced through-plane distortion relates reciprocally to the RF bandwidth, which is evaluated in vitro for a total knee arthroplasty. Clinically relevant sequences (TSE and SEMAC) with conventional and high RF pulse bandwidths and different contrasts are tested on eight patients with different types of knee implants. Distortion is rated by two radiologists. An additional analysis assesses the capability of a local spine transmit coil. Furthermore, B1 effects close to elongated metal structures are described by an analytical model comprising a water cylinder and a metal rod, which is verified numerically and experimentally. The dependence of the optimal polarization of the transmit B1 field, creating minimum shading, on the position of the metal is analyzed. In addition, the optimal polarization is determined for two patients; its benefit compared to circular polarization is assessed.
Phantom experiments confirm the relation of the RF bandwidth and the through-plane distortion, which can be reduced by up to 79% by exploitation of a commercial local transmit/receive knee coil at 3T. On average, artifacts are rated “hardly visible” for patients with joint arthroplasties, when high-bandwidth RF pulses and SEMAC are used, and for patients with titanium fixtures, when high-bandwidth RF pulses are used in combination with TSE. The benefits of the local spine transmit coil are less compared to the knee coil, but enable a bandwidth 3.9 times as high as the body coil. The modulation of B1 due to metal is approximated well by the model presented and the position of the metal has strong influence on this effect. The optimal polarization can mitigate shading substantially.
In conclusion, through-plane distortion and related artifacts can be reduced significantly by the application of high-bandwidth RF pulses by local transmit coils at 3T. Parallel transmission offers an option to substantially reduce shading close to long metal structures aligned with the magnetic field. Effective techniques dedicated for metal implant imaging at 3T are introduced in this work.
The use of human adipose-derived mesenchymal stem cells (ASCs) for cell-based therapeutic approaches, in terms of repair and regeneration of various tissues and organs, offers an alternative therapeutic tool in the field of regenerative medicine. The ability of ASCs to differentiate along mesenchymal lineages is not the only property that makes these cells particularly attractive for therapeutic purposes. Their promising functions in promoting angiogenesis, reducing inflammation as well as in functional tissue restoration are largely related to the trophic effects of a broad panel of secreted cytokines and growth factors. However, in cell-based approaches, the cell-loaded construct often is exposed to an ischemic microenvironment characterized by severe oxidative and nutritional stress after transplantation due to the initial lack of vascular connection, resulting in reduced cell viability and altered cell behaviour. Therefore, the effective use of ASCs in regenerative medicine first requires a comprehensive characterization of the cells in terms of their viability, differentiation capacity and especially their secretory capabilities under ischemia-mimicking conditions in order to better understand their beneficial role. Accordingly, in the first part of this work, ASCs were investigated under different ischemic conditions, in which cells were exposed to both glucose and oxygen deprivation, with respect to viability and secretory function. Using mRNA gene expression analysis, significantly higher expression of selected angiogenic, anti-apoptotic and immunomodulatory factors (IL-6, VEGF, STC-1) could be demonstrated under harsh ischemic conditions. These results were reflected at the protein expression level by a significantly increased secretion of these factors. For stanniocalcin-1 (STC-1), a factor not yet described in ASCs, a particularly high expression with significant secreted amounts of the protein could be demonstrated under harsh ischemic conditions. Thus, the first part of this work, in addition to the characterization of the viability, provided first insights into the secretory response of ASCs under ischemic conditions.
The response of ASCs to glucose deficiency in combination with severe hypoxia has been little explored to date. Thus, the focus of the second part of this work was on a more detailed investigation of the secretory response of ASCs under glucose and oxygen deprivation. For a more comprehensive analysis of the secretion profile, a cytokine antibody array was performed, which allowed the detection of a broad panel of secreted angiogenic factors
(IL-8, ANG), matrix-regulating proteins (TIMP-1, TIMP-2), chemokines (MCP-1/CCL2,
IP-10/CXCL 10) and other factors under ischemic conditions. To verify these results, selected factors were examined using ELISA. The analysis revealed that the secretion of individual factors (e.g., STC-1, VEGF) was significantly upregulated by the combination of glucose and oxygen deprivation compared to oxygen deprivation alone.
In order to investigate the impact of the secretome of ischemic ASCs on cell types involved in tissue regeneration, the effect of conditioned medium of ischemia-challenged ASCs on both endothelial cells and fibroblasts was investigated in subsequent experiments. Significantly increased viability and tube formation of endothelial cells as well as activated migration of fibroblasts by the secreted factors of ischemic ASCs could be demonstrated. A direct correlation of these effects to STC-1, which was significantly upregulated under ischemic conditions and has been described as a regulator of key cellular functions, could not be verified.
The particular secretory capacity of ASCs provides a valuable tool for cell-based therapies, such as cell-assisted lipotransfer (CAL), where by enriching fat grafts with isolated ASCs, a significantly improved survival rate of the transplanted construct is achieved with less resorption of the fat tissue as well as a reduction in adverse implications, such as fibrosis and cyst formation. In order to better understand the function of ASCs in CAL, an autologous transwell-based lipograft-ASC co-culture was established in the last part of this work, in which first investigations showed a markedly increased secretion of VEGF compared to lipografts without added ASCs. As the stability rate of the fat tissue and thus the success of CAL is presumably also dependent on the preparation of the tissue before transplantation, the conventional preparation method of fat tissue for vocal fold augmentation in laryngoplasty was additionally evaluated in vitro in a pilot experiment. By analyzing the viability and tissue structure of the clinically prepared injection material, a large number of dead cells and a clearly damaged tissue structure with necrotic areas could be demonstrated. In comparison, the preparation method of the fat tissue established in this work as small tissue fragments was able to provide a clearly intact, vital, and vascularized tissue structure. This type of adipose tissue preparation represents a promising alternative for clinical vocal fold augmentation.
In conclusion, the results of this work contribute to a comprehensive characterization of ASCs under ischemic conditions, such as those prevalent at the transplantation site or in tissue regeneration. The results obtained, especially on the secretory capacity of ASCs, provide new insights into how ASCs mediate regenerative effects in an ischemic milieu and why their use for therapeutic purposes is highly attractive and promising.
In the first part of his work, the causes for the sudden degradation of useable capacity of lithium-ion cells have been studied by means of complementary methods such as computed tomography, Post-Mortem studies and electrochemical analyses. The results obtained point unanimously to heterogeneous aging as a key-factor for the sudden degradation of cell capacity, which in turn is triggered by differences in local compression.
At high states of health, the capacity fade rate is moderate but some areas of the graphite electrode degrade faster than others. Still, the localized changes are hardly noticeable on cell level due to averaging effects. Lithium plating occurs first in unevenly compressed areas, creating patterns visible to the human eye. As lithium plating leads to rapid consumption of active lithium, a sudden drop in capacity is observed on cell level. Lithium plating appears to spread out from the initial areas over the whole graphite electrode, quickly consuming the remaining useful lithium and active graphite. It can be hypothesized that a self-amplifying circle of reciprocal acceleration of local lithium loss and material loss causes rapid local degradation.
Battery cell designers can improve cycle life by homogeneous pressure distribution in the cell and using negative active materials that are resilient to elevated discharge potentials such as improved carbons or lithium titanate. Also, a sufficiently oversized negative electrode and suitable electrolyte additives can help to avoid lithium plating. When packs are designed, care must be taken not to exert local pressure on parts of cells and to avoid both very high and low states of charge.
In the second part of this dissertation the resilience of cylindrical and pouchbag cells to shocks and different vibrations was investigated. Stresses inflicted by vibration and shock tests according to the widely recognized UN38.3 transport test were compared to a long-time test that exposed cells to a 186 days long ordeal of sine sweep vibrations with a profile based on real-world applications. All cells passed visual and electric inspection performed by TU München after the vibration tests. Only cylindrical cells subjected to long-term vibrations in axial direction showed an increase in impedance and a loss of capacity that could be recuperated in part.
The detailed analyses presented in this thesis gave more details on the damages inflicted by vibrations and shocks and revealed drastic damages in some cases. In cylindrical cells, only movement in axial direction caused damage. Long term vibrations were found to be especially detrimental.
No damage whatsoever could be detected for pouch cells, regardless of the test protocol and the direction of movement. The extreme resilience of pouchbag cells shows that the electrode stack of lithium-ion cells is resistant to vibrations, and that damages are caused by design imperfections that can be improved at low cost.
The findings of this work, and the general state of research show that it is most crucial to control the lithiation and thus potential of the graphite electrode.
In the last part of this work, a new, direct method for charge estimation based on changing transmission is presented. A correlation between transmission of short ultrasonic pulses and state of charge is found. This new technology allows direct measurement of the state of charge. The method is demonstrated for batteries with different positive active materials, showing its versatility. As the observed changes can be traced to the lithiation of graphite, it can be determined without a reference electrode. Already at this early stage of development, the found correlations allow estimation of state of charge. The present hysteresis in the signal height of the slow wave, which is unneglectable especially during discharging at higher currents, will be subject to further investigation.
The observed effects can be explained by effects on different length scales. Biot’s theory explains the second wave’s slowness based on the active material particles size in the range of 0.01 mm and electrolyte-filled pores. Lithiation of graphite changes the porosity of the electrode and thereby the velocity and wavelength of the impulse. When the wavelength approaches the length scale of the layers, 0.1 mm, scattering effects dampen the transmitted signal. Finally, the wavelength of the pulse should be shorter than the transducers diameter to obtain a homogeneous wave front.
To conclude, the new method allows the control of each individual cell in a pack independent from the electrical connections of the cells.
As the method shows great promise, further studies regarding factors such as long-term behavior, temperature and current rates should be conducted. In this thesis hysteresis was observed and a deeper understanding of the reasons behind it may allow further improvements of measurement precision.
In this work heterostructures based on the half-Heusler alloy NiMnSb have been fabricated and characterized. NiMnSb is a member of the half-metallic ferromagnets, which exhibit an electron spin-polarization of 100% at the Fermi-level. For fabrication of these structures InP substrates with surface orientations of (001),(111)A and (111)B have been used. The small lattice mismatch of NiMnSb to InP allows for pseudomorphic layers, the (111) orientation additionally makes the formation of a half-metallic interface possible. For the growth on InP(001), procedures for the substrate preparation, growth of the lattice matched (In,Ga)As buffer layer and of the NiMnSb layer have been developed. The effect of flux-ratios and substrate temperatures on the MBE growth of the buffer as well as of the NiMnSb layer have been investigated and the optimum conditions have been pointed out. NiMnSb grows in the layer-by-layer Frank-van der Merwe growth mode, which can be seen by the intensity oscillations of the RHEED specular spot during growth. RHEED and LEED measurements show a flat surface and a well-defined surface reconstruction. High resolution x-ray measurements support this statement, additionally they show a high crystalline quality. Measurements of the lateral and the vertical lattice constant of NiMnSb films on (001) oriented substrates show that layers above a thickness of 20nm exhibit a pseudomorphic as well as a relaxed part in the same layer. Whereas layers around 40nm show partly relaxed partitions, these partitions are totally relaxed for layers above 100nm. However, even these layers still have a pseudomorphic part. Depth-dependent x-ray diffraction experiments prove that the relaxed part of the samples is always on top of the pseudomorphic part. The formation and propagation of defects in these layers has been investigated by TEM. The defects nucleate early during growth and spread until they form a defect network at a thickness of about 40nm. These defects are not typical misfit dislocations but rather antiphase boundaries which evolve in the Mn/Sb sublattice of the NiMnSb system. Dependent on the thickness of the NiMnSb films different magnetic anisotropies can be found. For layers up to 15nm and above 25nm a clear uniaxial anisotropy can be determined, while the layers with thicknesses in between show a fourfold anisotropy. Notably the easy axis for the thin layers is perpendicular to the easy axis observed for the thick layers. Thin NiMnSb layers show a very good magnetic homogeneity, as can be seen by the very small FMR linewidth of 20Oe at 24GHz. However, the increase of the linewidth with increasing thickness shows that the extrinsic damping gets larger for thicker samples which is a clear indication for magnetic inhomogeneities introduced by crystalline defects. Also, the magnetic moment of thick NiMnSb is reduced compared to the theoretically expected value. If a antiferromagnetic material is deposited on top of the NiMnSb, a clear exchange biasing of the NiMnSb layer can be observed. In a further step the epitaxial layers of the semiconductor ZnTe have been grown on these NiMnSb layers, which enables the fabrication of NiMnSb/ZnTe/NiMnSb TMR structures. These heterostructures are single crystalline and exhibit a low surface and interface roughness as measured by x-ray reflectivity. Magnetic measurements of the hysteresis curves prove that both NiMnSb layers in these heterostructures can switch separately, which is a necessary requirement for TMR applications. If a NiMn antiferromagnet is deposited on top of this structure, the upper NiMnSb layer is exchange biased by the antiferromagnet, while the lower one is left unaffected. Furthermore the growth of NiMnSb on (111) oriented substrates has been investigated. For these experiments, InP substrates with a surface orientation of (111)A and (111)B were used, which were miscut by 1 to 2° from the exact orientation to allow for smoother surfaces during growth. Both the (In, Ga)As buffer as well as the NiMnSb layer show well defined surface reconstructions during growth. X-ray diffraction experiments prove the single crystalline structure of the samples. However, neither for the growth on (111)A nor on (111)B a perfectly smooth surface could be obtained during growth, which can be attributed to the formation of pyramid-like facets evolving as a result of the atomic configuration at the surface. A similar relaxation behavior as NiMnSb layers on (001) oriented InP could not be observed. RHEED and x-ray diffraction measurements show that above a thickness of about 10nm the NiMnSb layer begins to relax, but remnants of pseudomorphic parts could not be found. Magnetic measurements show that the misorientation of the substrate crystal has a strong influence on the magnetic anisotropies of NiMnSb(111) samples. In all cases a uniaxial anisotropy could be observed. The easy axis is always aligned parallel to the direction of the miscut of the substrate.
Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of infectious neurodegenerative diseases that are associated with misfolding of the cellular form of the cellular prion protein (PrPC) into a disease associated conformer (PrPSc). No therapy for prion diseases is available at present. So far, anti-PrPC vaccination is hampered by immunological tolerance of the mammalian immune system to endogenous PrPC. The aim of this thesis was to set up a new vaccination strategy based on virus-like particles (VLP) to induce anti-PrPC antibody responses in PrPC-competent mice. In a first step it was assessed whether VLP have the capacity to induce antibody responses that are protective against conventional pathogens. For this purpose, VLP displaying the vesicular stomatitis virus-gylcoprotein (VLP-VSV) were generated and tested for their immunogenicity. Similarly to live vesicular stomatitis virus (VSV), replication deficient VLP-VSV induced T help-independent VSV neutralizing IgM responses that switched to the IgG subclass in a T help-dependent manner. Furthermore, type I IFN receptor (IFNAR) triggering only marginally affected VLP-VSV induced neutralizing IgM responses, whereas it was critically required to promote the IgG switch. The analysis of conditional knockout mice with a lymphocyte-specific IFNAR deletion revealed that IFNAR triggering of lymphocytes did not play a crucial role, neither upon VLP-VSV nor VSV immunization. Collectively, these data verified the high immunogenicity of VLP. Therefore, in a next step VLP were generated displaying the C-terminal half of PrP (residues 121-231aa) fused to the platelet derived growth factor receptor (PDGFR) transmembrane region (VLP-PrPD111) for anti-PrPC immunization. On the surface of such retroparticles, PrPC was expressed at high levels as determined by electron microscopy. VLP-PrPD111 immunization of Prnp-deficient (Prnp0/0) mice resulted in antibody response specifically binding the cellular form of PrPC. Upon intravenous injection of wild-type mice, high PrPC-specific IgM responses were induced, whereas the T cell-dependent switch from the IgM to the IgG subclass was less pronounced. As a consequence, anti-PrPC titers were rather short-lived. The impaired subclass switch was probably related with host T cell tolerance to endogenous PrPC. Attempts to increase anti-PrPC IgG responses in wild-type mice via administration of VLP-PrPD111 emulsified in various different adjuvants failed. Nevertheless, in single individuals low IgG antibodies were induced after immunization of VLP-PrPD111 emulsified in CFA. To circumvent T cell tolerance in wild-type mice, a multitude of different immunization strategies was tested, including priming and boosting protocols with different types of VLP or VLP expressing PrPC together with foreign T helper epitopes. Overall, those efforts did not improve anti-PrPC IgG responses in wild-type mice. Interestingly, anti-PrPC antibodies induced in Prnp0/0 mice reduced PrPSc levels in prion infected cell cultures, whereas serum of vaccinated wild-type mice did not. To assess the protective capacity of VLP-PrPD111 induced immune responses, vaccinated wild-type mice were infected with scrapie (RML 5.0). Unfortunately, vaccinated mice did not show a significant delay in the onset of scrapie. In a last part of the thesis it was studied whether in the absence of T cell help activated “memory” B cells were able to produce anti-PrPC specific antibodies. To address this question, PrPC-specific memory B cells were sorted from vaccinated Prnp0/0 mice and adoptively transferred into wild-type recipient mice. Upon VLP-PrPD111 challenge, no PrPC-specific IgG titers were induced in the recipients. Nevertheless, several VLP-PrPD111 challenged recipient mice were protected against scrapie infection. In conclusion, VLP were characterized as highly immunogenic vaccines that were used to elucidate various questions concerning adaptive immune response and basic mechanisms of PrPC-specific tolerance vs. immunity. Remarkably, VLP-PrPD111 was able to induce native PrPC-specific antibodies in wild-type mice but major difficulties associated with PrPC-specific tolerance made efficacious scrapie vaccination impossible. New vaccination approaches are being tested to overcome these limitations.
The top quark plays an important role in current particle physics, from a theoretical point of view because of its uniquely large mass, but also experimentally because of the large number of top events recorded by the LHC experiments ATLAS and CMS, which makes it possible to directly measure the properties of this particle, for example its couplings to the other particles of the standard model (SM), with previously unknown precision. In this thesis, an effective field theory approach is employed to introduce a minimal and consistent parametrization of all anomalous top couplings to the SM gauge bosons and fermions which are compatible with the SM symmetries. In addition, several aspects and consequences of the underlying effective operator relations for these couplings are discussed. The resulting set of couplings has been implemented in the parton level Monte Carlo event generator WHIZARD in order to provide a tool for the quantitative assessment of the phenomenological implications at present and future colliders such as the LHC or a planned international linear collider. The phenomenological part of this thesis is focused on the charged current couplings of the top quark, namely anomalous contributions to the trilinear tbW coupling as well as quartic four-fermion contact interactions of the form tbff, both affecting single top production as well as top decays at the LHC. The study includes various aspects of inclusive cross section measurements as well as differential distributions of single tops produced in the t channel, bq → tq', and in the s channel, ud → tb. We discuss the parton level modelling of these processes as well as detector effects, and finally present the prospected LHC reach for setting limits on these couplings with 10 resp. 100 fb−1 of data recorded at √s = 14 TeV.
Remote sensing time series is the collection or acquisition of remote sensing data in a
fixed equally spaced time period over a particular area or for the whole world. Near
daily high spatial resolution data is very much needed for remote sensing applications
such as agriculture monitoring, phenology change detection, environmental
monitoring and so on. Remote sensing applications can produce better and accurate
results if they are provided with dense and accurate time series of data. The current
remote sensing satellite architecture is still not capable of providing near daily
or daily high spatial resolution images to fulfill the needs of the above mentioned
remote sensing applications. Limitations in sensors, high development, operational
costs of satellites and presence of clouds blocking the area of observation are some
of the reasons that makes near daily or daily high spatial resolution optical remote
sensing data highly challenging to achieve. With developments in the optical sensor
systems and well planned remote sensing satellite constellations, this condition
can be improved but it comes at a cost. Even then the issue will not be completely
resolved and thus the growing need for high temporal and high spatial resolution
data cannot be fulfilled entirely. Because the data collection process relies on satellites
which are physical system, these can fail unpredictably due to various reasons
and cause a complete loss of observation for a given period of time making a gap
in the time series. Moreover, to observe the long term trend in phenology change
due to rapidly changing environmental conditions, the remote sensing data from
the present is not just sufficient, the data from the past is also important. A better
alternative solution for this issue can be the generation of remote sensing time series
by fusing data from multiple remote sensing satellite which has different spatial and
temporal resolutions. This approach will be effective and efficient. In this method
a high temporal low spatial resolution image from a satellite such as Sentinel-2 can
be fused with a low temporal and high spatial resolution image from a satellite such
as the Sentinel-3 to generate a synthetic high temporal high spatial resolution data.
Remote sensing time series generation by data fusion methods can be applied to
the satellite images captured currently as well as the images captured by the satellites
in the past. This will provide the much needed high temporal and high spatial
resolution images for remote sensing applications. This approach with its simplistic
nature is cost effective and provides the researchers the means to generate the
data needed for their application on their own from the limited source of data available
to them. An efficient data fusion approach in combination with a well planned
satellite constellation can offer a solution which will ensure near daily time series of
remote sensing data with out any gap. The aim of this research work is to develop
an efficient data fusion approaches to achieve dense remote sensing time series.
The enhancement of electronic and optical properties of semiconductor nanostructures is known as a direct consequence of the spatial confinement of carriers. However, the physics of quantum confinement is still not entirely understood. This work focuses on a qualitative study of quasi-zero dimensional II-VI semiconductor nanostructures (quantum dots QDs). In particular, commercially available as-received and heat treated CdSxSe1-x QDs embedded in a dielectric matrix were investigated by means of linear and nonlinear spectroscopy techniques. Low wavenumber Raman in off-resonance scattering regime was applied in order to obtain key-properties of the nanocrystals, such as the QD's size and the distribution of the QD's size inside the inhomogeneous broadening. Moreover, by careful selection of the polarization geometries, different acoustic vibrational modes could be evidenced. In comparison to the bulk, 3D confinement of carriers leads to modifications in the energy distribution in a QD and as a consequence, the intensity of the acoustical phonons is enhanced. However, only 2 acoustic vibrational modes (labelled l=0 and l=2) are Raman-active, which were selectively excited using linear polarized laser light in parallel- and cross-polarized excitation geometries. The QD's size was determined using the dependence of the frequency of the acoustic vibrational mode on the diameter of the vibrating particle, whereas the QD's size distribution was estimated from the normalized full width at the half of the maximum (FWHM) of the symmetric acoustic vibrational mode. In order to study relaxation mechanisms, which in quantum confined systems occur on a ps time scale, ultrafast spectroscopy techniques using laser pulses in the fs range must be employed. To this purpose, fs-FWM and fs-PPT measurements were performed on CdS0.6Se0.4 QDs of 9.1 nm in diameter, embedded in a glass matrix. The laser pulses employed in these experiments were circularly polarized, careful selection of the polarization geometries making different nonlinear processes available to study. It was shown that the relaxation of polarization selection rules depend strongly on the symmetry of the nanocrystals under discussion. The investigated nanocrystals belong to the symmetry group C2v or lower and their hexagonal crystal shape could be evidenced. The relaxation of selection rules was explained in the framework of the 4-level system, including a ground state, two exciton states and a biexcitons state. The appearance of FWM and PPT signals in forbidden polarization geometries was shown to be due to exciton state splitting due to lowering of the QD’s symmetry and due to the strong Coulomb interaction between carriers belonging to the same nanocrystal. Moreover, the significant difference in the origin of the gratings created by two pulses having the same and opposite polarizations, respectively. The intensity of the FWM signals should be the square of the intensity of the PPT signals and therefore the PPT measurements were employed as a check method for the results yielded by the FWM technique. The efficiency of circularly polarized femtosecond FWM spectroscopy techniques was proved once more in the investigation of heat treated CdSe QDs embedded in a dielectric matrix. The role of non-phonon energy relaxation mechanisms in the exciton ground and excited state of the QDs ensemble was extensively studied. Moreover, the dependence of the crystal shape asymmetry on the particle size and on the growth conditions could be estimated. It was shown, that the most efficient procedure to grow high quality nanocrystals is a longer heat treating at lower temperatures. In this case, the particles have more time to "nucleate" and to adopt a more "symmetric" shape. Further, the relaxation of excitons was extensively investigated. It was shown, that the electron intraband dynamics depend strongly on the Coulomb interaction between electrons and holes. Even at low excitation density, the Auger processes cannot be ignored. Auger autoionization of excitons followed by capture of carriers in surface states and deep traps in the dielectric matrix slow down the exciton relaxation process leading to an exciton lifetime ranging on a ps time scale. The relaxation of excitons from higher lying energy levels occurs also on two paths. At the beginning of the relaxation process (t31 < 400 fs), Auger-like thermalization of carriers is responsible for relaxation of the electron from 1pe into its 1se state, while the hole relaxes rapidly through its dense spectrum of states in the valence band. This process is immediately followed by capturing of carriers in deep traps, situated at the semiconductor-dielectric heterointerface. The traps are a consequence of the QD's asymmetry: the more and the deeper the traps, the higher the asymmetry of the nanocrystals (the band offset  is larger). This work presents a complete characterization of CdSSe QDs embedded in a glass matrix. The most important properties of the nanocrystals like QD's size and size distribution inside the inhomogeneous broadening were determined by means of low wavenumber Raman spectroscopy. In order to draw a full picture of these nanoparticles further complementary nonlinear spectroscopy techniques were used. Invaluable conclusions were available as a result of TI-FWM techniques applied in the framework of transient grating on 3D confined nanocrystals embedded in a glass matrix. The polarized the TI-FWM measurements were successfully performed on different QDs ensembles in order to determine symmetry properties and to describe the ultrafast relaxation mechanisms. This work brings additional contribution concerning the preparation of high quality QDs by presenting the effect of different growth conditions on the QDs symmetry, thus indicating a way for efficient manufacturing of nanocrystals.
Environmental interlinked problems such as human-induced land cover change, water scarcity, loss in soil fertility, and anthropogenic climate change are expected to affect the viability of agriculture and increase food insecurity in many developing countries. Climate change is certainly the most serious of these challenges for the twenty-first century. The poorest regions of the world – tropical West Africa included – are the most vulnerable due to their high dependence on climate and weather sensitive activities such as agriculture, and the widespread poverty that limits the institutional and economic capacities to adapt to the new stresses brought about by climate change. Climate change is already acting negatively on the poor smallholders of tropical West Africa whose livelihoods dependent mainly on rain-fed agriculture that remains the cornerstone of the economy in the region. Adaptation of the agricultural systems to climate change effects is, therefore, crucial to secure the livelihoods of these rural communities. Since information is a key for decision-making, it is important to provide well-founded information on the magnitude of the impacts in order to design appropriate and sustainable adaptation strategies.
Considering the case of agricultural production in the Republic of Benin, this study aims at using large-scale climatic predictors to assess the potential impacts of past and future climate change on agricultural productivity at a country scale in West Africa. Climate signals from large-scale circulation were used because state-of-the art regional climate models (RCM) still do not perfectly resolve synoptic and mesoscale convective processes. It was hypothesised that in rain-fed systems with low investments in agricultural inputs, yield variations are widely governed by climatic factors. Starting with pineapple, a perennial fruit crops, the study further considered some annual crops such as cotton in the group of fibre crops, maize, sorghum and rice in the group of cereals, cowpeas and groundnuts belonging to the legume crops, and cassava and yams which are root and tuber crops. Thus the selected crops represented the three known groups of photosynthetic pathways (i.e. CAM, C3, and C4 plants).
In the study, use was made of the historical agricultural yield statistics for the Republic of Benin, observed precipitation and mean near-surface air temperature data from the Climatic Research Unit (CRU TS 3.1) and the corresponding variables simulated by the regional climate model (RCM) REMO. REMO RCM was driven at its boundaries by the global climate model ECHAM 5. Simulations with different greenhouse gas concentrations (SRES-A1B and B1 emission scenarios) and transient land cover change scenarios for present-day and future conditions were considered. The CRU data were submitted to empirical orthogonal functions analysis over the north hemispheric part of Africa to obtain large-scale observed climate predictors and associated consistent variability modes. REMO RCM data for the same region were projected on the derived climate patterns to get simulated climate predictors. By means of cross-validated Model Output Statistics (MOS) approach combined with Bayesian model averaging (BMA) techniques, the observed climate predictors and the crop predictand were further on used to derive robust statistical relationships. The robust statistical crop models perform well with high goodness-of-fit coefficients (e.g. for all combined crop models: 0.49 ≤ R2 ≤ 0.99; 0.28 ≤ Brier-Skill-Score ≤ 0.90).
Provided that REMO RCM captures the main features of the real African climate system and thus is able to reproduce its inter-annual variability, the time-independent statistical transfer functions were then used to translate future climate change signal from the simulated climate predictors into attainable crop yields/crop yield changes. The results confirm that precipitation and air temperature governed agricultural production in Benin in general, and particularly, pineapple yield variations are mainly influenced by temperature. Furthermore, the projected yield changes under future anthropogenic climate change during the first-half of the 21st century amount up to -12.5% for both maize and groundnuts, and -11%, -29%, -33% for pineapple, cassava, and cowpeas respectively. Meanwhile yield gain of up to +10% for sorghum and yams, +24% for cotton, and +39% for rice are expected. Over the time period 2001 – 2050, on average the future yield changes range between -3% and -13% under REMO SRES–B1 (GHG)+LCC, -2% and -11% under REMO SRES–A1B (GHG only),and -3% and -14% under REMO SRES–A1B (GHG)+LCC for pineapple, maize, sorghum, groundnuts, cowpeas and cassava. In the meantime for yams, cotton and rice, the average yield gains lie in interval of about +2% to +7% under REMO SRES–B1 (GHG)+LCC, +0.1% and +12% under REMO SRES–A1B (GHG only), and +3% and +10% under REMO SRES–A1B (GHG)+LCC. For sorghum, although the long-term average future yield depicts a reduction there are tendencies towards increasing yields in the future. The results also reveal that the increases in mean air temperature more than the changes in precipitation patterns are responsible for the projected yield changes. As well the results suggest that the reductions in pineapple yields cannot be attributed to the land cover/land use changes across sub-Saharan Africa. The production of groundnuts and in particular yams and cotton will profit from the on-going land use/land cover changes while the other crops will face detrimental effects.
Henceforth, policymakers should take effective measures to limit the on-going land degradation processes and all other anthropogenic actions responsible for temperature increase. Biotechnological improvement of the cultivated crop varieties towards development of set of seed varieties adapted to hotter and dry conditions should be included in the breeding pipeline programs. Amongst other solutions, application of appropriate climate-smart agricultural practices and conservation agriculture are also required to offset the negative impacts of climate change in agriculture.
The Amduat is one of the most important Netherworld Books which was recorded in various kinds of Ancient Egyptian sources since the beginning of the 18th dynasty, especially the walls of the royal tombs. The main theme of the Amduat is the journey of the sun god through the underworld where the solar bark and its crew is the central scene of the journey. The study focuses on finding the reasons of choosing the crew’s members who manage the sun bark’s journey in the Amduat. It also aims at illustrating the functions and responsibilities of each crew member. Following a historical approach, the study analyzes the Pyramid Texts and Coffin Texts as the most important documents before the New Kingdom, and proceeding to the inscriptions and writings of the monuments which contain portrayals and inscriptions of the Amduat in the New Kingdom. Furthermore, it sheds some light on the solar cycle’s main features and primary aspects, and tries to scrutinize the date, meaning, and symbolisms of the Amduat and its indications in the earlier sources.
Modulation of insulin-induced genotoxicity in vitro and genomic damage in gestational diabetes
(2019)
Diabetes mellitus is a global health problem, where the risk of diabetes increases rapidly
due to the lifestyle changes. Patients with type II diabetes have many complications
with increased risk of morbidity and mortality. High levels of insulin may lead to DNA
oxidation and damage. Several studies proposed that hyperinsulinemia may be an
important risk factor for various types of cancer. To investigate insulin signaling
pathway inducing oxidative stress and genomic damage, pharmaceutical and natural
compounds which can interfere with the insulin pathway including PI3K inhibitors,
resveratrol, lovastatin, and RAD-001 were selected due to their beneficial effects
against metabolic disorder. Thus, the anti-genotoxic potential of these compounds
regarding insulin-mediated oxidative stress were investigated in normal rat kidney cells
in vitro. Our compounds showed protective effect against genotoxic damage and
significantly decreased reactive oxygen specious after treatment of cells with insulin
with different mechanisms of protection between the compounds. Thus, these
compounds may be attractive candidates for future support of diabetes mellitus therapy.
Next, we explored the link between gestational diabetes mellitus and genomic damage
in cells derived from human blood. Moreover, we investigated the influence of
estradiol, progesterone, adrenaline and triiodothyronine on insulin-induced genomic
damage in vitro. First, we studied the effect of these hormones in human promyelocytic
leukemia cells and next ex vivo with non-stimulated and stimulated peripheral blood
mononuclear cells. In parallel, we also measured the basal genomic damage using three
conditions (whole blood, non-stimulated and stimulated peripheral blood mononuclear
cells) in a small patient study including non-pregnant controls with/without hormonal
contraceptives, with a subgroup of obese women, pregnant women, and gestational
diabetes affected women. A second-time point after delivery was also applied for
analysis of the blood samples. Our results showed that GDM subjects and obese
individuals exhibited higher basal DNA damage compared to lower weight nonpregnant
or healthy pregnant women in stimulated peripheral blood mononuclear cells
in both comet and micronucleus assays. On the other hand, the DNA damage in GDM
women had decreased at two months after birth. Moreover, the applied hormones also
showed an influence in vitro in the enhancement of the genomic damage in cells of the control and pregnant groups but this damage did not exceed the damage which existed
in obese and gestational diabetes mellitus patients with high level of genomic damage.
In conclusion, insulin can induce genomic damage in cultured cells, which can be
modulated by pharmaceutical and naturals substances. This may be for future use in the
protection of diabetic patients, who suffer from hyperinsulinemia during certain disease
stages. A particular form of diabetes, GDM, was shown to lead to elevated DNA
damage in affected women, which is reduced again after delivery. Cells of affected
women do not show an enhanced, but rather a reduced sensitivity for further DNA
damage induction by hormonal treatment in vitro. A potential reason may be an
existence of a maximally inducible damage by hormonal influences.
Quantitative Electron Paramagnetic Resonance Studies of Charge Transfer in Organic Semiconductors
(2020)
In the present work we investigated various charge transfer processes, as they appear in the versatile world of organic semiconductors by probing the spin states of the corresponding charge carrier species via electron paramagnetic resonance (EPR) spectroscopy. All studied material systems are carbon-based compounds, either belonging to the group of polymers, fullerenes, or single-wall carbon nanotubes (SWNTs).
In the first instance, we addressed the change of the open circuit voltage (Voc) with the fullerene blend stoichiometry in fullerene-based solar cells for organic photovoltaics (OPV). The voltage depends strongly on the energy separation between the lowest unoccupied molecular orbital (LUMO) of the donor and the highest occupied molecular orbital (HOMO) of the acceptor. By exploiting the Gaussian distribution of the charge carriers in a two-level system, and thus also their spins in the EPR experiment, it could be shown that the LUMOs get closer by a few to a few hundred meV when going from pure fullerene materials to a fullerene mixture. The reason for this strong energetic effect is likely the formation of a fullerene alloy.
Further, we investigated the chemical doping mechanism of SWNTs with a (6,5)-chirality and their behaviour under optical excitation. In order to determine the unintentional (pre)-doping of SWNTs, EPR spectra of the raw material as well as after different purification steps were recorded. This facilitated the determination of nanotube defects and atmospheric p-doping as the causes of the measured EPR signals. In order to deliberately transfer additional charge carriers to the nanotubes, we added the redox-active substance AuCl3 where we determined an associated doping-yield of (1.5±0.2)%. In addition, a statistical occupation model was developed which can be used to simulate the distribution of EPR active, i.e. unpaired and localised charge carriers on the nanotubes.
Finally, we investigated the charge transfer behaviour of (6,5)-SWNTs together with the polymer P3HT and the fullerene PC60BM after optical excitation.
Platelets are small anucleated cell fragments that originate from megakaryocytes (MKs), which are large cells located in the bone marrow (BM). MKs extend long cytoplasmic protrusions, a process which is called proplatelet formation, into the lumen of the sinusoidal vessels where platelets are sized by the bloodstream. During the process of platelet biogenesis, segments of the MK penetrate the endothelium and, through cytoskeletal remodeling inside the MK, proplatelet fragments are released. Rho GTPases, such as RhoA and RhoB, are critically involved in cytoskeletal rearrangements of both the actin and the tubulin cytoskeleton.
The first part of this thesis concentrated on the protein RhoB and its involvement in cytoskeletal organization in MKs and platelets. Single knockout (KO) mice lacking RhoB had a minor microthrombocytopenia, which means a smaller platelet size and reduced platelet number, accompanied by defects in the microtubule cytoskeleton in both MKs and platelets. In particular, tubulin organization and stability, which is regulated by posttranslational modifications of α-tubulin, were disturbed in RhoB-/- platelets. In contrast, RhoB-/- MKs produced abnormally shaped proplatelets but had unaltered posttranslational modifications of α-tubulin.
The second part focused on the influence of RhoA and RhoB on MK localization and platelet biogenesis in murine BM. Many intact RhoA-/- MKs are able to transmigrate through the endothelial layer and stay attached to the vessel wall, whereas only 1% of wildtype (wt) MKs are detectable in the intrasinusoidal space. Concomitant deficiency of RhoA and RhoB reverts this transmigration and results in macrothrombocytopenia, MK clusters around the vessel in the BM and defective MK development. The underlying mechanism that governs MKs to distinct localizations in the BM is poorly understood, thus this thesis suggests that this process may be dependent on RhoB protein levels, as RhoA deficiency is coincided with increased RhoB levels in MKs and platelets.
The third part of this thesis targeted the protein PDK1, a downstream effector of Rho GTPases, in regard to MK maturation and polarization throughout thrombopoiesis. MK- and platelet-specific KO in mice led to a significant macrothrombocytopenia, impaired actin cytoskeletal reorganization during MK spreading and proplatelet formation, with defective MK maturation. This was associated with decreased PAK activity and, subsequently, phosphorylation of its substrates LIMK and Cofilin. Together, the observations of this thesis highlight the importance of Rho GTPases and their downstream effectors on the regulation of the MK and platelet cytoskeleton.
Extreme value theory aims at modeling extreme but rare events from a probabilistic point of view. It is well-known that so-called generalized Pareto distributions, which are briefly reviewed in Chapter 1, are the only reasonable probability distributions suited for modeling observations above a high threshold, such as waves exceeding the height of a certain dike, earthquakes having at least a certain intensity, and, after applying a simple transformation, share prices falling below some low threshold. However, there are cases for which a generalized Pareto model might fail. Therefore, Chapter 2 derives certain neighborhoods of a generalized Pareto distribution and provides several statistical tests for these neighborhoods, where the cases of observing finite dimensional data and of observing continuous functions on [0,1] are considered. By using a notation based on so-called D-norms it is shown that these tests consistently link both frameworks, the finite dimensional and the functional one. Since the derivation of the asymptotic distributions of the test statistics requires certain technical restrictions, Chapter 3 analyzes these assumptions in more detail. It provides in particular some examples of distributions that satisfy the null hypothesis and of those that do not. Since continuous copula processes are crucial tools for the functional versions of the proposed tests, it is also discussed whether those copula processes actually exist for a given set of data. Moreover, some practical advice is given how to choose the free parameters incorporated in the test statistics. Finally, a simulation study in Chapter 4 compares the in total three different test statistics with another test found in the literature that has a similar null hypothesis. This thesis ends with a short summary of the results and an outlook to further open questions.
Atomic nanowires formed by self-assembled growth on semiconducting surfaces represent a feasible physical realization of quasi-1D electron systems and can be used to study fascinating 1D quantum phenomena. The system in the focus of this thesis, Si(553)-Au, is generated by Au adsorption onto a stepped silicon surface. It features two different chain types, interspersed with each other: A Au chain on the terrace, and a honeycomb chain of graphitic silicon located at the step edge. The silicon atoms at the exposed edges of the latter are predicted to be spin-polarized and charge-ordered [1], leading to an ordered array of local magnetic moments referred to as ``spin chains''.
The present thesis puts this spin chain proposal to an experimental test.
A detailed scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) scrutiny reveals a distinct unoccupied density of states (DOS) feature localized at every third Si step-edge atom, which aligns perfectly with the density functional theory (DFT) prediction.
This finding provides strong evidence for the formation of spin chains at the Si(553)-Au step edges, and simultaneously rules out the interpretation of previous studies which attributed the x3 step-edge superstructure to a Peierls instability.
To study the formation of spin chains in further detail, an additional member of the so-called Si(hhk)-Au family -- Si(775)-Au -- is analyzed.
Based on DFT modeling (performed by S.C. Erwin, Naval Research Laboratory, USA) and detailed STM and STS experiments, a new structure model for this surface is developed, and the absence of spin chains at the Si(775)-Au step edges is demonstrated.
The different step-edge charge distributions of all known Si(hhk)-Au surfaces are traced back to an electron transfer between the terrace and the step edge. Accordingly, an unintentional structure defect should create a localized spin at the Si(775)-Au step edge. This prediction is verified experimentally, and suggest that surface chemistry can be used to create and destroy Si spin chains.
Having clarified why spin chains form on some Si(hhk)-Au surfaces but not on others, various interaction effects of the Si(553)-Au spin chains are inspected.
A collaborative analysis by SPA-LEED (M. Horn-von Hoegen group, University of Duisburg-Essen, Germany), DFT (S.C. Erwin), and STM reveals strong lateral coupling between adjacent spin chains, bearing interesting implications for their magnetic ordering. The centered geometry uncovered leads to magnetic frustration, and may stabilize a 2D quantum spin liquid.
Moreover, a complex interplay between neighboring Au and Si chains is detected.
Specifically, the interaction is found effectively ``one-way'', i.e., the Si step edges respond to the Au chains but not vice versa.
This unidirectional effect breaks the parity of the Si chains, and creates two different configurations of step edges with opposite directionality.
In addition to the static properties of the Si(553)-Au surface mentioned above, the occurrence of solitons in both wire types is witnessed in real space by means of high-resolution STM imaging. The solitons are found to interact with one another such that both move in a coupled fashion along the chains. Likewise, STM experiments as a function of the tunneling current suggest an excitation of solitons along the step edge by the STM tunneling tip.
Solitons are also found to play an essential role in the temperature-dependent behavior of the Si(553)-Au step edges.
It is an accepted fact that the distinct x3 superstructure of the Si(553)-Au step edges vanishes upon heating to room temperature. As a first step in exploring this transition in detail over a large temperature range, a previously undetected, occupied electronic state associated with the localized step-edge spins is identified by means of angle-resolved photoemission spectroscopy (ARPES).
A tracking of this state as a function of temperature reveals an order-disorder-type transition. Complementary STM experiments attribute the origin of this transition to local, thermally activated spin site hops, which correspond to soliton-anitsoliton pairs.
Finally, a manipulation of the Si(553)-Au atomic wire array is achieved by the stepwise adsorption of potassium atoms. This does not only increase the filling of the Au-induced surface bands culminating in a metal-insulator transition (MIT), but also modifies the Si step-edge charge distribution, as indicated by STM and ARPES experiments.
[1] S. C. Erwin and F. Himpsel, Intrinsic magnetism at silicon surfaces, Nat. Commun. 1,
58 (2010).
The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit.
The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples.
In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity.
The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm.
These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data.
Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM.
In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.
In this work the energy transfer and excitonic coupling in different chromophore arrangements were investigated. A difference in the coupling strength was introduced by varring the connecting unit and the spacial orientation relative to each other.
The synthesis of the 2,7-substituted pyrene compounds could be optimised and good yields of HAB 1 and HAB 2 and small amounts of HAB 2 could be achieved by cobalt-catalysed trimerisation or Diels Alder reaction in the end. Absorption and fluorescence spectra reveal strong intramolecular interactions between the pyrene molecules in the HAB 1. Excitation spectra recorded at the high and low energy fluorescence suggest the contribution of two components to the spectra. One being similar to the ground state aggregate and a second species similar to undisturbed pyrene. All these feature can be accounted to two different fluorescent states which are due to electronical decoupling in the excited state. Due to the strong intramolecular coupling already in the ground state of the molecule, no energy transfer could be studied, as the six pyrene units cannot be seen as separate spectroscopic entities between which energy could be transferred.
In the second part of this thesis dye conjugates of different size and alignment were synthesised to study the interaction of the transition-dipole moments. Therefore a systematic investigation of Sonogashira conditions was performed in order to obtain good yields of the desired compounds and keep dehalogenation at a minimum level. Nevertheless only the symmetrical triads could be purified as the asymmeric triads and pentades proved to decompose during purification.
The pyrene containing triads Py2B and Py2SQB show small interactions already in the ground state represented by red shifts of the spectra and a broadening of the bands. Nevertheless, these interactions are in the weak coupling regime and energy transfer between the constituents is possible. On the contrary in the TA spectra it is obvious that always the whole triad, at least to some extend is excited. To question if the excitation of the high energy state is deactivated by energy transfer or rather IC in a superchromophore could not be distinguished in the course of this work. At present additional time-dependent calculations of the dynamics are in progress to get a deeper understanding of the photophysical processes taking place in the triads.
The dye conjugates B2SQB-3 and (SQB)2B-4 can be assigned to the strong interaction range and hence are describable by exciton theory. The transition-dipole moments proved to be more than additive and increase for both compounds from absorption to fluorescence. This can be explained by an enhancement of the coupling in the relaxed excited state compared to the absorption into the Franck-Condon state due to a more steep potential energy surface in the excited state and hence smaller fluctuations.
In the last part of this thesis the influence of disrupting electronical communication by implementing a rigid non-conjugated bridge in a bichromophoric trans-squaraine system was tested. While the flexible linked squaraines show complex spectra due to different conformers the SQA2Anth compound is rigified and no rotation is possible. This change in flexibility is represented in the steady-state spectra where just one main absorption and fluorescence band is present due to a single allowed excitonic state. The system proves to own an excited state that is completely delocalised over the whole molecule.
The human pathogen Chlamydia trachomatis is the main cause of sexually transmitted infections worldwide. The obligate intracellular bacteria are the causative agent of several diseases that reach from conjunctivitis causing trachoma and blindness as well as salpingitis and urethritis which can lead to infertility if left untreated.
In order to gain genetically engineered Chlamydia that inducible knock down specific gene expression, the CRISPRi system was established in C. trachomatis. In a proof of principle experiment it was shown that C. trachomatis pCRISPRi:gCdu1III target ChlaDUB1 expression and reduce the protein amount up to 50 %. Knock-down of the DUB did not influence protein levels of anti-apoptotic Mcl-1 and did not make cells susceptible for apoptosis. However, reduced dCas9 protein size, bacterial growth impairment and off target effects interfering with the GFP signal, form obstacles in CRISPRi system in Chlamydia. For routinely use of the CRISPRi method in C. trachomatis further investigation is needed.
Since the bacterial life cycle includes two morphological and functional distinct forms, it is essential for chlamydial spread to complete the development cycle and form infectious progeny. Therefore, Chlamydia has evolved strategies to evade the host immune system in order to stay undetected throughout the developmental cycle. The bacteria prevent host cell apoptosis via stabilization of anti-apoptotic proteins like Mcl-1, Survivin and HIF-1α and activate pro-survival pathways, inhibiting invasion of immune cells to the site of infection. The host cell itself can destroy intruders via cell specific defense systems that involve autophagy and recruitment of professional immune cells. In this thesis the role of the chlamydial deubiuqitinase ChlaDUB1 upon immune evasion was elucidated. With the mutant strain Ctr Tn-cdu1 that encodes for a truncated DUB due to transposon insertion, it was possible to identify ChlaDUB1 as a potent opponent of the autophagic system. Mutant inclusions were targeted by K48 and K63 chain ubiquitination. Subsequently the inclusion was recognized by autophagic receptors like p62, NBR1 and NDP52 that was reversed again by complementation with the active DUB. Xenophagy was promoted so far as LC3 positive phagosomes formed around the inclusion of Ctr Tn-cdu1, which did not fuse with the lysosome. The detected growth defect in human primary cells of Chlamydia missing the active DUB was not traced back to autophagy, but was due to impaired development and replication. It was possible to identify Ankib1, the E3 ligase, that ubiquitinates the chlamydial inclusion in a siRNA based screen. The activating enzyme Ube1 and the conjugating enzyme Ube2L3 are also essential in this process. Chlamydia have a reduced genome and depend on lipids and nutrients that are translocated from the host cell to the inclusion to proliferate. Recruitment of fragmented Golgi stacks to the inclusion surface was prevented when ChlaDUB1 was inactive, probably causing diminished bacterial growth. Additionally, the modification of the inclusion by Ankib1 and subsequent decoration by autophagic markers was not only present in human but also murine cells. Comparison of other Chlamydia strains and species revealed Ankib1 to be located at the proximity of the inclusion in C. trachomatis strains only but not in C. muridarum or C. pneumoniae, indicating that Ankib1 is specifically the E3 ligase of C. trachomatis. Moreover, the role of ChlaDUB1 in infected tissue was of interest, since ChlaDUB1 protein was also found in early EB stage and so might get in contact with invading immune cells after cell lysis. While bacteria spread and infect new host cells, Chlamydia can also infect immune cells. Infection of human neutrophils with Ctr Tn-cdu1 shows less bacterial survival and affirms the importance of the DUB for bacterial fitness in these cells.
Staphylococcus aureus (SA) causes nosocomial infections including life threatening sepsis by multi-resistant strains (MRSA). It has the ability to form biofilms to protect it from the host immune system and from anti staphylococcal drugs. Biofilm and planctonic life style is regulated by a complex Quorum-Sensing (QS) system with agr as a central regulator. To study biofilm formation and QS mechanisms in SA a Boolean network was build (94 nodes, 184 edges) including two different component systems such as agr, sae and arl. Important proteins such as Sar, Rot and SigB were included as further nodes in the model. System analysis showed there are only two stable states biofilm forming versus planctonic with clearly different subnetworks turned on. Validation according to gene expression data confirmed this. Network consistency was tested first according to previous knowledge and literature. Furthermore, the predicted node activity of different in silico knock-out strains agreed well with corresponding micro array experiments and data sets. Additional validation included the expression of further nodes (Northern blots) and biofilm production compared in different knock-out strains in biofilm adherence assays. The model faithfully reproduces the behaviour of QS signalling mutants. The integrated model allows also prediction of various other network mutations and is supported by experimental data from different strains. Furthermore, the well connected hub proteins elucidate how integration of different inputs is achieved by the QS network. For in silico as well as in vitro experiments it was found that the sae-locus is also a central modulator of biofilm production. Sae knock-out strains showed stronger biofilms. Wild type phenotype was rescued by sae complementation. To elucidate the way in which sae takes influence on biofilm formation the network was used and Venn-diagrams were made, revealing nodes regulated by sae and changed in biofilms. In these Venn-diagrams nucleases and extracellular proteins were found to be promising nodes. The network revealed DNAse to be of great importance. Therefore qualitatively the DNAse amount, produced by different SA mutants was measured, it was tried to dissolve biofilms with according amounts of DNAse and the concentration of nucleic acids, proteins and polysaccharides were measured in biofilms of different SA mutants.
With its thorough validation the network model provides a powerful tool to study QS and biofilm formation in SA, including successful predictions for different knock-out mutant behaviour, QS signalling and biofilm formation. This includes implications for the behaviour of MRSA strains and mutants. Key regulatory mutation combinations (agr–, sae–, sae–/agr–, sigB+, sigB+/sae–) were directly tested in the model but also in experiments. High connectivity was a good guide to identify master regulators, whose detailed behaviour was studied both in vitro and in the model. Together, both lines of evidence support in particular a refined regulatory role for sae and agr with involvement in biofilm repression and/or SA dissemination. With examination of the composition of different mutant biofilms as well as with the examination of the reaction cascade that connects sae to the biofilm forming ability of SA and also by postulating that nucleases might play an important role in that, first steps were taken in proving and explaining regulatory links leading from sae to biofilms. Furthermore differences in biofilms of different mutant SA strains were found leading us in perspective towards a new understanding of biofilms including knowledge how to better regulate, fight and use its different properties.
Data mining has proved its significance in various domains and applications. As an important subfield of the general data mining task, subgroup mining can be used, e.g., for marketing purposes in business domains, or for quality profiling and analysis in medical domains. The goal is to efficiently discover novel, potentially useful and ultimately interesting knowledge. However, in real-world situations these requirements often cannot be fulfilled, e.g., if the applied methods do not scale for large data sets, if too many results are presented to the user, or if many of the discovered patterns are already known to the user. This thesis proposes a combination of several techniques in order to cope with the sketched problems: We discuss automatic methods, including heuristic and exhaustive approaches, and especially present the novel SD-Map algorithm for exhaustive subgroup discovery that is fast and effective. For an interactive approach we describe techniques for subgroup introspection and analysis, and we present advanced visualization methods, e.g., the zoomtable that directly shows the most important parameters of a subgroup and that can be used for optimization and exploration. We also describe various visualizations for subgroup comparison and evaluation in order to support the user during these essential steps. Furthermore, we propose to include possibly available background knowledge that is easy to formalize into the mining process. We can utilize the knowledge in many ways: To focus the search process, to restrict the search space, and ultimately to increase the efficiency of the discovery method. We especially present background knowledge to be applied for filtering the elements of the problem domain, for constructing abstractions, for aggregating values of attributes, and for the post-processing of the discovered set of patterns. Finally, the techniques are combined into a knowledge-intensive process supporting both automatic and interactive methods for subgroup mining. The practical significance of the proposed approach strongly depends on the available tools. We introduce the VIKAMINE system as a highly-integrated environment for knowledge-intensive active subgroup mining. Also, we present an evaluation consisting of two parts: With respect to objective evaluation criteria, i.e., comparing the efficiency and the effectiveness of the subgroup discovery methods, we provide an experimental evaluation using generated data. For that task we present a novel data generator that allows a simple and intuitive specification of the data characteristics. The results of the experimental evaluation indicate that the novel SD-Map method outperforms the other described algorithms using data sets similar to the intended application concerning the efficiency, and also with respect to precision and recall for the heuristic methods. Subjective evaluation criteria include the user acceptance, the benefit of the approach, and the interestingness of the results. We present five case studies utilizing the presented techniques: The approach has been successfully implemented in medical and technical applications using real-world data sets. The method was very well accepted by the users that were able to discover novel, useful, and interesting knowledge.
Synthesis of (RS)-5-amino-3-aryl (methyl)-pentanoic acid hydrochlorides, 3 aminomethyl-5-chloro-benzoic acid hydrochloride and (RS)-4-amino-3-(4`-ethynyl(iodo)-phenyl)-butanoic acid hydrochlorides have been accomplished. The aim of their synthesis was to evaluate their GABABR agonist activity and to derive a model which will correlate their structure with the observed pEC50. The GABABR agonist activity of the prepared compounds has been determined in functional assay based on calcium measurement in vitro using tsA cells transfected with GABAB1b/GABAB2/Gαq-z5. Reviews on the neurotransmitter receptors (ligand-gated ion channel receptors and G protein-coupled receptors), their agonists and antagonists have been given in the general part of this work. A detailed discussion on the strategy followed for the synthesis of the designed compounds as well as the starting materials and intermediates has been described and illustrated in Schemes 2-6. The synthesized compounds were evaluated for their GABABR agonist activity. Furthermore, these compounds were docked in the available 3D homology model of GABABR using the program FlexiDock implemented in SYBYL software. Subsequently, we derived a predictive model which correlates the experimentally determined pEC50 with the calculated binding energy of certain baclofen analogues and homologues. In addition, we used the program DISCO (DIStance COmparisons) implemented in SYBYL software to find the pharmacophore features of GABAB agonists.
Regulation of pathogen-inducible volatile compounds in Arabidopsis and their role in plant defense
(2010)
Plants are constantly attacked by pathogenic microbes. As a result, they have evolved a plethora of constitutive and inducible defense responses to defend against attempted pathogen infection. Although volatile organic compounds have been implicated in plant defense, direct evidence of their function in plant resistance is still lacking. I have examined the role of VOCs in Arabidopsis defense against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. maculicola. The obtained results show that the vegetative parts of Arabidopsis produces and emits the volatile phenylpropanoid MeSA and three kinds of terpenoids, (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), alpha-ionon and beta-farnesen, upon avirulent and virulent P. syringae inoculation. Whereas the most abundant volatiles, MeSA and TMTT, are already produced at early stages of infection in the compatible and incompatible interaction, enhanced emission of alpha-ionon and beta-farnesen can only be detected in later stages of the compatible interaction. It was revealed that pathogen-induced synthesis of TMTT in Arabidopsis requires the JA signaling pathway but occurs independently of SA defense signaling. Similarly, the production of MeSA is dependent on JA signaling but not on the SA defense signaling pathway. Furthermore, production of MeSA is dependent on the function of ISOCHORISMATE SYNTHASE1, which produces its precursor SA. Upon inoculation with avirulent P. syringae, endogenously produced JA activates the JA signalling pathway to mediate MeSA and TMTT synthesis. By contrast, in the compatible Arabidopsis-Psm interaction, production of MeSA predominantly depends on the P. syringea the virulence factor coronatine, which activates JA downstream signaling. To learn more about the role of inducible VOCs in plant defense responses, I have identified an Arabidopsis T-DNA insertions line with a defect in the TERPENE SYNTHASE4 (TPS4) gene. Emission profiles from this mutant revealed that the induced production of TMTT but not of alpha-ionone, beta-farnesene or MeSA are abolished, demonstrating that TPS4 specifically regulates the P. syringae-induced synthesis of TMTT in Arabidopsis. The lack of TMTT in tps4 mutants, however, does not affect plant defense responses and resistance induction against P. syringae. This excludes a role of the terpenoid as an effective phytoalexin in Arabidopsis leaves against the bacterial pathogen. Moreover, tps4 mutant plants are still able to mount a SAR response, excluding a signaling function of TMTT during SAR. An important aim of our studies was to address the defensive role of MeSA, the major VOC emitted from P. syringae-inoculated Arabidopsis leaves. MeSA has been recently proposed as a critical long distance signal in the development of SAR. I found that two independent T-DNA insertions lines with defects in expression of the pathogen-inducible SA methyl transferase gene BSMT1 are completely devoid of pathogen-induced production of MeSA. However, bsmt1 mutant plants are capable to increase the level of SA in systemic, non-infected leaves of Arabodopsis and develop SAR like wild-type plants upon local P. syringae-inoculation. Thus, MeSA does not function as a critical SAR signal in Arabidopsis. Further experiments showed that SA accumulation in distant leaves occurs due to de novo synthesis through isochorismate synthase. In addition, we also ruled out a critical defensive role of MeSA at inoculation sites, because bsmt1 mutants are able to build up SA-dependent defense responses and local resistance in a wild-type-like manner. The conversion of SA to MeSA and subsequently emission of MeSA from the plant might help the plant to detoxify an excess of SA. This process is regulated by the JA pathway and might be one means to mediate negative crosstalk between JA and SA signaling. Moreover, the COR-triggered conversion of SA to MeSA and emission of the volatile methyl ester could be a way by which virulent P. syringae is able to attenuate the SA-defense pathway.
Scientific surveys provide sufficient evidence that anxiety disorders are one of the most common psy-chiatric disorders in the world. The lifetime prevalence rate of anxiety disorder is 28.8% (Kessler, et al., 2005). The most widely studied anxiety disorders are as follows panic disorder (PD), post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), social phobia (or social anxiety disorder), specific phobias, and generalized anxiety disorder (GAD). (NIMH Article, 2009). Classical conditioning is the stable paradigm used from the last one century to understand the neurobi-ology of fear learning. Neurobiological mechanism of fear learning is well documented with the condi-tioning studies. In the therapy of anxiety disorders, exposure based therapies are known to be the most effective approaches. Flooding is a form of exposure therapy in which a participant is exposed to the fear situation and kept in that situation until their fear dissipates. The exposure therapy is based on the phenomena of extinction; this means that a conditioned response diminishes if the conditioned stimulus (CS) is repeatedly presented without an unconditioned stimulus (UCS). One problem with extinction as well as with exposure-based therapy is the problem of fear return (for e.g. renewal, spontaneous recov-ery and reinstatement) after successful extinction. Therefore, extinction does not delete the fear memory trace. It has been well documented that memory processes can be modulated or disrupted using several sci-entific paradigms such as behavioral (for e.g. exposure therapy), pharmacological (for e.g. drug manipu-lation), non-invasive stimulation (for e.g. non-invasive stimulation such as electroconvulsive shock (ECS), transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), etc. However, modulation of memory processes after reactivation or via non-invasive stimulation is still not clear, which is the focus of the current study. In addition, study of genetic variant suggests that genetic differences play a vital role in the psychiatric disorder especially in fear learning. Hence, it is also one of the concerns of the current dissertation to investigate the interaction between gene and reconsolidation of memory. With respect to fear-conditioning, there are three findings in the current dissertation, which are as fol-lows: (i) In the first study we investigated that non-invasive weak electrical stimulation interferes with the consolidation process and disrupts the fear consolidation to attain stable form. This might offer an effective treatment in the pathological memories, for e.g. PTSD, PD, etc. (ii) In the second study we demonstrated whether a brief single presentation of the CS will inhibit the fear recovery. Like earlier studies we also found that reactivation followed by reconsolidation douses fear return. Attenuation of fear recovery was observed in the reminder group compared to the no-reminder group. (iii) Finally, in our third study we found a statistically significant role of brain derived neurotrophic factor (BDNF) polymorphism in reconsolidation. Results of the third study affirm the involvement of BDNF variants (Met vs. Val) in the modulation of conditioned fear memory after its reactivation. In summary, we were able to show in the current thesis modulation of associative learning and recon-solidation via transcranial direct current stimulation and genetic polymorphism.
The present cumulative dissertation summarizes three clinical studies, which examine
subgroups of patients within the fibromyalgia syndrome (FMS). FMS entails chronic pain and
associated symptoms, and its pathophysiology is incompletely understood (1). Previous studies
show that there is a subgroup of patients with FMS with objective histological pathology of the
small nerve fibers of the peripheral nervous system (PNS). Another subgroup of FMS patients
does not show any signs of pathological changes of the small nerve fibers. The aim of this
dissertation was to compare FMS patients with healthy controls, and these two FMS subgroups
for differences in the central nervous system (CNS) in order to explore possible interactions
between PNS and the CNS. Regarding the CNS, differences of FMS patients with healthy
controls have already been found in studies with small sample sizes, but no subgroups have yet
been identified. Another aim of this thesis was to test whether the subgroups show a different
response to different classes of pain medication. The methods used in this thesis are structural
and functional magnetic resonance imaging (MRI), magnetic resonance diffusion imaging and
magnetic resonance spectroscopy. For the evaluation of clinical symptoms, we used
standardized questionnaires. The subgroups with and without pathologies of the PNS were
determined by skin biopsies of the right thigh and lower leg based on the intraepidermal nerve
fiber density (IENFD) of the small nerve fibers.
1) In the first MRI study, 43 female patients with the diagnosis of FMS and 40 healthy
control subjects, matched in age and body mass index, were examined with different MRI
sequences. Cortical thickness was investigated by structural T1 imaging, white matter integrity
by diffusion tensor imaging and functional connectivity within neuronal networks by functional
resting state MRI. Compared to the controls, FMS patients had a lower cortical volume in
bilateral frontotemporoparietal regions and the left insula, but a higher cortical volume in the
left pericalcarine cortex. Compared to the subgroup without PNS pathology, the subgroup with
PNS pathology had lower cortical volume in both pericalcarine cortices. Diffusion tensor
imaging revealed an increased fractional anisotropy (FA) of FMS patients in corticospinal
pathways such as the corona radiata, but also in regions of the limbic systems such as the fornix
and cingulum. Subgroup comparison again revealed lower mean FA values of the posterior
thalamic radiation and the posterior limb of the left internal capsule in the subgroup with PNS
pathology. In the functional connectivity analysis FMS patients, compared to controls, showed
a hypoconnectivity between the right median frontal gyrus and the posterior cerebellum and
the right crus cerebellum, respectively. In the subgroup comparisons, the subgroup with PNS
pathology showed a hyperconnectivity between both inferior frontal gyri, the right posterior
parietal cortex and the right angular gyrus. In summary, these results show that differences in
brain morphology and functional connectivity exist between FMS patients with and without
PNS pathology. These differences were not associated with symptom duration or severity and,
in some cases, have not yet been described in the context of FMS. The differences in brain
morphology and connectivity between subgroups could also lead to a differential response to
treatment with centrally acting drugs. Further imaging studies with FMS patients should take
into account this heterogeneity of FMS patient cohorts.
2) Following the results from the first MRI study, drug therapies of FMS patients and
their treatment response were compared between PNS subgroups. As there is no licensed drug
for FMS in Europe, the German S3 guideline recommends amitriptyline, duloxetine and
pregabalin for temporary use. In order to examine the current drug use in FMS patients in
Germany on a cross-sectional basis, 156 patients with FMS were systematically interviewed.
The drugs most frequently used to treat pain in FMS were non-steroidal anti-inflammatory
drugs (NSAIDs) (28.9%), metamizole (15.4%) and amitriptyline (8.8%). Pain relief assessed by
patients on a numerical rating scale from 0-10 averaged 2.2 points for NSAIDs, 2.0 for
metamizole and 1.5 for amitriptyline. Drugs that were discontinued for lack of efficacy and not
for side effects were acetaminophen (100%), flupirtine (91.7%), selective serotonin reuptake
inhibitors (81.8%), NSAIDs (83.7%) and weak opioids (74.1%). Patients were divided into
subgroups with and without PNS pathology as determined by skin biopsies. We found no
differences in drug use and effect between the subgroups. Taken together, these results show
that many FMS patients take medication that is not in accordance with the guidelines. The
reduction of symptoms was best achieved with metamizole and NSAIDs. Further longitudinal
studies on medication in FMS are necessary to obtain clearer treatment recommendations.
3) Derived from previous pharmacological and imaging studies (with smaller case
numbers), there is a hypothesis in the FMS literature that hyperreactivity of the insular cortex
may have an impact on FMS. The hyperreactivity seems to be due to an increased concentration
of the excitatory neurotransmitter glutamate in the insular cortex of FMS patients. The
hypothesis is supported by magnetic resonance spectroscopy studies with small number of
cases, as well as results from pharmacological studies with glutamate-inhibiting medication.
Studies from animal models have also shown that an artificially induced increase in glutamate
in the insular cortex can lead to reduced skin innervation. Therefore, the aim of this study was
to compare glutamate and GABA concentrations in the insular cortex of FMS patients with
those of healthy controls using magnetic resonance imaging. There was no significant
difference of both neurotransmitters between the groups. In addition, there was no correlation
between the neurotransmitter concentrations and the severity of clinical symptoms. There
were also no differences in neurotransmitter concentrations between the subgroups with and
without PNS pathology. In conclusion, our study could not show any evidence of a correlation
of glutamate and GABA concentrations with the symptoms of FMS or the pathogenesis of
subgroups with PNS pathologies.
This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.
Adjuvants are compounds added to an agrochemical spray formulation to improve or modify the action of an active ingredient (AI) or the physico-chemical characteristics of the spray liquid. Adjuvants can have more than only one distinct mode of action (MoA) during the foliar spray application process and they are generally known to be the best tools to improve agrochemical formulations. The main objective for this work was to elucidate the basic MoA of adjuvants by uncoupling different aspects of the spray application. Laboratory experiments, beginning from retention and spreading characteristics, followed by humectant effects concerning the spray deposit on the leaf surface and ultimately the cuticular penetration of an AI, were figured out to evaluate overall in vivo effects of adjuvants which were also obtained in a greenhouse spray test. For this comprehensive study, the surfactant classes of non-ionic sorbitan esters (Span), polysorbates (Tween) and oleyl alcohol polyglycol ether (Genapol O) were generally considered because of their common promoting potential in agrochemical formulations and their structural diversity.
The reduction of interfacial tension is one of the most crucial physico-chemical properties of surfactants. The dynamic surface tension (DST) was monitored to characterise the surface tension lowering behaviour which is known to influence the droplet formation and retention characteristics. The DST is a function of time and the critical time frame of droplet impact might be at about 100 ms. None of the selected surfactants were found to lower the surface tension sufficiently during this short timeframe (chapter I). At ca. 100 ms, Tween 20 resulted in the lowest DST value. When surfactant monomers are fully saturated at the droplet-air-interface, an equilibrium surface tension (STeq) value can be determined which may be used to predict spreading or run-off effects. The majority of selected surfactants resulted in a narrow distribution of STeq values, ranging between 30 and 45 mN m- 1. Nevertheless, all surfactants were able to decrease the surface tension considerably compared to pure water (72 mN m- 1). The influence of different surfactants on the wetting process was evaluated by studying time-dependent static contact angles on different surfaces and the droplet spread area on Triticum aestivum leaves after water evaporation. The spreading potential was observed to be better for Spans than for Tweens. Especially Span 20 showed maximum spreading results. To transfer laboratory findings to spray application, related to field conditions, retention and leaf coverage was measured quantitatively on wheat leaves by using a variable track sprayer. Since the retention process involves short time dynamics, it is well-known that the spray retention on a plant surface is not correlated to STeq but to DST values. The relationship between DST at ca. 100 ms and results from the track sprayer showed increasing retention results with decreasing DST, whereas at DST values below ca. 60 mN m- 1 no further retention improvement could be observed.
Under field conditions, water evaporates from the droplet within a few seconds to minutes after droplet deposition on the leaf surface. Since precipitation of the AI must essentially being avoided by holding the AI in solution, so-called humectants are used as tank-mix adjuvants. The ability of pure surfactants to absorb water from the surrounding atmosphere was investigated comprehensively by analysing water sorption isotherms (chapter II). These isotherms showed an exponential shape with a steep water sorption increase starting at 60% to 70% RH. Water sorption was low for Spans and much more distinct for the polyethoxylated surfactants (Tweens and Genapol O series). The relationship between the water sorption behaviour and the molecular structure of surfactants was considered as the so-called humectant activity. With an increasing ethylene oxide (EO) content, the humectant activity increased concerning the particular class of Genapol O. However, it could be shown that the moisture absorption across all classes of selected surfactants correlates rather better with their hydrophilic-lipophilic balance values with the EO content.
All aboveground organs of plants are covered by the cuticular membrane which is therefore the first rate limiting barrier for AI uptake. In vitro penetration experiments through an astomatous model cuticle were performed to study the effects of adjuvants on the penetration of the lipophilic herbicide Pinoxaden (PXD) (chapter III). In order to understand the influence of different adjuvant MoA like humectancy, experiments were performed under three different humidity levels. No explicit relationship could be found between humidity levels and the PXD penetration which might be explained by the fact that humidity effects would rather affect hydrophilic AIs than lipophilic ones. Especially for Tween 20, it became obvious that a complex balance between multiple MoA like spreading, humectancy and plasticising effects have to be considered.
Greenhouse trials, focussing the adjuvant impact on in vivo action of PXD, were evaluated on five different grass-weed species (chapter III). Since agrochemical spray application and its following action on living plants also includes translocation processes in planta and species dependent physiological effects, this investigation may help to simulate the situation on the field. Even though the absolute weed damage was different, depending both on plant species and also on PXD rates, adjuvant effects in greenhouse experiments displayed the same ranking as in cuticular penetration studies: Tween 20 > Tween 80 > Span 20 ≥ Span 80.
Thus, the present work shows for the first time that findings obtained in laboratory experiments can be successfully transferred to spray application studies on living plants concerning adjuvant MoA. A comparative analysis, using radar charts, could demonstrate systematic derivations from structural similarities of adjuvants to their MoA (summarising discussion and outlook). Exemplarily, Tween 20 and Tween 80 cover a wide range of selected variables by having no outstanding MoA improving one distinct process during foliar application, compared to non-ethoxylated Span 20 and Span 80 which primarily revealed a surface active action. Most adjuvants used in this study represent polydisperse mixtures bearing a complex distribution of EO and aliphatic chains. From this study it seems alike that adjuvants having a wide EO distribution offer broader potential than adjuvants with a small EO distribution. It might be a speculation that due to this broad distribution of single molecules, all bearing their individual specific physico-chemical nature, a wide range of properties concerning their MoA is covered.
Kinetics and timing of IL-12 production by dendritic cells for Th1 polarization \(in\) \(vivo\)
(2020)
Dendritic cell (DC) based vaccines rely on the quality of DC maturation to induce antigen presentation, co-stimulation, lymph node migration and the release of heterodimeric IL-12p70 in case of T helper type-1 cell (Th1) polarization. In contrast, DCs that cannot secrete IL-12p70 (e.g. after cytokine cocktail maturation) readily induce Th1 cells when injected into mice and humans. Since it was also previously suggested that DCs are capable of activating other DCs in a bystander fashion, we tested here for the DC source of IL-12p70 for Th1 polarization in a murine DC vaccination model. Migration of the injected murine bone marrow-derived DCs (BM-DCs) was essential for antigen delivery to the lymph node. However, they contributed only partially to antigen presentation, and induced a non-polarized Th0 state of the cognate T cells producing IL-2 but no IFN-. Instead, endogenous dermal migratory XCR1+ cDC1s underwent re-programming by the injected BM-DCs to acquire bystander antigen presentation and IL-12 release for Th1 polarization in the lymph node. Genetic deficiency of migratory DCs and specifically of XCR1+ migratory DCs completely abolished Th1 priming. The kinetic of cell interactions in the draining lymph nodes appeared step-wise as i) injected DCs with cognate T cells, ii) injected DCs with bystander XCR1+ DCs, and iii) bystander XCR1+ DCs with T cells. The transcriptome of the bystander DCs showed a down-regulation of Treg and Th2/Th9 inducing genes, and up-regulation of genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory BM-DCs direct T cell priming and bystander DC activation, but not Th1 polarization which is mediated by endogenous IL-12p70+ XCR1+ migratory bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.
This thesis describes the inclusion of dynamical effects in the theoretical calculation of Electron Paramagnetic Resonance (EPR) spectroscopic parameters. The studies were performed using Density Functional Theory (DFT) methodology and a perturbation-theoretical approach to g-tensor calculations. Hydrogen atoms trapped in octasilasesquioxane cages display unexpectly high, positive g-values. Computational simulation of these systems successfully reproduced the positive g-values and found them to arise from spin-orbit coupling around the oxygen nuclei. Dynamical effects were estimated by calculating the potential well in which the hydrogen atom moves. Semiquinone radical anions are important bioradicals that play a role in photosynthesis and respiration. The simplest and most prototypical, benzosemiquinone anion, was simulated both in the gas phase and in aqueous solution by Car-Parrinello Molecular Dynamics (CPMD). The neutral benzoquinone was also simulated for comparison. The solvation environments of both the anionic and neutral molecules were analysed and compared. EPR parameters were calculated for the semiquinone, providing the first example of full inclusion of dynamic effects in g-tensor calculation. The effects of different solvation interactions on the g-tensor and hyperfine interactions were extensively examined. Additionally, static calculations (i.e., calculations not incorporating any dynamical effects) were performed. Comparison between these (and prior computational studies) and the dynamical system allowed an assessment of the effects of dynamics on solvation and EPR parameters. Ubisemiquinone radical anion, one of the most widely-occurring semiquinone radicals, was simulated in the aqueous phase using CPMD. The solvation environment was analysed and EPR parameters were calculated. The motion of the side-chain, and its effects on solvation and EPR parameters, were examined.
While teleoperation of technical highly sophisticated systems has already been a wide field of research, especially for space and robotics applications, the automation industry has not yet benefited from its results. Besides the established fields of application, also production lines with industrial robots and the surrounding plant components are in need of being remotely accessible. This is especially critical for maintenance or if an unexpected problem cannot be solved by the local specialists.
Special machine manufacturers, especially robotics companies, sell their technology worldwide. Some factories, for example in emerging economies, lack qualified personnel for repair and maintenance tasks. When a severe failure occurs, an expert of the manufacturer needs to fly there, which leads to long down times of the machine or even the whole production line. With the development of data networks, a huge part of those travels can be omitted, if appropriate teleoperation equipment is provided.
This thesis describes the development of a telemaintenance system, which was established in an active production line for research purposes. The customer production site of Braun in Marktheidenfeld, a factory which belongs to Procter & Gamble, consists of a six-axis cartesian industrial robot by KUKA Industries, a two-component injection molding system and an assembly unit. The plant produces plastic parts for electric toothbrushes.
In the research projects "MainTelRob" and "Bayern.digital", during which this plant was utilised, the Zentrum für Telematik e.V. (ZfT) and its project partners develop novel technical approaches and procedures for modern telemaintenance. The term "telemaintenance" hereby refers to the integration of computer science and communication technologies into the maintenance strategy. It is particularly interesting for high-grade capital-intensive goods like industrial robots. Typical telemaintenance tasks are for example the analysis of a robot failure or difficult repair operations. The service department of KUKA Industries is responsible for the worldwide distributed customers who own more than one robot. Currently such tasks are offered via phone support and service staff which travels abroad. They want to expand their service activities on telemaintenance and struggle with the high demands of teleoperation especially regarding security infrastructure. In addition, the facility in Marktheidenfeld has to keep up with the high international standards of Procter & Gamble and wants to minimize machine downtimes. Like 71.6 % of all German companies, P&G sees a huge potential for early information on their production system, but complains about the insufficient quality and the lack of currentness of data.
The main research focus of this work lies on the human machine interface for all human tasks in a telemaintenance setup. This thesis provides own work in the use of a mobile device in context of maintenance, describes new tools on asynchronous remote analysis and puts all parts together in an integrated telemaintenance infrastructure. With the help of Augmented Reality, the user performance and satisfaction could be raised. A special regard is put upon the situation awareness of the remote expert realized by different camera viewpoints. In detail the work consists of:
- Support of maintenance tasks with a mobile device
- Development and evaluation of a context-aware inspection tool
- Comparison of a new touch-based mobile robot programming device to the former teach pendant
- Study on Augmented Reality support for repair tasks with a mobile device
- Condition monitoring for a specific plant with industrial robot
- Human computer interaction for remote analysis of a single plant cycle
- A big data analysis tool for a multitude of cycles and similar plants
- 3D process visualization for a specific plant cycle with additional virtual information
- Network architecture in hardware, software and network infrastructure
- Mobile device computer supported collaborative work for telemaintenance
- Motor exchange telemaintenance example in running production environment
- Augmented reality supported remote plant visualization for better situation awareness
Information on the state of the terrestrial vegetation cover is important for several ecological, economical, and planning issues. In this regard, vegetation properties such as the type, vitality, or density can be described by means of continuous biophysical parameters. One of these parameters is the leaf area index (LAI), which is defined as half the total leaf area per unit ground surface area. As leaves constitute the interface between the biosphere and the atmosphere, the LAI is used to model exchange processes between plants and their environment. However, to account for the variability of ecosystems, spatially and temporally explicit information on LAI is needed both for monitoring and modeling applications.
Remote sensing aims at providing such information. LAI is commonly derived from remote sensing data by empirical-statistical or physical models. In the first approach, an empirical relationship between LAI measured in situ and the corresponding canopy spectral signature is established. Although this method achieves accurate LAI estimates, these relationships are only valid for the place and time at which the field data were sampled, which hampers automated LAI derivation. The physical approach uses a radiation transfer model to simulate canopy reflectance as a function of the scene’s geometry and of leaf and canopy parameters, from which LAI is derived through model inversion based on remote sensing data. However, this model inversion is not stable, as it is an under-determined and ill-posed problem.
Until now, LAI research focused either on the use of coarse resolution remote sensing data for global applications, or on LAI modeling over a confined area, mostly in forest and crop ecosystems, using medium to high spatial resolution data. This is why to date no study is available in which high spatial resolution data are used for LAI mapping in a heterogeneous, natural landscape such as alpine grasslands, although a growing amount of high spatial and temporal resolution remote sensing data would allow for an improved environmental monitoring. Therefore, issues related to model parameterization and inversion regularization techniques improving its stability have not yet been investigated for this ecosystem.
This research gap was taken up by this thesis, in which the potential of high spatial resolution remote sensing data for grassland LAI estimation based on statistical and radiation transfer modeling is analyzed, and the achieved accuracy and robustness of the two approaches is compared. The objectives were an ecosystem-adapted radiation transfer model set-up and an optimized LAI derivation in mountainous grassland areas. Multi-temporal LAI in situ measurements as well as time series of RapidEye data from 2011 and 2012 over the catchment of the River Ammer in the Bavarian alpine upland were used. In order to obtain accurate in situ data, a comparison of the LAI derivation algorithms implemented in the LAI-2000 PCA instrument with destructively measured LAI was performed first. For optimizing the empirical-statistical approach, it was then analyzed how the selection of vegetation indices and regression models impacts LAI modeling, and how well these models can be transferred to other dates. It was shown that LAI can be derived
with a mean accuracy of 80 % using contemporaneous field data, but that the accuracy decreases to on average 51 % when using these models on remote sensing data from other dates. The combined use of several data sets to create a regression which is used for LAI derivation at different points in time increased the LAI estimation accuracy to on average 65 %. Thus, reduced field measurement labor comes at the cost of LAI error rates being increased by 10 - 30 % as long as at least two campaigns are conducted. Further, it was shown that the use of RapidEye’s red edge channel improves the LAI derivation by on average 5.4 %.
With regard to physical LAI modeling, special interest lay in assessing the accuracy improvements that can be achieved through model set-up and inversion regularization techniques. First, a global sensitivity analysis was applied to the radiation transfer model in order to identify the most important model parameters and most sensitive spectral features. After model parameterization, several inversion regularizations, namely the use of a multiple sample solution, the additional use of vegetation indices, and the addition of noise, were analyzed. Further, an approach to include the local scene’s geometry in the retrieval process was introduced to account for the mountainous topography. LAI modeling accuracies of in average 70 % were achieved using the best combination of regularization techniques, which is in the upper range of accuracies that were achieved in the few existing other grassland studies based on in situ or air-borne measured hyperspectral data. Finally, further physically derived vegetation parameters and inversion uncertainty measures were evaluated in detail to identify challenging modeling conditions, which was mostly neglected in other studies. An increased modeling uncertainty for extremely high and low LAI values was observed. This indicates an insufficiently wide model parameterization and a canopy deviation from model assumptions on some fields. Further, the LAI modeling accuracies varied strongly between the different scenes. From this observation it can be deduced that the radiometric quality of the remote sensing data, which might be reduced by atmospheric effects or unexpected surface reflectances, exerts a high influence on the LAI modeling accuracy.
The major findings of the comparison between the empirical-statistical and physical LAI modeling approaches are the higher accuracies achieved by the empirical-statistical approach as long as contemporaneous field data are available, and the computationally efficiency of the statistical approach. However, when no or temporally unfitting in situ measurements are available, the physical approach achieves comparable or even higher accuracies. Furthermore, radiation transfer modeling enables the derivation of other leaf and canopy variables useful for ecological monitoring and modeling applications, as well as of pixel-wise uncertainty measures indicating the robustness and reliability of the model inversion and LAI derivation procedure. The established look-up tables can be used for further LAI derivation in Central European grassland also in other years.
The use of high spatial resolution remote sensing data for LAI derivation enables a reliable land cover classification and thus a reduced LAI mapping error due to misclassifications. Furthermore, the RapidEye pixels being smaller than individual fields allow for a radiation transfer model inversion over homogeneous canopies in most cases, as canopy gaps or field parcels can be clearly distinguished. However, in case of unexpected local surface conditions such as blooming, litter, or canopy gaps, high spatial resolution data show corresponding strong deviations in reflectance values and hence LAI estimation, which would be reduced using coarser resolution data through the balancing effect of the surrounding surface reflectances. An optimal pixel size with regard to modeling accuracy hence depends on the canopy and landscape structure. Furthermore, a reduced spatial resolution would enable a considerable acceleration of the LAI map derivation.
This illustration of the potential of RapidEye data and of the challenges associated to LAI derivation in heterogeneous grassland areas contributes to the development of robust LAI estimation procedures based on new and upcoming, spatially and temporally high resolution remote sensing imagery such as Landsat 8 and Sentinel-2.
The human gut is home for thousands of microbes that are important for human life. As most of these cannot be cultivated, metagenomics is an important means to understand this important community. To perform comparative metagenomic analysis of the human gut microbiome, I have developed SMASH (Simple metagenomic analysis shell), a computational pipeline. SMASH can also be used to assemble and analyze single genomes, and has been successfully applied to the bacterium Mycoplasma pneumoniae and the fungus Chaetomium thermophilum. In the context of the MetaHIT (Metagenomics of the human intestinal tract) consortium our group is participating in, I used SMASH to validate the assembly and to estimate the assembly error rate of 576.7 Gb metagenome sequence obtained using Illumina Solexa technology from fecal DNA of 124 European individuals. I also estimated the completeness of the gene catalogue containing 3.3 million open reading frames obtained from these metagenomes. Finally, I used SMASH to analyze human gut metagenomes of 39 individuals from 6 countries encompassing a wide range of host properties such as age, body mass index and disease states. We find that the variation in the gut microbiome is not continuous but stratified into enterotypes. Enterotypes are complex host-microbial symbiotic states that are not explained by host properties, nutritional habits or possible technical biases. The concept of enterotypes might have far reaching implications, for example, to explain different responses to diet or drug intake. We also find several functional markers in the human gut microbiome that correlate with a number of host properties such as body mass index, highlighting the need for functional analysis and raising hopes for the application of microbial markers as diagnostic or even prognostic tools for microbiota-associated human disorders.
African trypanosomes are unicellular parasites that cause nagana and sleeping sickness in livestock and man, respectively. The major pathogens for the animal disease include Trypanosoma vivax, T. congolense, and T. brucei brucei, whereas T. b. gambiense and T. b. rhodesiense are responsible for human infections. Given that the bloodstream form (BSF) of African trypanosomes is exclusively extracellular, its cell surface forms a critical boundary with the host environment. The cell surface of the BSF African trypanosomes is covered by a dense coat of immunogenic variant surface glycoproteins (VSGs). This surface protein acts as an impenetrable shield that protects the cells from host immune factors and is also involved in antibody clearance and antigenic variation, which collectively ensure that the parasite stays ahead of the host immune system. Gene expression in T. brucei is markedly different from other eukaryotes: most genes are transcribed as long polycistronic units, processed by trans-splicing a 39-nucleotide mini exon at the 5′ and polyadenylation at the 3′ ends of individual genes to generate the mature mRNA.
Therefore, gene expression in T. brucei is regulated post-transcriptionally, mainly by the action of RNA binding proteins (RBPs) and conserved elements in the 3′ untranslated regions (UTR) of transcripts. The expression of VSGs is highly regulated, and only a single VSG gene is expressed at a time from one of the ~15 subtelomeric domains termed bloodstream expression sites (BES). When cells are engineered to simultaneously express two VSGs, the total VSG mRNA do not exceed the wild type amounts. This suggests that a robust VSG mRNA balancing mechanism exists in T. brucei. The present study uses inducible and constitutive expression of ectopic VSG genes to show that the endogenous VSG mRNA is regulated only if the second VSG is properly targeted to the ER. Additionally, the endogenous VSG mRNA response is triggered when high amounts of the GFP reporter with a VSG 3′UTR is targeted to the ER. Further evidence that non-VSG ER import signals can efficiently target VSGs to the ER is presented. This study suggests that a robust trans-regulation of the VSG mRNA is elicited at the ER through a feedback loop to keep the VSG transcripts in check and avoid overshooting the secretory pathway capacity.
Further, it was shown that induction of expression of the T. vivax VSG ILDat1.2 in T. brucei causes a dual cell cycle arrest, with concomitant upregulation of the protein associated with differentiation (PAD1) expression. It could be shown that T. vivax VSG ILDat1.2 can only be sufficiently expressed in T. brucei after replacing its native GPI signal peptide with that of a T. brucei VSG. Taken together, these data indicate that inefficient VSG GPI anchoring and expression of low levels of the VSG protein can trigger differentiation from slender BSF to stumpy forms. However, a second T. vivax VSG, ILDat2.1, is not expressed in T. brucei even after similar modifications to its GPI signals. An X-ray crystallography approach was utilized to solve the N-terminal domain (NTD) structure of VSG ILDat1.2. This is first structure of a non-T. brucei VSG, and the first of a surface protein of T. vivax to be solved. VSG ILDat1.2 NTD maintains the three-helical bundle scaffold conserved in T. brucei surface proteins. However, it is likely that there are variations in the architecture of the membrane proximal region of the ILDat1.2 NTD and its CTD from T. brucei VSGs. The tractable T. brucei system is presented as a model that can be used to study surface proteins of related trypanosome species, thus creating avenues for further characterization of trypanosome surface coats.
Oxygen-centered radicals are important intermediates in photobiological, mechanistic and synthetic studies. The majority of precursors of reactive oxyl radicals are labile and thus delicate to handle. Therefore N-(alkoxy)-pyridinethiones and N-(Alkoxy)-thiazolethiones have attracted attention as "mild'' photochemical source of alkoxyl radicals, in the last few years. A disadvantage of the pyridine compounds, is their sensibility to daylight. Despite of their similarities, both molecules behave surprisingly different, if photolyzed in the absence of trapping reagents. The pyridinethione compounds undergo highly efficient radical chain reactions under such conditions while the corresponding thiazolethiones react surprisingly sluggish and give rise to several unwanted side products. The properties of both compounds should be understood and optimized in the frame of this work. Additionally new compounds should be suggested that can also be applied in the photochemical alkoxyl radical generation. Some background information about the generation and application of alkoxyl radicals is provided in chapter 2. Electronic excitations and UV/vis spectroscopy together with a description of quantum chemical approaches that are able to calculate such phenomena are outlined in chapter 3. Chapter 4 deals with the description of the vertical excitation spectra. During the validation CASSCF, CASPT2, TD-DFT and RI-CC2 were tested with respect to their ability to describe the vertical excitations in both compounds. The CASPT2 approach gives accurate descriptions of the electronic excitation spectra of all compounds. The time-dependent DFT results are very sensitive on the choice of the functional and a validation of the results should be always done. On the basis of these computations the spectroscopic visible absorption bands of both compounds were assigned to a pi-->pi* transition in the thiohydroxamic acid functionality. In chapter 5 the mechanism of the thermally and the photochemically induced N,O homolysis in both compounds is unveiled. The near UV-induced N,O homolysis will start from the S2 state. The expected relaxation from the S2- to the S1-state and the dissociation process is expected to be very fast in the case of the thiazolethione compound. The potential surfaces of the pyridine compound in contrast point to a slower N,O bond dissociation. Due to the resulting faster dissociation process the excess energy which results from the photochemical activation is quenched only to small amounts. The maximal possible excess energy of the fragments is lower and a quenching is much more likely in the case of the pyridinethione compounds. This explaines the different reactivities of both compounds. For the also already successfully applied precursor system N-(alkoxy)-pyridineones the computed dissociation paths show courses that clearly predict a slow bond dissociation process. Chapter 6 deals with the tuning of the initial excitation wave length of the known pyridinethiones und thiazolethiones. In the first part the effects of substituents on the thiazolethione heterocycle was examined. The UV/vis spectra of 4 and 5 substituted thiazolethiones can be interpreted like the spectrum of the parent compound. The second part of chapter 6 deals with the identification of a substitution pattern on the pyridine heterocycle which induces a blue shift of the photo active band. The computations showed that electron rich and electron poor substituents result the same effects on the electronic excitation spectra. These substituent effects are additive, but the steric orientation of the substituents has to be taken into account. Chapter 7 describes a computer aided design of new alkoxyl radical precursors. Combining the advantages of both compounds the radical formation should be initiated by an irradiation with light at about 350 nm, and the amount of side products during the radical formation process should be small. To achieve this 18 test candidates were obtained by a systematic variation of the parent compound of the thiazolethione precursor. To identify the promising new precursor systems a screening of the lower electronic excitations of all resulting 18 systems was performed with TD-DFT. For promising systems the N,O or P,O dissociation paths, respectively, were analyzed according to the developed model. N-(methoxy)-azaphospholethione and N-(methoxy)-pyrrolethione seem to be the most promising candidates. The computations predict a strong absorption at about 350 nm respectively 320 nm. Due to the amounts of maximal excess energy and the shapes of the potential surfaces of the N,O bond dissociation paths their reactivity should resemble more the behavior of the pyridinethiones.
Nature-based tourism and ecotourism experienced a dynamic development over the past decade. While originally often described as specialized post-Fordist niche markets for ecologically aware and affluent target groups, in many regions they are nowadays characterized by a heterogeneous structure and the presence of a wide product range, from individual travels to package tours.
The present dissertation analyzes the structure and economic importance of tourism in two highly frequented protected areas in middle income countries, the Sian Ka’an Biosphere Reserve (SKBR) in Mexico and the Souss-Massa National Park (SMNP) in Morocco. Both areas are situated in close proximity to the most important package tour destinations Cancún (Mexico) and Agadir (Morocco) and are subject to high touristic use and development pressure. So far, the planning of a more sustainable tourism development is hampered by the lack of reliable data.
Based on demand-side surveys and income multipliers calculated with the help of regionalized input-output models, the visitor structure and economic impact of tourism in both protected areas are described. With regional income effects of approximately 1 million USD (SKBR) and approximately 1.9 million USD (SMNP), and resulting income equivalents of 1,348 and 5,218 persons, both the SKBR and the SMNP play an important—and often undervalued—role for the regional economies in underdeveloped rural peripheral regions of the countries.
Detailed analyses of the visitor structures show marked differences with regard to criteria such as travel organization, nature/protected area affinity and expenditures. With regard to planning and marketing of nature-based tourism, protected area managers and political decision-takers are advised to focus on ecologically and economically attractive visitor groups. Based on the results of the two case studies as well as existing tourism typologies from the literature, a classification scheme is presented that may be used for a more target-oriented development and marketing of nature-based tourism products.
The presented thesis deals with the investigation of the characteristic physical properties of lead-free double perovskites. For this purpose lead-free double perovskite single crystals were grown from solution. In order to assess the influence of growth temperature on tail states in the material, the crystals were studied using Photoluminescence Excitation (PLE) and Transmission measurements. Additionally, lead-free double perovskite solar cells and thin films were investigated to address the correlation of precursor stoichiometry and solar cell efficiency. In a last step a new earth abundant lead-free double perovskite was introduced and its physical properties were studied by photoluminescene and absorptance. Like this it was possible to assess the suitability of this material for solar cell applications in the future.
Merocyanine Dyes as Organic Semiconductors for Vacuum-processed Solar Cell and Transistor Devices
(2015)
The present thesis comprises the synthesis of new functional merocyanine dyes, the study of their electro-optical properties as well as solid state packing and their application as p-type semiconductor materials in transistor and solar cell devices. The absorption properties of the obtained compounds could be modified by variation of the donor unit, the introduction of electron-withdrawing substituents in the acceptor unit or elongation of the polymethine chain. For a particular dye, the absorption band could be shifted by more than 160 nm by increasing the solvent polarity due to a conformational switch between a merocyanine-like and a cyanine-like structure. Single crystal analyses revealed that the studied dyes tend to pack either in an antiparallel fashion forming dimers with no overall dipole moment or in a staircase-like pattern where the dipole moments point to the same direction and are only balanced by another staircase oriented in the opposite direction (stair dimer). With respect to application as semiconductor materials, the latter packing arrangement resulted most favorable for charge carrier mobility. We concluded that this packing motif is preserved in the solar cell devices, where the selenium-containing dye afforded the highest performance of this series for an optimized planar-mixed heterojunction solar cell (6.2 %).
A rising percentage of women with risk factors for cardiovascular disease (CVD) reach menopause and experience postmenopausal symptoms. In consequence they require assessment concerning the appropriate combination and safety of a hormone replacement therapy. Clinical trials using the combination of equine estrogens and medroxyprogesterone acetate (MPA) reported an increased risk of thromboembolic events and no cardiovascular protective effects in women receiving this type of hormone replacement therapy. However unopposed estradiol and different regimes estrogens/progestins in vitro and in animal studies have proved to be beneficial for the cardiovascular system. Thus it is possible that the negative outcomes of the clinical trials are an exclusive feature of the regime equine estrogens plus MPA. The present study was initiated to evaluate the cardiovascular effects and possible mechanism of damage of the regime MPA plus 17ß-estradiol in comparison to unopposed 17ß-estradiol during cardiac disease. The role of 17ß-estradiol and MPA during left ventricular dysfunction and chronic heart failure was studied in female Wistar rats that received myocardial infarction. After 8 weeks of treatment the combination of MPA plus estradiol aggravated left ventricular remodelling and dysfunction as judged by increased heart weight, elevated left ventricular end diastolic pressure and decreased left ventricular fractional shortening, effects that were accompanied by increase left ventricular oxidative stress and expression of rac 1 and p67phox regulatory subunits of the NADPH oxidase. In contrast ovariectomy as well as 17ß- estradiol supplementation conferred neutral effects on cardiac function and remodelling post myocardial infarction. Suggesting that the aggravating symptoms of the regime MPA plus 17ß –estradiol are inherent to this pharmacological regime and are not a class effect of the progesterone receptor ligands and are neither due to inhibition of estradiol beneficial effects. Considering that aldosterone plays an important role in the development and aggravation of cardiovascular disease the cardiovascular effects of MPA plus 17ß –estradiol was studied in a model of mineralocorticoid receptor activation and compared to the effects of regimes based in drospirenone, a new progestin with antimineralocorticoid properties. The complex pattern of cardiovascular injury in ovariectomized Wistar rats induced by 8 weeks of continuous chronic aldosterone infusion and high-salt diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17 ß-estradiol in ovariectomized animals. The beneficial role of 17 ß-estradiol on blood pressure, cardiac hypertrophy, vascular osteopontin expression and perivascular fibrosis was completely abrogated by coadministration of MPA. In contrast, drospirenone was either neutral or additive to 17 ß-estradiol in protecting against aldosterone salt-induced cardiovascular injury and inflammation. Taking into account that the kidney plays a major role for the development and aggravation of hypertension a further characterization of fluid balance, renal morphology and renal gene expression in the aldosterone salt treated rats was conducted. Aldo-salt treatment resulted in remnant kidney hypertrophy without structural damage, effects that were not modified by 17 ß-estradiol. However combination of MPA with 17 ß-estradiol enhanced kidney hypertrophy, fluid turnover, renal sodium retention and potassium excretion and was associated with increased renal ENaC expression, extensive renal lesions, tubular damage and enhanced p67phox expression and protein tyrosin nitrosylation. Different to the protective effects of drospirenone that included a complete blockade of kidney hypertrophy and sodium retention and enhanced renal expression of angiotensin II type-2 receptors. Therefore the loss of 17 ß-estradiol cardiovascular beneficial effects and the renal harmful effects in the aldosterone salt treated rats receiving MPA can not be extrapolated to other progestins. Indeed drospirenone conferred protective effects due to its antimineralocorticoid properties. In conclusion, the choice of specific synthetic progestins has profound implications on the development of cardiovascular and renal injury; MPA aggravated cardiac disease, which contributes to explain the adverse outcomes of clinical trials on the prevention of cardiovascular disease by combined estrogen and MPA treatment.
The taphonomic and paleoecologic aspects of the Upper Hauterivian to Lower Barremian Agua de la Mula Member of the Agrio Formation (Neuquén Basin, Argentina) were studied in the frame of the sequence stratigraphic paradigm. The Agua de la Mula Member, a ca. 600 m thick succession of highly cyclic marine sediments was surveyed at two localities. Detailed bed-by-bed sedimentologic, stratigraphic, ichnologic, taphonomic and paleoecologic data collection allowed a precise paleoenvironmental, stratigraphic, taphonomic and synecologic interpretation, in a controlled sequence stratigraphic framework. The main architectural stratigraphic component is the Starvation-Dilution Sequence, interpreted as a the effect of a sixth-order, Milankovitch precession-driven cycle. Dilution hemisequences are siliciclastic-dominated and show evidence of depth changes. Starvation hemisequences show a diverse variation of mixed carbonate-siliciclastic facies that is linked to sequence stratigraphy. Ammonite-based biostratigraphy was revised and new knowledge proposed. The stratigraphic framework was improved by combining biostratigraphy, sequence stratigraphy and event stratigraphy. Nine main sequences were described, linked to other stratigraphic markers and correlated with other sequence stratigraphic charts. Several orders of cyclicity were inferred. Third- and fourth-order sequences are the major sequences, not subordinated to higher hierarchies (lower order). Precession, obliquity, and short and long eccentricity cycles of the Milankovitch band are proposed. Among the different sequence stratigraphic models the transgression-regression model fits the majority of the sequences described in this work. The depositional-sequence model could be applied only to the first third-order sequence, in which the true sequence boundary is identifiable. Starvation-dilution sequences, however, are composed by to components that are not completely explained by those models. Starvation hemisequences developed in intermediate to deep settings record the transgressive phase as well as the earLy regressive one without visible stratigraphic boundaries. 112 samples with 22,572 individuals were grouped into fifteen fossil associations and one assemblage that reflect the interaction of different factors: age, position in major, medium and starvation dilution sequences and, linked to sequence stratigraphy, depth, oxygen availability, rate of terrigenous input, water agitation, and substrate conditions. Temporary possible reduction in oxygen content is inferred based on all sources of available evidence. Organic buildups are briefly described and their development interpreted in terms of the sequence stratigraphic framework. Vertical patterns of replacement of fossil associations are described and related to sequence stratigraphy. Five types of skeletal concentrations represent the diversity of coquinas decribed in this study. Type 1, 2, 4 and 5 correspond to starvation hemisequences deposited in progressively shallower settings, from basin to inner ramp. Type 3 is embedded into dilution hemisequences and inferred to be linked to shell bed type I of Kidwell (1985). Types 1 and 2 correspond to transgression, maximum flooding and early regression without distinction. Type 4A as well as Type 5 are interpreted as onlap shell beds (Kidwell 1991a) or early TST shell beds (Fürsich and Pandey 2003). Type 4B corresponds to the MFZ shell bed (Fürsich and Pandey 2003) or mid-cycle shell bed (Abbott 1997), while Type 4C to the downlap shell bed (Kidwell 1991a). Time-averaging of shell beds was assessed with precision as the time involved in the deposition of the starvation hemisequences could be inferred. All shell beds comprise within-habitat assemblages forming within a few thousand years, with little environmental condensation. The fossilization of the marine calcareous shells is modelled as a series of steps called windows: environmental, destructional, burial and diagenetic. The “diagenetic window” is the most relevant. Connected to this it is proposed that carbonate dissolution is the primary control on the development of shell beds, as has been proposed before (Fürsich 1982; Fürsich and Pandey 2003). The interpretative power resulting from combining several lines of evidence, e.g., facies analysis, sequence stratigraphy, biostratigraphy, trace fossil analysis, paleoecology and taphonomy, and unravelling their multiple relationships, are the most relevant conclusions of this study.
Serotonin (5-HT) has been implicated in the regulation of emotions as well as in its pathological states, such as anxiety disorders and depression. Mice with targeted deletion of genes encoding various mediators of central serotonergic neurotransmission therefore provides a powerful tool in understanding contributions of such mediators to homeostatic mechanisms as well as to the development of human emotional disorders. Within this thesis a battery of electrophysiological recordings were conducted in the dorsal raphe nucleus (DRN) and the hippocampus of two murine knockout lines with deficient serotonergic systems. Serotonin transporter knockout mice (5-Htt KO), which lack protein responsible for reuptake of 5-HT from the extracellular space and tryptophan hydroxylase 2 knockout (Tph2 KO) mice, which lack the gene encoding the neuronal 5-HT-synthesising enzyme. First, 5-HT1A receptor-mediated autoinhibition of serotonergic neuron firing in the DRN was assessed using the loose-seal cell-attached configuration. Stimulation of 5-HT1A receptors by a selective agonist, R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT), showed a mild sensitisation and a marked desensitisation of these receptors in Tph2 KO and 5-Htt KO mice, respectively. While application of tryptophan, a precursor of 5-HT and a substrate of Tph2, did not cause autoinhibition in Tph2 KO mice due to the lack of endogenously produced 5-HT, data from 5-Htt KO mice as well as heterozygous mice of both KO mice lines demonstrated the presence of autoinhibitory mechanisms as normal as seen in wildtype (WT) controls. When the Tph2-dependent step in the 5-HT synthesis pathway was bypassed by application of 5-hydroxytryptophan (5-HTP), serotonergic neurons of both Tph2 KO and 5-Htt KO mice showed decrease in firing rates at lower concentrations of 5-HTP than in WT controls. Elevated responsiveness of serotonergic neurons from Tph2 KO mice correspond to mild sensitisation of 5-HT1A receptors, while responses from 5-Htt KO mice suggest that excess levels of extracellular 5-HT, created by the lack of 5-Htt, stimulates 5-HT1A receptors strong enough to overcome desensitisation of these receptors. Second, the whole-cell patch clamp recording data from serotonergic neurons in the DRN showed no differences in basic electrophysiological properties between Tph2 KO and WT mice, except lower membrane resistances of neurons from KO mice. Moreover, the whole-cell patch clamp recording from CA1 pyramidal neurons in the hippocampus of 5-Htt KO mice showed increased conductance both at a steady state and at action potential generation. Lastly, magnitude of long-term potentiation (LTP) induced by the Schaffer collateral/commissural pathway stimulation in the ventral hippocampus showed no differences among Tph2 KO, 5-Htt KO, and WT counterparts. Taken together, lack and excess of extracellular 5-HT caused sensitisation and desensitisation of autoinhibitory 5-HT1A receptors, respectively. However, this may not directly translate to the level of autoinhibitory regulation of serotonergic neuron firing when these receptors are stimulated by endogenously synthesised 5-HT. In general, KO mice studied here showed an astonishing level of resilience to genetic manipulations of the central serotonergic system, maintaining overall electrophysiological properties and normal LTP inducibility. This may further suggest existence of as-yet-unknown compensatory mechanisms buffering potential alterations induced by genetic manipulations.
Understanding the causal relationship between genotype and phenotype is a major objective in biology. The main interest is in understanding trait architecture and identifying loci contributing to the respective traits. Genome-wide association mapping (GWAS) is one tool to elucidate these relationships and has been successfully used in many different species. However, most studies concentrate on marginal marker effects and ignore epistatic and gene-environment interactions. These interactions are problematic to account for, but are likely to make major contributions to many phenotypes that are not regulated by independent genetic effects, but by more sophisticated gene-regulatory networks. Further complication arises from the fact that these networks vary in different natural accessions. However, understanding the differences of gene regulatory networks and gene-gene interactions is crucial to conceive trait architecture and predict phenotypes.
The basic subject of this study – using data from the Arabidopsis 1001 Genomes Project – is the analysis of pre-mature stop codons. These have been incurred in nearly one-third of the ~ 30k genes. A gene-gene interaction network of the co-occurrence of stop codons has been built and the over and under representation of different pairs has been statistically analyzed. To further classify the significant over and under- represented gene-gene interactions in terms of molecular function of the encoded proteins, gene ontology terms (GO-SLIM) have been applied. Furthermore, co- expression analysis specifies gene clusters that co-occur over different genetic and phenotypic backgrounds. To link these patterns to evolutionary constrains, spatial location of the respective alleles have been analyzed as well. The latter shows clear patterns for certain gene pairs that indicate differential selection.
Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely.
In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a Förster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA.
Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.
Part 1 of this work describes the development of accurate physically grounded force fields for
intermolecular Cation-π interactions based on SAPT energy decomposition analysis.
The presented results demonstrate the benefits of the used DFT-SAPT method to describe non-bonding
interactions. First of all, this method is able to reproduce the high level CCSD(T) energy values
but using much less computational time. Second it provides the possibility to separate the total
intermolecular interaction energy into several physically meaningful contributions. The relative
contributions of the dimers investigated can be seen in Fig. 6.16. In Tab. 6.3 the percentage
contribution of the attractive energy parts to the stabilization energy is shown. The polarization
energy is important for the NH+...C6H6 interaction, whereas it becomes less crucial
considering other dimers. The dispersion energy contribution is large in the case of
the C6H6...H2O dimers, whereas it is relatively less important for the NH+...C6H6
interaction. The electrostatic energy contributes a large amount of stabilizing energy
in all considered dimer interactions. ...
New experimental methods have drastically accelerated the pace and quantity at which biological data is generated. High-throughput DNA sequencing is one of the pivotal new technologies. It offers a number of novel applications in various fields of biology, including ecology, evolution, and genomics. However, together with those opportunities many new challenges arise. Specialized algorithms and software are required to cope with the amount of data, often requiring substantial training in bioinformatic methods. Another way to make those data accessible to non-bioinformaticians is the development of programs with intuitive user interfaces.
In my thesis I developed analyses and programs to tackle current problems with high-throughput data in biology. In the field of ecology this covers the establishment of the bioinformatic workflow for pollen DNA meta-barcoding. Furthermore, I developed an application that facilitates the analysis of ecological communities in the context of their traits. Information from multiple public databases have been aggregated and can now be mapped automatically to existing community tables for interactive inspection. In evolution the new data are used to reconstruct phylogenetic trees from multiple genes. I developed the tool bcgTree to automate this process for bacteria. Many plant genomes have been sequenced in current years. Sequencing reads of those projects also contain data from the chloroplasts. The tool chloroExtractor supports the targeted extraction and analysis of the chloroplast genome. To compare the structure of multiple genomes specialized software is required for calculation and visualization of the relationships. I developed AliTV to address this. In contrast to existing programs for this task it allows interactive adjustments of produced graphics. Thus, facilitating the discovery of biologically relevant information. Another application I developed helps to analyze transcriptomes even if no reference genome is present. This is achieved by aggregating the different pieces of information, like functional annotation and expression level, for each transcript in a web platform. Scientists can then search, filter, subset, and visualize the transcriptome.
Together the methods and tools expedite insights into biological systems that were not possible before.
LIM and SH3 protein 1 (LASP1) is a nucleocytoplasmic scaffolding protein. LASP1 interacts with various cytoskeletal proteins via its domain structure and is known to participate in physiological processes of cells. In the present study, a detailed investigation of the expression pattern of LASP1 protein in normal skin, melanocytic nevi and melanoma was carried out and the melanocyte–specific function of LASP1 was analyzed. LASP1 protein was identified in stratum basale of skin epidermis and a very high level was detected in nevi, the benign tumor of melanocyte. In the highly proliferative basal cells, an additional distinct nuclear localization of the protein was noted. In different tumor entities, an elevated LASP1 expression and nuclear localization, correlated positively with malignancy and tumor grade. However, LASP1 level was determined to be very low in melanoma and even reduced in metastases. Melanoma is distinguished as the first tumor tested to date – that displayed an absence of elevated LASP1 expression. In addition no significant relation was observed between LASP1 protein expression and clinicopathological parameters in melanoma.
The epidermal melanin unit of skin comprises of melanocytes and keratinocytes. Melanocytes are specialized cells that synthesize the photo protective coloring pigment, melanin inside unique organelles called melanosomes. The presence of LASP1 in melanocytes is reported for the first time through this study and the existence was confirmed by immunoblotting analysis in cultured normal human epidermal melanocyte (NHEM) and in melanoma cell lines, along with the immunohistostaining imaging in normal skin and in melanocytic nevi. LASP1 depletion in MaMel2 cells revealed a moderate increase in the intracellular melanin level independently of de novo melanogenesis, pointing to a partial hindrance in melanin release. Immunofluorescence images of NHEM and MaMel2 cells visualized co-localization of LASP1 with dynamin and tyrosinase concomitant with melanosomes at the dendrite tips of the cells. Melanosome isolation experiments by sucrose density gradient centrifugation clearly demonstrated the presence of LASP1 and the melanosome specific markers tyrosinase and TRP1 in late stage melanosomes.
The study identified LASP1 and dynamin as novel binding partners in melanocytes and provides first evidence for the existence of LASP1 and dynamin (a protein well–known for its involvement in vesicle formation and budding) in melanosomes. Co-localization of LASP1 and dynamin along the dendrites and at the tips of the melanocytes indicates a potential participation of the two proteins in the membrane vesicle fission at the plasma membrane.
In summary, a possible involvement of LASP1 in the actin–dynamin mediated membrane fission and exocytosis of melanin laden melanosome vesicles into the extracellular matrix is suggested.
While beneficial sponge-microbe associations have received much attention in recent years, less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic microorganisms. Thus, the aim of this study was to examine two selected Caribbean disease conditions, termed “Sponge Orange Band” and “Sponge White Patch”, via ecological and molecular methods. Sponge Orange Band (SOB) disease affects the prominent Caribbean barrel sponge Xestospongia muta that is counted among the high-microbial-abundance (HMA) sponges, whereas Sponge White Patch (SWP) disease affects the abundant rope sponge Amphimedon compressa that belongs to the low-microbial-abundance (LMA) sponges. I have documented for both Caribbean sponge diseases a disease progression going along with massive tissue destruction as well as loss of the characteristic microbial signatures. Even though new bacteria were shown to colonize the bleached areas, the infection trials revealed in both cases no indication for the involvement of a microbial pathogen as an etiologic agent of disease leaving us still in the dark about the cause of Sponge Orange Band as well as Sponge White Patch disease.
Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses
(2021)
Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks.
In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply.
Infrared photodissociation spectroscopy of ionic hydrocarbons : microsolvation and protonation sites
(2007)
This work has presented a spectroscopic analysis of three types of hydrocarbon cations: two ionized aromatic hydrocarbons, two protonated aromatic hydrocarbons and the cation of a fundamental radical hydrocarbon. The experiments were centered on the proton stretch vibrations of mass-selected complexes of these systems and polar (H2O) and non polar (Ar, N2, CO2) ligands. The experiments have been done in a tandem mass spectrometer coupled with an electron impact ionization ion source; an OPO laser system was used as tunable IR light source. All the proposed dimer structures have been also modeled using quantum chemical calculations (QCC). These calculations have consistently been matched with the experimental results and have enabled clear identification of the spectral features observed. This has enabled the evaluation of thermochemical properties which could not be extracted directly from experiment. The experiments done on complexes of 1-Np+ and Im+ have allowed for the acidity of their various groups to be probed: the shifts in the frequency as well as the enhancement in the intensity of the OH and NH stretch vibrations resulting from the complexation have yielded dependences on both the species (L) and the number (n) of the ligands. OH bound 1-Np+···Ar has been detected for the first time, showing that the REMPI-IRPD method is severely limited with respect to the production of the most stable isomer of a given cationic complex. The detection of c-1-Np+···(N2)n corresponds to the first observation of c-1-Np+ complexes and enables thus direct comparison of both 1-Np+ rotamers. The shift of the NH vibration of Im+···N2(H) yielded the first experimental estimate for the PA of the imidazyl radical. It was also found that the most stable 1-Np+···Ar and Im+···Ar structures differ qualitatively from that of the corresponding neutral dimers (H-bound vs pi-bound), emphasizing the large impact of ionization on the interaction potential and the preferred recognition motif between acidic aromatic molecules (A) and nonpolar ligands. The IRPD spectra of 1-Np+···Ln and Im+···Ln yielded spectroscopic information about the CH, NH and OH stretch vibrations of bare 1-Np+ and Im+. The dependence of the shifts in the frequency of the OH and NH stretch vibrations allows for creating microsolvation models. The spectroscopic results obtained on size-selected 1-NpH+···Ln show that, in the output of the presently used ion source, three classes of 1-NpH+ isomers can be identified: oxonium ions (1-Np protonated at the O atom); carbenium ions obtained by protonation in the para and ortho positions with respect to the OH functional group; carbenium ions obtained by the addition of a proton to well-defined sites on the second naphthalene ring. The spectral identification of these three classes of protonation sites is supported by their different photofragmentation patterns. It was demonstrated that the spectroscopic monitoring of the microsolvation of ImH+ in Ar and N2 together with the QCCs paint a very detailed picture of the microsolvation process, evidencing clear differences between the microsolvation models as function of the PA of the ligands. Important differences have also been identified between the various binding sites, enabling the creation of a clear scale of priorities for occupation of the binding sites during microsolvation. The application of IRPD to the study of microhydrated ImH+ provided for the first time direct spectroscopic information on the properties of the N-H bonds of this biomolecular building block under controlled microhydration. It was demonstrated that, as protonation enhances the acidity of the NH groups, the ability for proton conductivity of ImH+ increases. A very important result is derived from the IRPD spectroscopy of C2H5+···L (L = Ar, N2, CO2, CH4) dimers. The equilibrium geometry of the C2H5+ has long been debated. Now, IRPD spectra were recorded over the range of the CH stretch fundamentals (covering possible sp3 and sp2 hybridization of C). Depending on the ligand species, the spectra are found to be dominated by the fingerprint of two largely different dimer geometries. Using the experimental C2H5+···Ar spectrum and the corresponding QCCs, the structure of the (weakly perturbed) C2H5+ was found to be the nonclassical one, with one proton straddling across the C=C bond of the H2C=CH2. On the other hand, ligands like N2 and CH4 are strongly influencing the geometry, as seen in the spectral signatures of the C2H5+···N2 and C2H5+···CH4, which correspond to the classical [H2CCH3]+. It was thus demonstrated that while the nonclassical C2H5+ is the global minimum on the PES of the free [C2,H5]+, the structure of the C2H5+ can be strongly influenced by the chemical properties of the environment.
In order to survive, organisms avoid threats and seek rewards. Classical conditioning is a simple model to explain how animals and humans learn associations between events that allow them to predict threats and rewards efficiently. In the classical conditioning paradigm, a neutral stimulus is paired with a biologically significant event (the unconditioned stimulus – US). In virtue of this association, the neutral stimulus acquires affective motivational properties, and becomes a conditioned stimulus (CS+). Defensive responses emerge for pairings with an aversive US (e.g., pain), and appetitive responses emerge for pairing with an appetitive event (e.g., reward). It has been observed that animals avoid a CS+ when it precedes an aversive US during a training phase (CS+ US; forward conditioning); whereas they approach a CS+ when it follows an aversive US during the training phase (US CS+; backward conditioning). These findings indicate that the CS+ acquires aversive properties after a forward conditioning, whereas acquires appetitive properties after a backward conditioning. It is thus of interest whether event timing also modulates conditioned responses in such an opponent fashion in humans, who are capable of explicit cognition about the associations. For this purpose, four experiments were conducted in which a discriminative conditioning was applied in groups of participants that only differed in the temporal sequence between CS+ onset and US onset (i.e., the interstimulus interval – ISI). During the acquisition phase (conditioning), two simple geometrical shapes were presented as conditioned stimuli. One shape (CS+) was always associated with a mild painful electric shock (i.e., the aversive US) and the other one (CS-) was never associated with the shock. In a between-subjects design, participants underwent either forward or backward conditioning. During the test phase (extinction), emotional responses to CS+ and CS- were tested and the US was never presented. Additionally, a novel neutral shape (NEW) was presented as control stimulus. To assess cognitive components, participants had to rate both the valence (the degree of unpleasantness or pleasantness) and the arousal (the degree of calmness or excitation) associated with the shapes before and after conditioning. In the first study, startle responses, an ancestral defensive reflex consisting of a fast twitch of facial and body muscles evoked by sudden and intense stimuli, was measured as an index of stimulus implicit valence. Startle amplitude was potentiated in the presence of the forward CS+ whilst attenuated in the presence of the backward CS+. Respectively, the former response indicates an implicit negative valence of the CS+ and an activation of the defensive system; the latter indicated an implicit positive valence of the CS+ and an activation of the appetitive system. In the second study, the blood-oxygen level dependent (BOLD) response was measured by means of functional magnetic resonance imaging (fMRI) to investigate neural responses after event learning. Stronger amygdala activation in response to forward CS+ and stronger striatum activation in response to backward CS+ were found in comparison to CS-. These results support the notion that the defensive motivational system is activated after forward conditioning since the amygdala plays a crucial role in fear acquisition and expression. Whilst the appetitive motivational system is activated after backward conditioning since the striatum plays a crucial role in reward processing. In the third study, attentional processes underlying event learning were observed by means of steady-state visual evoked potentials (ssVEPs). This study showed that both forward and backward CS+ caught attentional resources. More specifically, ssVEP amplitude was higher during the last seconds of forward CS+ that is just before the US, but during the first seconds of backward CS+ that is just after the US. Supposedly, attentional processes were located at the most informative part of CS+ in respect to the US. Participants of all three studies rated both forward and backward CS+ more negative and arousing compared to the CS-. This indicated that event timing did not influence verbal reports similarly as the neural and behavioral responses indicating a dissociation between the explicit and implicit responses. Accordingly, dual process theories propose that human behavior is determined by the output of two systems: (1) an impulsive implicit system that works on associative principles, and (2) a reflective explicit system that functions on the basis of knowledge about facts and values. Most importantly, these two systems can operate in a synergic or antagonistic fashion. Hence, the three studies of this thesis congruently suggest that the impulsive and the reflective systems act after backward association in an antagonistic fashion. In sum, event timing may turn punishment into reward in humans even though they subjectively rate the stimulus associated with aversive events as being aversive. This dissociation might contribute to understand psychiatric disorders, like anxiety disorders or drug addiction.
Large-scale anatomical and functional analyses of the connectivity in both invertebrate and mammalian brains have gained intense attention in recent years. At the same time, the understanding of synapses on a molecular level still lacks behind. We have only begun to unravel the basic mechanisms of how the most important synaptic proteins regulate release and reception of neurotransmitter molecules, as well as changes of synaptic strength. Furthermore, little is known regarding the stoichiometry of presynaptic proteins at different synapses within an organism. An assessment of these characteristics would certainly promote our comprehension of the properties of different synapse types. Presynaptic proteins directly influence, for example, the probability of neurotransmitter release as well as mechanisms for short-term plasticity. We have examined the strength of expression of several presynaptic proteins at different synapse types in the central nervous system of Drosophila melanogaster using immunohistochemistry. Clear differences in the relative abundances of the proteins were obvious on different levels: variations in staining intensities appeared from the neuropil to the synaptic level. In order to quantify these differences, we have developed a ratiometric analysis of antibody stainings. By application of this ratiometric method, we could assign average ratios of presynaptic proteins to different synapse populations in two central relays of the olfactory pathway. In this manner, synapse types could be characterized by distinct fingerprints of presynaptic protein ratios. Subsequently, we used the method for the analysis of aberrant situations: we reduced levels of Bruchpilot, a major presynaptic protein, and ablated different synapse or cell types. Evoked changes of ratio fingerprints were proportional to the modifications we had induced in the system. Thus, such ratio signatures are well suited for the characterization of synapses. In order to contribute to our understanding of both the molecular composition and the function of synapses, we also characterized a novel synaptic protein. This protein, Drep-2, is a member of the Dff family of regulators of apoptosis. We generated drep-2 mutants, which did not show an obvious misregulation of apoptosis. By contrast, Drep-2 was found to be a neuronal protein, highly enriched for example at postsynaptic receptor fields of the input synapses of the major learning centre of insects, the mushroom bodies. Flies mutant for drep-2 were viable but lived shorter than wildtypes. Basic synaptic transmission at both peripheral and central synapses was in normal ranges. However, drep-2 mutants showed a number of deficiencies in adaptive behaviours: adult flies were locomotor hyperactive and hypersensitive towards ethanol-induced sedation. Moreover, the mutant animals were heavily impaired in associative learning. In aversive olfactory conditioning, drep-2 mutants formed neither short-term nor anaesthesia-sensitive memories. We could demonstrate that Drep-2 is required in mushroom body intrinsic neurons for normal olfactory learning. Furthermore, odour-evoked calcium transients in these neurons, a prerequisite for learning, were reduced in drep-2 mutants. The impairment of the mutants in olfactory learning could be fully rescued by pharmacological application of an agonist to metabotropic glutamate receptors (mGluRs). Quantitative mass spectrometry of Drep-2 complexes revealed that the protein is associated with a large number of translational repressors, among them the fragile X mental retardation protein FMRP. FMRP inhibits mGluR-mediated protein synthesis. Lack of this protein causes the fragile X syndrome, which constitutes the most frequent monogenic cause of autism. Examination of the performance of drep-2 mutants in courtship conditioning showed that the animals were deficient in both short- and long-term memory. Drep-2 mutants share these phenotypes with fmrp and mGluR mutants. Interestingly, drep-2; fmrp double mutants exhibited normal memory. Thus, we propose a model in which Drep-2 antagonizes FMRP in the regulation of mGluR-dependent protein synthesis. Our hypothesis is supported by the observation that impairments in synaptic plasticity can arise if mGluR signalling is imbalanced in either direction. We suggest that Drep-2 helps in establishing this balance.
It has been proposed that different features of a face provide a source of information for separate perceptual and cognitive processes. Properties of a face that remain rather stable over time, so called invariant facial features, yield information about a face’s identity, and changeable aspects of faces transmit information underlying social communication such as emotional expressions and speech movements. While processing of these different face properties was initially claimed to be independent, a growing body of evidence suggests that these sources of information can interact when people recognize faces with whom they are familiar. This is the case because the way a face moves can contain patterns that are characteristic for that specific person, so called idiosyncratic movements. As a face becomes familiar these idiosyncratic movements are learned and hence also provide information serving face identification. While an abundance of experiments has addressed the independence of invariant and variable facial features in face recognition, little is known about the exact nature of the impact idiosyncratic facial movements have on face recognition. Gaining knowledge about the way facial motion contributes to face recognition is, however, important for a deeper understanding of the way the brain processes and recognizes faces. In the following dissertation three experiments are reported that investigate the impact familiarity of changeable facial features has on processes of face recognition. Temporal aspects of the processing of familiar idiosyncratic facial motion were addressed in the first experiment via EEG by investigating the influence familiar facial movement exerts on event-related potentials associated to face processing and face recognition. After being familiarized with a face and its idiosyncratic movement, participants viewed familiar or unfamiliar faces with familiar or unfamiliar facial movement while their brain potentials were recorded. Results showed that familiarity of facial motion influenced later event-related potentials linked to memory processes involved in face recognition. The second experiment used fMRI to investigate the brain areas involved in processing familiar facial movement. Participants’ BOLD-signal was registered while they viewed familiar and unfamiliar faces with familiar or unfamiliar idiosyncratic movement. It was found that activity of brain regions, such as the fusiform gyrus, that underlie the processing of face identity, was modulated by familiar facial movement. Together these two experiments provide valuable information about the nature of the involvement of idiosyncratic facial movement in face recognition and have important implications for cognitive and neural models of face perception and recognition. The third experiment addressed the question whether idiosyncratic facial movement could increase individuation in perceiving faces from a different ethnic group and hence reduce impaired recognition of these other-race faces compared to own-race faces, a phenomenon named the own-race bias. European participants viewed European and African faces that were each animated with an idiosyncratic smile while their attention was either directed to the form or the motion of the face. Subsequently recognition memory for these faces was tested. Results showed that the own-race bias was equally present in both attention conditions indicating that idiosyncratic facial movement was not able to reduce or diminish the own-race bias. In combination the here presented experiments provide further insight into the involvement of idiosyncratic facial motion in face recognition. It is necessary to consider the dynamic component of faces when investigating face recognition because static facial images are not able to provide the full range of information that leads to recognition of a face. In order to reflect the full process of face recognition, cognitive and neural models of face perception and recognition need to integrate dynamic facial features as a source of information which contributes to the recognition of a face.
Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted:
1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI
2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis
3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics
Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice.
The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition.
In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly – at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe−/− mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD.
Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis.
Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models – two parameters highly influencing plaque development and progression – there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis.
Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development.
Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis.
Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models.
In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.
Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily (TNFSF) and is as such initially expressed as type II class transmembrane glycoprotein from which a soluble ligand form can be released by proteolytic processing. While the expression of TWEAK has been detected at the mRNA level in various cell lines and cell types, its cell surface expression has so far only been documented for dendritic cells, monocytes and interferon-γ stimulated NK cells. The fibroblast growth factor-inducible-14 (Fn14) is a TRAF2-interacting receptor of the TNF receptor superfamily (TNFRSF) and is the only receptor for TWEAK. The expression of Fn14 is strongly induced in a variety of non-hematopoietic cell types after tissue injury. The TWEAK/Fn14 system induces pleiotropic cellular activities such as induction of proinflammatory genes, stimulation of cellular angiogenesis, proliferation, differentiation, migration and in rare cases induction of apoptosis. On the other side, Toll-like receptor3 (TLR3) is one of DNA- and RNA-sensing pattern recognition receptors (PRRs), plays a crucial role in the first line of defense against virus and invading foreign pathogens and cancer cells. Polyinosinic-polycytidylic acid poly(I:C) is a synthetic analog of dsRNA, binds to TLR3 which acts through the adapter TRIF/TICAM1, leading to cytokine secretion, NF-B activation, IRF3 nuclear translocation, inflammatory response and may also elicit the cell death. TWEAK sensitizes cells for TNFR1-induced apoptosis and necroptosis by limiting the availability of protective TRAF2-cIAP1 and TRAF2-cIAP2 complexes, which interact with the TNFR1-binding proteins TRADD and RIPK1. In accordance with the fact that poly(I:C)-induced signaling also involves these proteins, we found enhanced necroptosis-induction in HaCaT and HeLa-RIPK3 by poly(I:C) in the presence of TWEAK (Figure 24). Analysis of a panel of TRADD, FADD, RIPK1 and caspase-8 knockout cells revealed furthermore similarities and differences in the way how these molecules act in cell death signaling by poly(I:C)/TWEAK and TNF and TRAIL. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for these responses in TNF and TRAIL signaling. Lack of FADD protein abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis. Moreover, we observed that both long and short FLIP rescued HaCaT and HeLa-RIPK3 cells from poly(I:C)-induced apoptosis or necroptosis.
To sum up, our results demonstrate that TWEAK, which is produced by interferon stimulated myeloid cells, controls the induction of apoptosis and necroptosis by the TLR3 ligand poly(I:C) and may thus contribute to cancer or anti-viral immunity treatment.
The one electron oxidation potential of ten TAAs with all permutations of Cl , OMe- and Me-substituents in the three p-positions were determined by CV. The half wave potential of the first oxidation wave correlates linearly with the number of Cl- and OMe-substituents. AM1-CISD derived values of the absorption energies are in good agreement with the experiments but differ strongly for the oscillator strengths as well as for neutral compounds and their corresponding mono radical cations. The small solvent dependence of the experimental UV/Vis spectra in CH2Cl2 and MeCN reflects a minor charge transfer character of the electronic transitions. The UV/Vis/NIR spectra of the series of TAAs and their corresponding radical cations and the AM1 computations reveal that even small substituents may lead to strong symmetry breaking and to a modified electronic structure. The spectroscopic properties of a series of four bis-TAA donor-bridge-donor X-B-X dimers, composed of two asymmetric TAA chromophores (monomers) were investigated. UV/vis-, fluorescence and transient absorption spectra were recorded and compared with those of the corresponding X-B monomers. The excited states of the dimers are described as MV states which show, depending on the chemical nature of the bridge, a varying amount of interactions. It was found that superradiant emission only proceeds in the case of weak and medium coupling. Whether the first excited state potential energy surface of the dimers is a single minimum or a double minimum potential depends on the solvent polarity and the electronic coupling. In the latter case, the dimer relaxes in a symmetry broken CT state. The [2.2]paracyclophane bridged dimer is an example for a weakly coupled system, because the spectroscopic behavior is very similar to the corresponding p xylene monomer. In contrast, anthracene as well as p-xylene bridges mediate a stronger coupling and reveal a significant cooperative influence on the optical properties. A series of [2.2]paracylophane bridged bis-TAA MV radical cations X-B-X+ were analyzed by a GMH three-level model which takes two transitions into account: the IV-CT band and the bridge band. From the GMH analysis, one can conclude that the [2.2]paracyclophane moiety is not the limiting factor which governs the intramolecular charge transfer. The electronic interactions are of course smaller than direct conjugation but from the order of magnitude of the couplings of the [2.2]paracyclophane MV species it can be assumed that this bridge is able to mediate significant through-space and through-bond interactions. From the exponential dependence of the electronic coupling V between the two TAA localized states on the distance r between the two redox centers, it was inferred that the HT proceeds via superexchange mechanism. The analysis reveals that even significantly longer conjugated bridges should still mediate significant electronic interactions, because the decay constant of a series of conjugated MV species is small. The absorption properties of a series of bis-TAA-[2.2]paracyclophane dications X+-B-X+ were presented. The localized and the CT transitions of these dications are explained and analyzed by an exciton coupling model which also considers the photophysical properties of the monomeric TAA radical cations. Together with AM1-CISD calculated transition moments, experimental transition moments and transition energies of the bis-TAA dications were used to calculate electronic couplings by a GMH approach. These couplings are a measure for interactions of the excited MV CT states. The modification of the diabatic states reveals similarities of the GMH three-level model and the exciton coupling model. Comparison of the two models shows that the transition moment between the excited mixed-valence states of the dimer equals the dipole moment difference of the ground and the excited bridge state of the corresponding monomer. Thianthrenophane (1) has a cavity which offers enough room to potentially enable endohedral coordination to small ions or molecules. For the complexation of silver(I) perchlorate, the complex stability constants of thianthrenophane logK1=5.45 and of thianthrene logK2=9.16 were determined by UV/Vis titration. Single competition transport experiments with ten metal salts demonstrate a very high selectivity of thianthrenophane as a carrier for silver(I) and a distinctly higher transport rate compared to carriers such as thianthrene and 14-ane-S4. Although the X-ray crystal structure analysis of the polymeric [Ag(1)]ClO4 shows an exohedral coordination to silver(I), the formation of an endohedral [Ag(1)]+ complex is suggested to be the explanation for the unusual carrier selectivity of silver(I) by 1 in bulk liquid membrane.
To grow larger, insects must shed their old rigid exoskeleton and replace it with a new one. This process is called molting and the motor behavior that sheds the old cuticle is called ecdysis. Holometabolic insects have pupal stages in between their larval and adult forms, during which they perform metamorphosis. The pupal stage ends with eclosion, i.e., the emergence of the adult from the pupal shell. Insects typically eclose at a specific time during the day, likely when abiotic conditions are at their optimum. A newly eclosed insect is fragile and needs time to harden its exoskeleton. Hence, eclosion is regulated by sophisticated developmental and circadian timing mechanisms.
In Drosophila melanogaster, eclosion is limited to a daily time window in the morning, regarded as the “eclosion gate”. In a population of laboratory flies entrained by light/dark cycles, most of the flies eclose around lights on. This rhythmic eclosion pattern is controlled by the circadian clock and persists even under constant conditions.
Developmental timing is under the control of complex hormonal signaling, including the steroid ecdysone, insulin-like peptides, and prothoracicotropic hormone (PTTH). The interactions of the central circadian clock in the brain and a peripheral clock in the prothoracic gland (PG) that produces ecdysone are important for the circadian timing of eclosion. These two clocks are connected by a bilateral pair of peptidergic PTTH neurons (PTTHn) that project to the PG. Before each molt, the ecdysone level rises and then falls shortly before ecdysis. The falling ecdysone level must fall below a certain threshold value for the eclosion gate to open. The activity of PTTHn is inhibited by short neuropeptide F (sNPF) from the small ventrolateral neurons (sLNvs) and inhibition is thought to lead to a decrease in ecdysone production.
The general aim of this thesis is to further the understanding of how the circadian clock and neuroendocrinal pathways are coordinated to drive eclosion rhythmicity and to identify when these endocrinal signaling pathways are active. In Chapter I, a series of conditional PTTHn silencing-based behavioral assays, combined with neuronal activity imaging techniques such as non-invasive ARG-Luc show that PTTH signaling is active and required shortly before eclosion and may serve to phase-adjust the activity of the PG at the end of pupal development. Trans-synaptic anatomical stainings identified the sLNvs, dorsal neurons 1 (DN1), dorsal neurons 2 (DN2), and lateral posterior neurons (LPNs) clock neurons as directly upstream of the PTTHn.
Eclosion motor behavior is initiated by Ecdysis triggering hormone (ETH) which activates a pair of ventromedial (Vm) neurons to release eclosion hormone (EH) which positively feeds back to the source of ETH, the endocrine Inka cells. In Chapter II trans-synaptic tracing showed that most clock neurons provide input to the Vm and non-canonical EH neurons. Hence, clock can potentially influence the ETH/EH feedback loop. The activity profile of the Inka cells and Vm neurons before eclosion is described. Vm and Inka cells are active around seven hours before eclosion. Interestingly, all EH neurons appear to be exclusively peptidergic.
In Chapter III, using chemoconnectomics, PTTHns were found to express receptors for sNPF, allatostatin A (AstA), allatostatin C (AstC), and myosuppressin (Ms), while EH neurons expressed only Ms and AstA receptors. Eclosion assays of flies with impaired AstA, AstC, or Ms signaling do not show arrhythmicity under constant conditions. However, optogenetic activation of the AstA neurons strongly suppresses eclosion.
Chapter IV focuses on peripheral ventral’ Tracheal dendrite (v’Td) and class IV dendritic arborization (C4da) neurons. The C4da neurons mediate larval light avoidance through endocrine PTTH signaling. The v’Td neurons mainly receive O2/CO2 input from the trachea and are upstream of Vm neurons but are not required for eclosion rhythmicity. Conditional ablation of the C4da neurons or torso (receptor of PTTH) knock-out in the C4da neurons impaired eclosion rhythmicity. Six to seven hours before eclosion, PTTHn, C4da, and Vm neurons are active based on ARG-Luc imaging. Thus, C4da neurons may indirectly connect the PTTHn to the Vm neurons.
In summary, this thesis advances our knowledge of the temporal activity and role of PTTH signaling during pupal development and rhythmic eclosion. It further provides a comprehensive characterization of the synaptic and peptidergic inputs from clock neurons to PTTHn and EH neurons. AstA, AstC, and Ms are identified as potential modulators of eclosion circuits and suggest an indirect effect of PTTH signaling on EH signaling via the peripheral sensory C4da neurons.
Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite’s proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.
In the present thesis the MBE growth and sample characterization of HgTe structures is investigated
and discussed. Due to the first experimental discovery of the quantum Spin Hall effect
(QSHE) in HgTe quantum wells, this material system attains a huge interest in the spintronics
society. Because of the long history of growing Hg-based heterostructures here at the Experimentelle
Physik III in Würzburg, there are very good requirements to analyze this material
system more precisely and in new directions. Since in former days only doped HgTe quantum
wells were grown, this thesis deals with the MBE growth in the (001) direction of undoped
HgTe quantum wells, surface located quantum wells and three dimensional bulk layers. All
Hg-based layers were grown on CdTe substrates which generate strain in the layer stack and
provide therefore new physical effects. In the same time, the (001) CdTe growth was investigated
on n-doped (001) GaAs:Si because the Japanese supplier of CdTe substrates had a
supply bottleneck due to the Tohoku earthquake and its aftermath in 2011.
After a short introduction of the material system, the experimental techniques were demonstrated
and explained explicitly. After that, the experimental part of this thesis is displayed.
So, the investigation of the (001) CdTe growth on (001) GaAs:Si is discussed in chapter 4.
Firstly, the surface preparation of GaAs:Si by oxide desorption is explored and analyzed.
Here, rapid thermal desorption of the GaAs oxide with following cool down in Zn atmosphere
provides the best results for the CdTe due to small holes at the surface, while e.g. an atomic
flat GaAs buffer deteriorates the CdTe growth quality. The following ZnTe layer supplies the
(001) growth direction of the CdTe and exhibits best end results of the CdTe for 30 seconds
growth time at a flux ratio of Zn/Te ~ 1/1.2. Without this ZnTe layer, CdTe will grow in the
(111) direction. However, the main investigation is here the optimization of the MBE growth
of CdTe. The substrate temperature, Cd/Te flux ratio and the growth time has to be adjusted
systematically. Therefore, a complex growth process is developed and established. This optimized
CdTe growth process results in a RMS roughness of around 2.5 nm and a FWHM value
of the HRXRD w-scan of 150 arcsec. Compared to the literature, there is no lower FWHM
value traceable for this growth direction. Furthermore, etch pit density measurements show
that the surface crystallinity is matchable with the commercial CdTe substrates (around 1x10^4
cm^(-2)). However, this whole process is not completely perfect and offers still room for improvements.
The growth of undoped HgTe quantum wells was also a new direction in research in contrast
to the previous n-doped grown HgTe quantum wells. Here in chapter 5, the goal of very low
carrier densities was achieved and therefore it is now possible to do transport experiments in
the n - and p - region by tuning the gate voltage. To achieve this high sample quality, very precise
growth of symmetric HgTe QWs and their HRXRD characterization is examined. Here,
the quantum well thickness can now determined accurate to under 0.3 nm. Furthermore, the transport analysis of different quantum well thicknesses shows that the carrier density and
mobility increase with rising HgTe layer thickness. However, it is found out that the band
gap of the HgTe QW closes indirectly at a thickness of 11.6 nm. This is caused by the tensile
strained growth on CdTe substrates. Moreover, surface quantum wells are studied. These
quantum wells exhibit no or a very thin HgCdTe cap. Though, oxidization and contamination
of the surface reduces here the carrier mobility immensely and a HgCdTe layer of around 5 nm
provides the pleasing results for transport experiments with superconductors connected to the
topological insulator [119]. A completely new achievement is the realization of MBE growth
of HgTe quantum wells on CdTe/GaAs:Si substrates. This is attended by the optimization of
the CdTe growth on GaAs:Si. It exposes that HgTe quantum wells grown in-situ on optimized
CdTe/GaAs:Si show very nice transport data with clear Hall plateaus, SdH oscillations, low
carrier densities and carrier mobilities up to 500 000 cm^2/Vs. Furthermore, a new oxide etching
process is developed and analyzed which should serve as an alternative to the standard
HCl process which generates volcano defects at some time. However, during the testing time
the result does not differ in Nomarski, HRXRD, AFM and transport measurements. Here,
long-time tests or etching and mounting in nitrogen atmosphere may provide new elaborate
results.
The main focus of this thesis is on the MBE growth and standard characterization of HgTe bulk
layers and is discussed in chapter 6. Due to the tensile strained growth on lattice mismatched
CdTe, HgTe bulk opens up a band gap of around 22 meV at the G-point and exhibits therefore
its topological surface states. The analysis of surface condition, roughness, crystalline quality,
carrier density and mobility via Nomarski, AFM, XPS, HRXRD and transport measurements
is therefore included in this work. Layer thickness dependence of carrier density and mobility
is identified for bulk layer grown directly on CdTe substrates. So, there is no clear correlation
visible between HgTe layer thickness and carrier density or mobility. So, the carrier density is
almost constant around 1x10^11 cm^(-2) at 0 V gate voltage. The carrier mobility of these bulk
samples however scatters between 5 000 and 60 000 cm^2/Vs almost randomly. Further experiments
should be made for a clearer understanding and therefore the avoidance of unusable
bad samples.But, other topological insulator materials show much higher carrier densities and
lower mobility values. For example, Bi2Se3 exhibits just density values around 1019 cm^(-2)
and mobility values clearly below 5000 cm2/Vs. The carrier density however depends much
on lithography and surface treatment after growth. Furthermore, the relaxation behavior and
critical thickness of HgTe grown on CdTe is determined and is in very good agreement with
theoretical prediction (d_c = 155 nm). The embedding of the HgTe bulk layer between HgCdTe
layers created a further huge improvement. Similar to the quantum well structures the carrier
mobility increases immensely while the carrier density levels at around 1x10^11 cm^(-2) at 0
V gate voltage as well. Additionally, the relaxation behavior and critical thickness of these
barrier layers has to be determined. HgCdTe grown on commercial CdTe shows a behavior as
predicted except the critical thickness which is slightly higher than expected (d_c = 850 nm).
Otherwise, the relaxation of HgCdTe grown on CdTe/GaAs:Si occurs in two parts. The layer
is fully strained up to 250 nm. Between 250 nm and 725 nm the HgCdTe film starts to relax
randomly up to 10 %. The relaxation behavior for thicknesses larger than 725 nm occurs than
linearly to the inverse layer thickness. A explanation is given due to rough interface conditions
and crystalline defects of the CdTe/GaAs:Si compared to the commercial CdTe substrate. HRXRD and AFM data support this statement. Another point is that the HgCdTe barriers protect the active HgTe layer and because of the high carrier mobilities the Hall measurements provide new transport data which have to be interpreted more in detail in the future. In addition, HgTe bulk samples show very interesting transport data by gating the sample from the top and the back. It is now possible to manipulate the carrier densities of the top and bottom surface states almost separately. The back gate consisting of the n-doped GaAs substrate and the thick insulating CdTe buffer can tune the carrier density for Delta(n) ~ 3x10^11 cm^(-2). This is sufficient to tune the Fermi energy from the p-type into the n-type region [138].
In this thesis it is shown that strained HgTe bulk layers exhibit superior transport data by embedding between HgCdTe barrier layers. The n-doped GaAs can here serve as a back gate.
Furthermore, MBE growth of high crystalline, undoped HgTe quantum wells shows also new
and extended transport output. Finally, it is notable that due to the investigated CdTe growth
on GaAs the Hg-based heterostructure MBE growth is partially independent from commercial
suppliers.
The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research.
To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake– feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time.
In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.
Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems.
The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine.
First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research.
At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces.
Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases.
Fremdschämen or Fremdscham, a negative emotion which arises while observing someone behave inappropriately, comes to fame after the turn of the millennium in german speaking countries. There, they name it literally „other‘s shame“ and it becomes obvious that this emotion happens most commonly while watching TV: reality shows, talent shows and bad comedies. The word even makes it to the dictionaries starting 2009, as its use increases unstoppably in everyday language, starting to get used in more and more situations, seemingly as a synonym of embarrassing or shameful. Still, a look in the emotional research on the subject returns exactly zero results as of 2011, leaving open the question as of what this emotion might be, and what it is not. The present wort aims at explaining not only the phenomenon of Fremdschämen, but also the Emotion behind it - Embarrassment -, at a process level.
The superfamily of G protein-coupled receptors (GPCRs) comprises more than 800 members, which are divided into five families based on phylogenetic analyses (GRAFS classification): Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin. The adhesion G protein-coupled receptor (aGPCR) family forms with 33 homologs in Mammalia the second largest and least investigated family of GPCRs. The general architecture of an aGPCR comprises the GPCR characteristics of an extracellular region (ECR), a seven transmembrane (7TM) domain and an intracellular region (ICR). A special feature of aGPCRs is the extraordinary size of the ECR through which they interact with cellular and matricellular ligands via adhesion motif folds. In addition, the ECR contains a so-called GPCR autoproteolysis-inducing (GAIN) domain, which catalyzes autoproteolytic cleavage of the protein during maturation. This cleavage leads to the formation of an N-terminal (NTF) and a C-terminal fragment (CTF), which build a unit by means of hydrophobic interactions and therefore appear as a heterodimeric receptor at the cell surface. In the past, it has been shown that the first few amino acids of the CTF act as a tethered agonist (TA) that mediates the activation of the receptor through the interaction with the 7TM domain. However, the molecular mechanism promoting the TA-7TM domain interaction remains elusive. This work reveals a novel molecular mechanism that does not require the dissociation of the NTF-CTF complex to promote release of the TA and thus activation of the aGPCR. The introduction of bioorthogonal labels into receptorsignaling- relevant regions of the TA of various aGPCRs demonstrated that the TA is freely accessible within the intact GAIN domain. This suggests a structural flexibility of the GAIN domain, which allows a receptor activation independent of the NTF-CTF dissociation, as found in cleavage-deficient aGPCR variants. Furthermore, the present study shows that the cellular localization and the conformation of the 7TM domain depends on the activity state of the aGPCR, which in turn indicates that the TA mediates conformational changes through the interaction with the 7TM domain, which ultimately regulates the receptor activity. In addition, biochemical analyses showed that the GAIN domain-mediated autoproteolysis of the human aGPCR CD97 (ADGRE5/E5) promotes further cleavage events within the receptor. This suggests that aGPCRs undergo cleavage cascades, which are initialized by the autoproteolytic reaction of the GAIN domain. Thus, it can be assumed that aGPCRs are subject to additional proteolytic events. Finally, the constitutive internalization of the NTF and the CTF of E5 was demonstrated by various labeling methods. It was possible to label both fragments independently and to follow their subcellular location in vitro. In summary, these obtained results contribute to a better understanding about the molecular mechanisms of activity and signaling of aGPCRs.
This work developed during the first funding period of the subproject B05 in the framework of the interdisciplinary research consortium TRR 225 ‘From the Fundamentals of Biofabrication toward functional Tissue Models’ and was part of a cooperation between the Orthopedic Department represented by Prof. Dr. Regina Ebert and the Institute of Organic Chemistry represented by Prof. Dr. Jürgen Seibel.
This project dealed with cellular behavior during the bioprinting process and how to influence it by modifying the cell glycocalyx with functional target molecules. The focus was on the impact of potential shear stress, that cells experience when they get processed in thermoresponsive bioinks, and a way to increase the cell stiffness via metabolic glycoengineering to attenuate shear forces. For the characterization of the metabolic glycoengineering, four different peracetylated and four non-acetylated modified monosaccharides (two mannose and two sialic acid sugars) were tested in primary human mesenchymal stromal cells (hMSC) and telomerase-immortalized hMSC (hMSC-TERT). Viability results demonstrated a dose-dependent correlation for all sugars, at which hMSC-TERT seemed to be more susceptible leading to lower viability rates. The assessment of the incorporation efficiencies was performed by click chemistry using fluorescent dyes and revealed also a dose-dependent correlation for all mannose and sialic acid sugars, while glucose and galactose variants were not detected in the glycocalyx. However, incorporation efficiencies were highest when using mannose sugars in the primary hMSC. A subsequent analysis of the temporal retention of the incorporated monosaccharides showed a constant declining fluorescence signal up to 6 d for azido mannose in hMSC-TERT, whereas no signal could be detected for alkyne mannose after 2 d. Investigation of the differentiation potential and expression of different target genes revealed no impairment after incubation with mannose sugars, indicating a normal phenotype for hMSC-TERT. Following the successful establishment of the method, either a coumarin derivative or an artificial galectin 1 ligand were incorporated into the cell glycocalyx of hMSC-TERT as functional target molecule. The biophysical analysis via shear flow deformation cytometry revealed a slightly increased cell stiffness and lowered fluidity for both molecules. A further part of this project aimed to control lectin-mediated cell adhesion by artificial galectin 1 ligands. As that hypothesis was settled in the work group of Prof. Dr. Jürgen Seibel, this work supported with an initial characterization of galectin 1 as part of the hMSC biology. A stable galectin 1 expression at gene and protein level in both hMSC and hMSC-TERT could be confirmed, at which immunocytochemical stainings could detect the protein only in the glycocalyx. The treatment of hMSC-TERT with a galectin 1 ligand in different concentrations did not show an altered gene expression of galectin 1. However, these first data in addition to the investigation of stiffness confirmed the applicability of specific and artificial
IV
galectin 1 ligands in biofabrication approaches to alter cell properties of hMSC. To conclude, metabolic glycoengineering has been successfully implemented in hMSC and hMSC-TERT to introduce glycocalyx modifications which reside there for several days. A proof of concept was carried out by the increase of cell stiffness and fluidity by the incorporation of a coumarin derivative or an artificial galectin 1 ligand.
For the characterization of shear stress impact on cells after printing in thermoresponsive bioinks, the processing of hMSC-TERT (mixing or additionally printing) with Pluronic F127 or Polyoxazoline-Polyoxazine (POx-POzi) polymer solution was investigated. While there were no changes in viability when using POx-POzi bioink, processing with Pluronic F127 indicated slightly lower viability and increased apoptosis activity. Assessment of cellular responses to potential shear stress showed no reorganization of the cytoskeleton independent of the bioink, but highly increased expression of the mechanoresponsive proto-oncogene c Fos which was more pronounced when using Pluronic F127 and just mixed with the bioinks. Interestingly, processing of the mechanoresponsive reporter cell line hMSC-TERT-AP1 revealed slightly elevated mechanotransduction activity when using POx-POzi polymer and just mixed with the bioinks as well. In conclusion, hMSC-TERT embedded in thermoresponsive bioinks might shortly experience shear stress during the printing process, but that did not lead to remarkable cell damage likely due to the rheological properties of the bioinks. Furthermore, the printing experiments also suggested that cells do not sense more shear stress when additionally printed.
Sustainability has become a critical topic in all areas of supply chain management. As discussed earlier, drivers for this development can be identified as both internal and external phenomena. Since customers are one of the key stakeholders in supply chain management, special attention is paid to the impact of costumers´ behavior on sustainable supply chain design decisions. In this context, two main research questions were analyzed:
1.What is the appropriate way to design a supply chain according to environmentally-oriented requirements of customers?
2.What is the impact of customer´s behavior regarding both usage and return of products on supply chain design decisions in an environmentally conscious closed-loop supply chain environment?
Therefore, three different optimization models with various main aspects are developed. To illustrate how the presented models can be applied in practical problem cases, guidelines for implementing an environmentally supply chain design project are presented.
In this work we wanted to investigate the role of NFATc1 in lymphocyte physiology and in pathological conditions (eg. psoriasis). NFATc1 is part of the signal transduction
pathways that regulates B cells activation and function. NFATc1 has different isoforms that are due to different promoters (P1 and P2), polyadenylation and alternative splicing. Moreover, we tried to elucidate the points of interactions between the NFAT and the NF-κB pathways in
activated B-cell fate. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA binding domain. We used mice which over-express a constitutive active version of NFATc1/α in their B cells with -or without- an ablated IRF4. IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt’s lymphoma cells and
induces terminal differentiation toward plasma cells. Our experiments showed that a ‘double hit’ in factors affecting B cell activation (NFATc1 in this case) and late B cell Differentiation (IRF4 in this case) alter the development of the B cells, lead to increase in their numbers and increase in stimulation induced proliferation. Therefore, the overall picture indicates a link between these 2 genes and probable carcinogenic alterations that may occur in B cells.
We also show that in splenic B cells, c-Rel (of the NF-κB canonical pathway) Support the induction of NFATc1/αA through BCR signals. We also found evidence that the lack of NFATc1 affects the expression of Rel-B (of the NF-κB non-canonical pathway). These data suggest a tight interplay between NFATc1 and NF-κB in B cells, influencing the competence of B cells and their functions in peripheral tissues.
We also used IMQ-induced psoriasis-like inflammation on mice which either lack NFATc1 from B cell. Psoriasis is a systemic chronic immunological disease characterized
primarily by abnormal accelerated proliferation of the skin keratinocytes. In psoriasis, the precipitating event leads to immune cell activation. Our experiments showed that NFATc1 is needed for the development of psoriasis. It also showed that IL-10 is the link that enables NFAT
from altering the B cell compartment (eg Bregs) in order to affect inflammation. The important role of B cell in psoriasis is supported by the flared up psoriasis-like inflammation in mice that lack B cells. Bregs is a special type of B cells that regulate other B cells and T cells; tuning the immunological response through immunomodulatory cytokines.
Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor.
The work presented in this thesis was mainly targeted at exploring the capabilities of evaporation based LC detectors as well as further alternatives for the control of impurities in substances not exhibiting a suitable chromophore for UV-detection. In the course of the work carried out, several new methods for the identification, impurities control and composition testing of APIs were elaborated. An evaporation based detector that entered into the field of pharmaceutical analysis in the recent years was the Evaporative Light Scattering Detector (ELSD). However, non-reproducible spikes were reported when injecting concentrated test solutions as they are usually required for the control of impurities. The reasons, for the appearance of these spikes as well as possibilities for their avoidance were explored in a systematic study. Moreover, the dependence of the detector sensitivity on different eluent composition, eluent flow-rate and ELSD settings was investigated. In the course of the revision of the Ph.Eur. monographs for aspartic acid and alanine, a C18 reversed phase ion-pair LC method using 1 mmol/L of perfluoroheptanoic acid as an ion-pair reagent and a charged aerosol detector (CAD) was developed and fully validated for the purity control of Asp. The method was capable of separating the organic acids and major amino acids known to occur as process related impurities. With a slight modification, the method was also applicable for the purity control of Ala. Based on the developed LC-CAD method for the impurity control of alanine, a comparative study of the performance characteristics of different evaporation based LC detectors, i.e. ELSD, CAD and the recently developed Nano Quantity Analyte Detector (NQAD) was carried out. Additionally, an MS detector and qNMR were included in this study. It was found that the control of impurities in Alanine at an ICH conform level could be ensured using LC coupled to CAD, MSD and NQAD detection as well as by the use of qNMR. In terms of performance, prize and ease of use CAD and NQAD were found to be the most suitable alternatives. In terms of repeatability and sensitivity, the CAD appeared slightly superior to the NQAD. The quality of streptomycin sulfate is not sufficiently controlled by the current Ph.Eur. monograph in that an appropriate test for the control of the related substances is missing. A study was carried out to develop a C18 reversed phase ion-pair LC method using pentafluoropropionic acid as an ion-pair reagent and a CAD for the identification and control of the related substances. The developed method allowed the separation of 21 impurities from streptomycin. Moreover, coupling of the method to MS allowed the identification of the separated impurities. The method was shown to be sufficiently sensitive to control the related substances with a disregard limit of 0.1% as it is normally applied in the Ph.Eur. for products derived from fermentation. Currently, the aescin content of horse-chestnut standardized dry extract is determined using a complex and laborious photometric determination. A more selective LC-UV assay determination for beta-aescin has been proposed for the Ph.Eur. draft monograph of horse-chestnut standardized dry extract. Possibilities were explored to further improve the LC-method using detection by CAD. It was demonstrated that by the use of a modified LC-CAD method several problems related to the differences in the UV-response of the various components contained in the active aescin fraction could be eliminated. Moreover the proposed reference standard strategy was reviewed. Eventually, it was demonstrated on the example of two different clusters of pharmacologically active peptides how low energy collision induced dissociation mass spectrometry (low energy CID-MS) can successfully be used for identification testing in pharmacopoeial monographs. In this respect, the combination of a direct confirmation of the molecular mass via the m/z-ratio of the molecule ions with structural sequence information obtained by low energy CID-MS experiments was found to deliver a higher degree of certainty of the identity of a given substance than the set of tests currently described in the monographs. A significant gain in efficiency and throughput and important reduction of the amount of sample consumed during testing were identified as being additional advantages of this approach. Taken together, it could be demonstrated on various examples how recent technological advancements in the field of analytical chemistry can contribute to improve the quality control of APIs.
Introduction.
Mobile health (mHealth) integrates mobile devices into healthcare, enabling remote monitoring, data collection, and personalized interventions. Machine Learning (ML), a subfield of Artificial Intelligence (AI), can use mHealth data to confirm or extend domain knowledge by finding associations within the data, i.e., with the goal of improving healthcare decisions. In this work, two data collection techniques were used for mHealth data fed into ML systems: Mobile Crowdsensing (MCS), which is a collaborative data gathering approach, and Ecological Momentary Assessments (EMA), which capture real-time individual experiences within the individual’s common environments using questionnaires and sensors. We collected EMA and MCS data on tinnitus and COVID-19. About 15 % of the world’s population suffers from tinnitus.
Materials & Methods.
This thesis investigates the challenges of ML systems when using MCS and EMA data. It asks: How can ML confirm or broad domain knowledge? Domain knowledge refers to expertise and understanding in a specific field, gained through experience and education. Are ML systems always superior to simple heuristics and if yes, how can one reach explainable AI (XAI) in the presence of mHealth data? An XAI method enables a human to understand why a model makes certain predictions. Finally, which guidelines can be beneficial for the use of ML within the mHealth domain? In tinnitus research, ML discerns gender, temperature, and season-related variations among patients. In the realm of COVID-19, we collaboratively designed a COVID-19 check app for public education, incorporating EMA data to offer informative feedback on COVID-19-related matters. This thesis uses seven EMA datasets with more than 250,000 assessments. Our analyses revealed a set of challenges: App user over-representation, time gaps, identity ambiguity, and operating system specific rounding errors, among others. Our systematic review of 450 medical studies assessed prior utilization of XAI methods.
Results.
ML models predict gender and tinnitus perception, validating gender-linked tinnitus disparities. Using season and temperature to predict tinnitus shows the association of these variables with tinnitus. Multiple assessments of one app user can constitute a group. Neglecting these groups in data sets leads to model overfitting. In select instances, heuristics outperform ML models, highlighting the need for domain expert consultation to unveil hidden groups or find simple heuristics.
Conclusion.
This thesis suggests guidelines for mHealth related data analyses and improves estimates for ML performance. Close communication with medical domain experts to identify latent user subsets and incremental benefits of ML is essential.
In a three-year study the current aeolian transportation processes were examined in a linear dune area previously used for grazing near Nizzana at the Israeli-Egyptian border. The research area was subject to heavy grazing across the border, which led to the total destruction of the natural vegetation in the period of 1967 to 1982. As a consequence, intensified aeolian activity and significant changes of the morphology of the dunes were observed. After the end of the grazingg on the Israeli side, a rapid return of the vegetation in the interdune corridors and on the footslopes of the dunes took place. In addition also a reduction of obviously active areas on the dune crests was observed. The situation on Egyptian territory west the border remained unchanged until today. This study is aimed at understanding the changed aeolian morphodynamics east the border. The emphasis was placed on the investigation of the spatial and temporal distribution of aeolian sand transport as well as on the influencing factors morphology, surface condition and vegetation.
Background and Aims: PMA is a recently described rare tumor entity occuring most often in young children. Due the worse outcome of PMA-patients as compared to children with pilocytic astrocytoma (PA), it has received a grade II assignment in the latest WHO classification. Nevertheless, increasing evidence suggests that the two tumor types are indeed pathologically and genetically related. The radiological differentiation of PMAs from PAs is challenging and the limited available data could not yet provide unequivocal distinguishing imaging features. Furthermore, it is not completely clarified whether PMA cases are associated with a higher rate of CSF dissemination compared to similarly young patients with PA. The aim of our study was firstly to compare MR/CT imaging features of these tumors, and secondly, to evaluate the occurrence of CSF dissemination.
Material and Methods: The study population included 15 children with PMA and 32 children with PA. A third group consisted of eight children with PAs with focal pilomyxoid features. All cases had been registered in the German multicenter SIOP/HIT-LGG trials. The initial MRIs (and CT scans, if available) at establishing the diagnosis were retrospectively analyzed according to standardized criteria and the findings compared between PMAs and PAs. Furthermore, we compared the occurrence of imaging evidences of CSF tumor dissemination between children with PMA and PA, respectively.
Results: The imaging appearance of PMAs and PAs was very similar. However, PAs tended to show more frequently cystic components (p=0.03). As opposed to PAs, PMAs did not have large tumor cysts. We did not find differences with respect to tumor size and tumor margin. Gadolinium enhancement of PMAs was significantly more frequently homogeneous (p=0.006). PMAs appeared to show more often intratumoral hemorrhages (p=0.047). Furthermore, suprasellar PMAs tended to have a more homogeneus texture on T2-weighted MR images (p=0.026). Within the subgroup < 6 years of age the PMA histology tended to have a larger effect on the occurrence of CSF dissemination than the age (p=0.05 vs.0.12).
Conclusions: Although the radiological appearance of PMAs and PAs is similar, some imaging features, like enhancement pattern or presence of cysts or hemorrhage may help differentiating these low-grade gliomas. Our results corroborate previous scarce data suggesting higher rate of CSF dissemination in PMAs, even in the youngest patient population. Thus, in young children with a chiasmatic-hypothalamic tumor suggestive of a PMA, an intensive search for CSF dissemination along the entire neuraxis should be performed.
The continuously increase in resistance of human pathogenic microorganisms to the known antibiotics leads to the necessity for searching new sources for production of new active antimicrobial compounds from different natural sources especially plants, since many plants have been found to be able to produce antimicrobial compounds as a defense phenomenon against invading microorganisms. The aim of this work is to screen cultures for production of antimicrobial activity against representative of human pathogenic microorganisms and selection the most active cell culture producing antimicrobial protein(s) which are active against these pathogenic microorganisms and also isolation ,purification of the active protein(s) and cloning of its/their genes. Ten different plant suspension cultures have been screened in presence of nine elicitors for their antimicrobial activity against five selected human pathogenic microorganisms, and it has been found that the heterotrophic cultures are more active against the tester isolates than the autotrophic ones. The intracellular fraction of the mixotrophic Arabidopsis thaliana culture elicited with salicylic acid showed the highest antimicrobial activity against the tester isolates. The presence of proteinous antimicrobial activity has been elucidated by testing the activity of ammonium sulphate precipitate against Candida maltosa. High speed centrifugation technique has been used for partial purification of the active protein. The proteinous nature of the isolated compound has been confirmed by using bioautography technique and its molecular weight could be estimated to be around 26KDa. The active protein has been purified using gel filtration, and using mass spectrometry technique, for microsequencing of the active protein, it has been found that the function of the protein is unknown and we have termed it as AtPDP1 according to Arabidopsis thaliana Plat-Domain Protein1, since it contains a plant stress domain termed PLAT domain. It has been found that a second protein from the same plant with high homology level to AtPDP1 with the same domain, we termed it as AtPDP2. Genes for AtPDP1 and AtPDP2 have been cloned in E. coli using PGEM-T easy vector. The expression of both genes have been tested using Digital Northern program, and it has been observed that both genes are induced by different pathogens, chemicals known to induce defense in plant cells and also different hormones. We tried to clone the gene for AtPDP1 in PBI121 binary vector under the control of an elicitor inducible promoter of a proteinase inhibitor gene, to test its function in plant by overexpression, but we did not succeeded. Also the work aims to cloning the different known thaumatin genes from Arabidopsis thaliana for future work which represented by testing their expression under different stimuli, since most thaumatins have antimicrobial activity and some of them are active against Candida spp..Thirteen genes of known thaumatins from Arabidopsis thaliana have been cloned in PGEM-Teasy vector in DH5-alpha cells. coli cells. The expression of the thirteen genes has been done using Digital Northern program and it has been found that different genes show different expressions under different stimuli and the expression of At1g75800 gene was the maximum under all stimuli. The minimum expression of genes was for At1g75050. The rest of thaumatin genes showed moderate expressions under different stimuli.
Mini Unmanned Aerial Vehicles (MUAVs) are becoming popular research platform and
drawing considerable attention, particularly during the last decade due to their afford- ability and multi-dimensional applications in almost every walk of life. MUAVs have obvious advantages over manned platforms including their much lower manufacturing and operational costs, risk avoidance for human pilots, flying safely low and slow, and realization of operations that are beyond inherent human limitations. The advancement in Micro Electro-Mechanical System (MEMS) technology, Avionics and miniaturization of sensors also played a significant role in the evolution of MUAVs. These vehicles range from simple toys found at electronic supermarkets for entertainment purpose to highly sophisticated commercial platforms performing novel assignments like offshore wind power station inspection and 3D modelling of buildings etc. MUAVs are also more environment friendly as they cause less air pollution and noise. Unmanned is therefore unmatched. Recent research focuses on use of multiple inexpensive vehicles flying together, while maintaining required relative separations, to carry out the tasks efficiently compared to a single exorbitant vehicle. Redundancy also does away the risk of loss of a single whole-mission dependent vehicle. Some of the valuable applications in the domain of cooperative control include joint load transportation, search and rescue, mobile communication relays, pesticide spraying and weather monitoring etc. Though realization of multi-UAV coupled flight is complex, however obvious advantages justify
the laborious work involved...
Pro-migratory signals mediated by the tumor microenvironment contribute to the cancer progression cascade, including invasion, metastasis and resistance to therapy. Derived from in vitro studies, isolated molecular steps of cancer invasion programs have been identified but their integration into the tumor microenvironment and suitability as molecular targets remain elusive. The purpose of the study was to visualize central aspects of tumor progression, including proliferation, survival and invasion by real-time intravital microscopy. The specific aims were to monitor the kinetics, mode, adhesion and chemoattraction mechanisms of tumor cell invasion, the involved guidance structures, and the response of invasion zones to anti-cancer therapy. To reach deeper tumor regions by optical imaging with subcellular resolution, near-infrared and infrared excited multiphoton microscopy was combined with a modified dorsal skinfold chamber model. Implanted HT-1080 fibrosarcoma and B16/F10 and MV3 melanoma tumors developed zones of invasive growth consisting of collective invasion strands that retained cell-cell contacts and high mitotic activity while invading at velocities of up to 200 μm per day. Collective invasion occurred predominantly along preexisting tissue structures, including blood and lymph vessels, collagen fibers and muscle strands of the deep dermis, and was thereby insensitive to RNAi based knockdown and/or antibody-based treatment against β1 and β3 integrins, chemokine (SDF-1/CXCL12) and growth factor (EGF) signaling. Therapeutic hypofractionated irradiation induced partial to complete regression of the tumor main mass, yet failed to eradicate the collective invasion strands, suggesting a microenvironmentally privileged niche. Whereas no radiosensitization was achieved by interference with EGFR or doxorubicin, the simultaneous inhibition of β1 and β3 integrins impaired cell proliferation and survival in spontaneously growing tumors and strongly enhanced the radiation response up to complete eradication of both main tumor and invasion strands. In conclusion, collective invasion in vivo is a robust process which follows preexisting tissue structures and is mainly independent of established adhesion and chemoattractant signaling. Due to its altered biological response to irradiation, collective invasion strands represent a microenvironmentally controlled and clinically relevant resistance niche to therapy. Therefore supportive regimens, such as anoikisinduction by anti-integrin therapy, may serve to enhance radio- and chemoefficacy and complement classical treatment regimens.
Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
Despite its precise agreement with the experiment, the validity of the standard model (SM) of elementary particle physics is ensured only up to a scale of several hundred GeV so far. Even more, the inclusion of gravity into an unifying theory poses a problem which cannot be solved by ordinary quantum field theory (QFT). String theory, which is the most popular ansatz for a unified theory, predicts QFT on noncommutative space-time as a low energy limit. Nevertheless, independently of the motivation given by string theory, the nonlocality inherent to noncommutative QFT opens up the possibility for the inclusion of gravity. There are no theoretical predictions for the energy scale Lambda_NC at which noncommutative effects arise and it can be assumed to lie in the TeV range, which is the energy range probed by the next generation of colliders. Within this work we study the phenomenological consequences of a possible realization of QFT on noncommutative space-time relying on this assumption. The motivation for this thesis was given by the gap in the range of phenomenological studies of noncommutative effects in collider experiments, due to the absence in the literature of Large Hadron Collider (LHC) studies regarding noncommutative QFTs. In the first part we thus performed a phenomenological analysis of the hadronic process pp -> Z gamma -> l^+l^- gamma at the LHC and of electron-positron pair annihilation into a Z boson and a photon at the International Linear Collider (ILC). The noncommutative extension of the SM considered within this work relies on two building blocks: the Moyal-Weyl star-product of functions on ordinary space-time and the Seiberg-Witten maps. The latter relate the ordinary fields and parameters to their noncommutative counterparts such that ordinary gauge transformations induce noncommutative gauge transformations. This requirement is expressed by a set of inhomogeneous differential equations (the gauge equivalence equations) which are solved by the Seiberg-Witten maps order by order in the noncommutative parameter Theta. Thus, by means of the Moyal-Weyl star-product and the Seiberg-Witten maps a noncommutative extension of the SM as an effective theory as expansion in powers of Theta can be achieved, providing the framework of our phenomenological studies. A consequence of the noncommutativity of space-time is the violation of rotational invariance with respect to the beam axis. This effect shows up in the azimuthal dependence of cross sections, which is absent in the SM as well as in other models beyond the SM. Thus, the azimuthal dependence of the cross section is a typical signature of noncommutativity and can be used in order to discriminate it against other new physics effects. We have found this dependence to be best suited for deriving the sensitivity bounds on the noncommutative scale Lambda_NC. By studying pp -> Z gamma -> l^+l^- gamma to first order in the noncommutative parameter Theta, we show in the first part of this work that measurements at the LHC are sensitive to noncommutative effects only in certain cases, giving bounds on the noncommutative scale of Lambda_NC > 1.2 TeV. Our result improved the bounds present in the literature coming from past and present collider experiments by one order of magnitude. In order to explore the whole parameter range of the noncommutativity, ILC studies are required. By means of e^+e^- -> Z gamma -> l^+l^- gamma to first order in Theta we have shown that ILC measurements are complementary to LHC measurements of the noncommutative parameters. In addition, the bounds on Lambda_NC derived from the ILC are significantly higher and reach Lambda_NC > 6 TeV. The second part of this work arose from the necessity to enlarge the range of validity of our model towards higher energies. Thus, we expand the neutral current sector of the noncommutative SM to second order in $\theta$. We found that, against the general expectation, the theory must be enlarged by additional parameters. The new parameters enter the theory as ambiguities of the Seiberg-Witten maps. The latter are not uniquely determined and differ by homogeneous solutions of the gauge equivalence equations. The expectation was that the ambiguities correspond to field redefinitions and therefore should vanish in scattering matrix elements. However, we proved that this is not the case, and the ambiguities do affect physical observables. Our conjecture is, that every order in Theta will introduce new parameters to the theory. However, only the experiment can decide to what extent efforts with still higher orders in Theta are reasonable and will also give directions for the development of theoretical models of noncommutative QFTs.
The bacterial pathogen Legionella pneumophila replicates intracellularly in protozoa, but can also cause severe pneumonia, called Legionnaires' disease. The bacteria invade and proliferate in the alveolar macrophages of the human lung. L. pneumophila bacteria exhibit a biphasic life cycle: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Primarily aim of this thesis was to evaluate the impact of the regulatory proteins FleQ, FleR, and RpoN in flagellar gene regulation. Phenotypic analysis, Western blot and electron microscopy of regulatory mutants in the genes coding for FleQ, RpoN and FleR demonstrated that flagellin expression is strongly repressed and that these mutants are non-flagellated in transmissive phase. Transcriptomic studies of these putative flagellar gene expression regulators demonstrated that fleQ controls the expression of numerous flagellar biosynthetic genes. Together with RpoN, FleQ controls transcription of 14 out of 31 flagellar class II genes, coding for the basal body, hook, and regulatory proteins. Unexpectedly, 7 out of 15 late flagellar genes class III and IV) are expressed dependent on FleQ but independent of RpoN. Thus, in contrast to the commonly accepted view that enhancer binding proteins as FleQ always interact with RpoN to initiate transcription, our results strongly indicate that FleQ of L. pneumophila regulates gene expression RpoN-dependent as well as RpoN-independent. Moreover, transcriptome analysis of a fleR mutant strain elucidated that FleR does not regulate the flagellar class III genes as previously suggested. Instead FleR regulates together with RpoN numerous protein biosynthesis and metabolic genes. Based on these experimental results our modified model for the transcriptional regulation of flagellar genes in L. pneumophila is that flagellar class II genes are controlled by FleQ and RpoN, while flagellar class III and IV genes are controlled in a fleQ-dependent but rpoN-independent manner. Although all L. pneumophila strains share the same complex life style, various pathotypes have evolved. This is reflected by the genomes, which contain e.g. genomic islands. The genomic island Trb-1 of L. pneumophila Corby, carries all genes necessary for a type-IV conjugation system, an integrase gene and a putative oriT site. The second aim of this thesis was to investigate the implication of this genomic island in conjugative DNA transfer. Using conjugation assays we showed that the oriT site located on Trb-1 is functional and contributes to conjugation between different L. pneumophila strains. As this is the first oriT site of L. pneumophila known to be functional our results provide evidence that conjugation is a major mechanism for the evolution of new pathotypes in L. pneumophila.
Telemedicine uses telecommunication and information technology to provide health care services over spatial distances. In the upcoming demographic changes towards an older average population age, especially rural areas suffer from a decreasing doctor to patient ratio as well as a limited amount of available medical specialists in acceptable distance. These areas could benefit the most from telemedicine applications as they are known to improve access to medical services, medical expertise and can also help to mitigate critical or emergency situations. Although the possibilities of telemedicine applications exist in the entire range of healthcare, current systems focus on one specific disease while using dedicated hardware to connect the patient with the supervising telemedicine center.
This thesis describes the development of a telemedical system which follows a new generic design approach. This bridges the gap of existing approaches that only tackle one specific application. The proposed system on the contrary aims at supporting as many diseases and use cases as possible by taking all the stakeholders into account at the same time. To address the usability and acceptance of the system it is designed to use standardized hardware like commercial medical sensors and smartphones for collecting medical data of the patients and transmitting them to the telemedical center. The smartphone can also act as interface to the patient for health questionnaires or feedback.
The system can handle the collection and transport of medical data, analysis and visualization of the data as well as providing a real time communication with video and audio between the users.
On top of the generic telemedical framework the issue of scalability is addressed by integrating a rule-based analysis tool for the medical data. Rules can be easily created by medical personnel via a visual editor and can be personalized for each patient. The rule-based analysis tool is extended by multiple options for visualization of the data, mechanisms to handle complex rules and options for performing actions like raising alarms or sending automated messages.
It is sometimes hard for the medical experts to formulate their knowledge into rules and there may be information in the medical data that is not yet known. This is why a machine learning module was integrated into the system. It uses the incoming medical data of the patients to learn new rules that are then presented to the medical personnel for inspection. This is in line with European legislation where the human still needs to be in charge of such decisions.
Overall, we were able to show the benefit of the generic approach by evaluating it in three completely different medical use cases derived from specific application needs: monitoring of COPD (chronic obstructive pulmonary disease) patients, support of patients performing dialysis at home and councils of intensive-care experts. In addition the system was used for a non-medical use case: monitoring and optimization of industrial machines and robots. In all of the mentioned cases, we were able to prove the robustness of the generic approach with real users of the corresponding domain. This is why we can propose this approach for future development of telemedical systems.
In the context of quantum mechanical calculations, the properties of non-adiabatic coupling in a small system, the Shin-Metiu model, is investigated.
The transition from adiabatic to non-adiabatic dynamics is elucidated in modifying the electron-nuclear interaction. This allows the comparison of weakly correlated electron-nuclear motion with the case where the strong correlations determine the dynamics.
The studies of the model are extended to include spectroscopical transitions being present in two-dimensional and degenerate four-wave mixing spectroscopy.
Furthermore, the quantum and classical time-evolution of the coupled motion in the complete electron-nuclear phase space is compared for the two coupling cases.
Additionally, the numerically exact electron flux within the weak coupling case is compared to the Born-Oppenheimer treatment.
In the last part of the thesis, the model is extended to two dimensions.
The system then possesses potential energy surfaces which exhibit a typical 'Mexican hat'-like structure and a conical intersection in the adiabatic representation.
Thus, it is possible to map properties of the system onto a vibronic coupling (Jahn-Teller) hamiltonian. Exact wave-packet propagations as well as nuclear wave-packet dynamics in the adiabatic and diabatic representation are performed.
Two phases of reef sampling were carried out. The first included regular samples taken along the coastline of Aqaba (27km long) at depths of 4-15m, and used to determine spatial distribution of pollution. The second phase included three 20cm-deep cores obtained from within the industrial zone. These cores were drilled from pre-dated communities, where the growth rate was determined earlier to be 10mm y-1, therefore the core obtained represented a period of 20 years (i.e. 1980-2000). The cores were used to reconstruct the metal pollution history at the most heavily used site along the coast (industrial zone).All samples were examined with respect to their metal content of Cd, Pb, Cu, Zn, Ni, and Cr. Almost all of them have shown records above the calculated background values. Mean values of Cd, Pb, Cu, Zn, Ni and Cr recorded along the coast were 1,25; 4,26; 9,76; 11,40; 2,29 and 10,522, µg g-1 respectively, and for core samples 1.4; 4.2; 5.7; 6.4; 2.3 and 8.21 µg g-1 respectively. Spatial distribution of metal enrichment in reef samples have shown a general and clear increasing trend towards the south. Same increasing trend was also in core samples where the six metals have shown a prominent increasing trend towards the core surface indicating an increase of coastal activities during the last twenty years. High and relatively high values were recorded at the oil port, the industrial area and main port, and thus categorized as highly impacted areas. Intermediate metal content were recorded in samples of the north beach, and thus classified as being relatively impacted, where the lowest metal concentrations were observed at the marine reserve, the least impacted site along the coast. The high enrichment of metal is attributed mainly to anthropogenic impacts. The natural inputs of the six metals studied in the Gulf of Aqaba are generally very low, due to the geographic positions and the absence of wadi discharge and as a result of low rainfall. Several potential sources of heavy metals were investigated. The industrial-related activities, port operations and phosphate dust were among the main sources currently threatening the marine ecosystem in Aqaba. Applying the Principle Components Analysis method (PCA) to all samples taken along the coastline has resulted in categorizing three different groups according to their metal enrichment, the first is composed of samples taken from the north beach and the main port with intermediate to high enrichment, the second joined the samples of the marine park and the marine reserve with low and relatively low enrichment, and the last group joined samples of the industrial zone and the oil port with high enrichment. The Principle Component Scores were also utilized to confirm the spatial distribution and relationships of the examined heavy metals along the coast. Two models (interpolated by SURFER  7.0 and ArcView 3.2a) were developed, the first was based on the PC scores of the first component, and shows clearly the positive anomalies in metal concentrations along the coast. The second model was developed by plotting the second factor scores on a landuse map of Aqaba. According to these models, it has shown that the positive anomalies are associated with three different zones; industrial area, the main port and the oil port. The results have shown that coral reefs can be used as good environmental indicator for assessments and monitoring processes, and they can provide data and information on both the spatial distribution of pollution and their history. The present work is the first to document the environmental status along the whole coast of Aqaba and the first to use coral reef as a tool/ indicator.
Cardiovascular diseases are considered the leading cause of death worldwide according to the World Health Organization. Heart failure is the last stage of most of these diseases, where loss of myocardium leads to architectural and functional decline.
The definitive treatment option for patients with CVDs is organ or tissue transplantation, which relies on donor availability. Therefore, generating an autologous bioengineered myocardium or heart could overcome this limitation. In addition, generating cardiac patches will provide ventricular wall support and enable reparative stem cells delivery to damaged areas. Although many hurdles still exist, a good number of researches have attempted to create an engineered cardiac tissue which can induce endogenous cardiac repair by replacing damaged myocardium.
The present study provided cardiac patches in two models, one by a detergent coronary perfusion decellularization protocol that was optimized, and the other that resulted in a 3D cell-free extracellular matrix with intact architecture and preserved s-glycosaminoglycan and vasculature conduits. Perfusion with 1% Sodium dodecyle sulfate (SDS) under constant pressure resulted in cell-free porcine scaffold within two and cell-free rat scaffold in 7 days, whereas scaffold perfused with 4% sodium deoxycholate (SDO) was not able to remove cells completely. Re-reendothelialization of tissue vasculature was obtained by injecting human microvascular endothelial cell and human fibroblast in 2:1 ratio in a dynamic culture. One-week later, CD31 positive cells and endothelium markers were observed, indicating new blood lining. Moreover, functionality test of re-endothelialized tissue revealed improvement in clotting seen in decellularized tissues. When the tissue was ready to be repopulated, porcine induced pluripotent stem cells (PiPSc) were generated by transfected reprogramming of porcine skin fibroblast and then differentiated to cardiac cells following a robust protocol, for an autologous cardiac tissue model. However, due to the limitation in the PiPSc cell number, alternatively, human induced pluripotent stem cells generated cardiac cells were used.
For reseeding a coculture of human iPSc generated cardiac cells, human mesenchymal stem cells and human fibroblast in 2:1:1 ratio respectively were used in a dynamic culture for 6-8 weeks. Contractions at different areas of the tissue were recorded at an average beating rate of 67 beats/min. In addition, positive cardiac markers (Troponin T), Fibroblast (vemintin), and mesenchymal stem cells (CD90) were detected. Not only that, but by week 3, MSC started differentiating to cardiac cells progressively until few CD90 positive cells were very few by week 6 with increasing troponin t positive cells in parallel. Electrophysiological and drug studies were difficult to obtain due to tissue thickness and limited assessment sources. However, the same construct was established using small intestine submucosa (SISer) scaffold, which recorded a spontaneous beating rate between 0.88 and 1.2 Hz, a conduction velocity of 23.9 ± 0.74 cm s−1, and a maximal contraction force of 0.453 ± 0.015 mN. Moreover, electrophysiological studies demonstrated a drug-dependent response on beating rate; a higher adrenalin frequency was revealed in comparison to the untreated tissue and isoproterenol administration, whereas a decrease in beating rate was observed with propranolol and untreated tissue.
The present study demonstrated the establishment of vascularized cardiac tissue, which can be used for human clinical application.
The coast of Aqaba and the Aqaba region (Jordan) were investigated on their hydrogeo-ecosystem. The results of the research were translated into digits to build a geo-spatial data base. The fillings of the graben aquifer receive indirect type of recharge through the side wadis which drain the highlands. Surface water balance was modeled for a period of 20 years of daily climate records using MODBIL program which attributes direct recharge to wet years only. The hydrodynamic fresh water/seawater interface in the coastal zones was investigated by applying vertical geoelectric surveys and models of several methods to confirm its coincidence with the aquifer’s flow amounts, where human impacts in terms of over-pumping allowed more encroachment of seawater into land, and unintended recharge which led to seaward interface migration. A groundwater balance and solute transport were approached by developing a flow model from the hydrogeological and hydrochemical data. The nature of soil cover and aquifer whose physical properties enhance human impacts indicated the vulnerability of groundwater to pollution. This certainly threatens the marine ecology which forms the sink where the in-excess flow ends. The constructed digital background was exported into GIS to sub-zone the study area in terms of the aquifer’s vulnerability to pollution risks using DRASTIC index. However, it was unable to meet all geo-spatial factors that proved to have significant impacts on the vulnerability. Consequently, a comprehensive index -SALUFT- was developed. This suggests the suitable land use units for each zone in the light of vulnerability grades aiming at protecting the available groundwater resources.
In this dissertation the electronic and high-energy optical properties of thin nanoscale
films of the magnetic topological insulator (MTI) (V,Cr)y(BixSb1-x)2-yTe3 are studied
by means of X-ray photoelectron spectroscopy (XPS) and electron energy-loss
spectroscopy (EELS). Magnetic topological insulators are presently of broad interest
as the combination of ferromagnetism and spin-orbit coupling in these materials
leads to a new topological phase, the quantum anomalous Hall state (QAHS), with
dissipation less conduction channels. Determining and controlling the physical
properties of these complex materials is therefore desirable for a fundamental understanding
of the QAHS and for their possible application in spintronics. EELS can
directly probe the electron energy-loss function of a material from which one can
obtain the complex dynamic dielectric function by means of the Kramers-Kronig
transformation and the Drude-Lindhard model of plasmon oscillations.
The XPS core-level spectra in (V,Cr)y(BixSb1-x)2-yTe3 are analyzed in detail with
regards to inelastic background contributions. It is shown that the spectra can be
accurately described based on the electron energy-loss function obtained from an
independent EELS measurement. This allows for a comprehensive and quantitative
analysis of the XPS data, which will facilitate future core-level spectroscopy studies
in this class of topological materials. From the EELS data, furthermore, the bulk and
surface optical properties were estimated, and compared to ab initio calculations
based on density functional theory (DFT) performed in the GW approximation
for Sb2Te3. The experimental results show a good agreement with the calculated
complex dielectric function and the calculated energy-loss function. The positions of
the main plasmon modes reported here are expected to be generally similar in other
materials in this class of nanoscale TI films. Hence, the present work introduces
EELS as a powerful method to access the high-energy optical properties of TI
thin films. Based on the presented results it will be interesting to explore more
systematically the effects of stoichiometry, magnetic doping, film thickness and
surface morphology on the electron-loss function, potentially leading to a better
understanding of the complex interplay of structural, electronic, magnetic and
optical properties in MTI nanostructures.
This thesis is devoted to numerical verification of optimality conditions for non-convex optimal control problems. In the first part, we are concerned with a-posteriori verification of sufficient optimality conditions. It is a common knowledge that verification of such conditions for general non-convex PDE-constrained optimization problems is very challenging. We propose a method to verify second-order sufficient conditions for a general class of optimal control problem. If the proposed verification method confirms the fulfillment of the sufficient condition then a-posteriori error estimates can be computed. A special ingredient of our method is an error analysis for the Hessian of the underlying optimization problem. We derive conditions under which positive definiteness of the Hessian of the discrete problem implies positive definiteness of the Hessian of the continuous problem. The results are complemented with numerical experiments. In the second part, we investigate adaptive methods for optimal control problems with finitely many control parameters. We analyze a-posteriori error estimates based on verification of second-order sufficient optimality conditions using the method developed in the first part. Reliability and efficiency of the error estimator are shown. We illustrate through numerical experiments, the use of the estimator in guiding adaptive mesh refinement.
We examined the regulation of NFATc1 in different lymphomas and observed an inversed correlation between the methylation status and expression of NFATc1. Our data demonstrate that aberrant DNA methylation associated with chromatin remodeling within nfatc1 locus is a major mechanism for the repression of NFATc1 expression, suggesting that the DNA methylation-mediated transcriptional silencing of NFATc1 may be a critical event in the tumorogenesis of ALCLs and cHLs. Furthermore, the DNA methylation of human nfatc1 promoter region could be used as a novel biomarker of tumor progression. Our results indicate a close link between the loss of immunoreceptor signaling and NFATc1 expression in human lymphomas. For both ALCLs and cHLs, defects in immunoreceptor signaling have been described which result in a loss of receptor-mediated gene expression programs (Schwering et al., 2003; Bonzheim et al., 2004; Marafioti et al., 2004). In T cells, one indicator gene of these programs appears to be the nfatc1 gene whose expression is controlled by TCR signals (Chuvpilo et al., 2002a). In contrast, in T cells NFATc1 expression is unaffected by TCR signals, and NFATc2 was found to be expressed at normal levels in ALCLs and cHLs (L.K., unpubl. data). Moreover, the activity of NF-kappaB factors which can bind to certain NFAT binding sites and share a distantly-related DNA binding domain with NFATs is strongly elevated in cHL cells (Bargou et al., 1997; Hinz et al., 2001; Hinz et al., 2002) suggesting that NFATs and NF-kappaBs exert very different effects on generation and maintenance of Hodgkin’s lymhomas. However, it should be mentioned that in Burkitt’s and further B cell lymphomas in which NFATc1 proteins are strongly expressed and controlled by receptor signals (Kondo et al., 2003), they could exert a promoting function in tumor development. The genes of p53 family members p63 and p73 are prominent examples for mammalian genes whose products can act both as oncoproteins and tumor suppressor genes (Hibi et al., 2000; Stiewe and Putzer, 2002), and it is likely that more genes exist which encode both tumor suppressors and oncoproteins. It remains to be shown whether the nfatc1 gene is one of them.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods of data analysis, which have until now mainly been used to model neural responses of electrophysiological recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing
scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize
statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared
data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods
of data analysis, which have until now mainly been used to model neural responses of electrophysiological
recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-
Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were
determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the
complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with
regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current
scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were
discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
Research on the deployment and use of technology to assist learning has seen a significant
rise over the last decades (Aparicio et al., 2017). The focus on course quality, technology,
learning outcome and learner satisfaction in e-learning has led to insufficient attention by
researchers to individual characteristics of learners (Cidral et al., 2017 ; Hsu et al., 2013). The current work aims to bridge this gap by investigating characteristics identified by previous works and backed by theory as influential individual differences in e-learning. These learner characteristics have been suggested as motivational factors (Edmunds et al., 2012) in decisions by learners to interact and exchange information (Luo et al., 2017).
In this work e-learning is defined as interaction dependent information seeking and sharing enabled by technology. This is primarily approached from a media psychology perspective. The role of learner characteristics namely, beliefs about the source of knowledge (Schommer, 1990), learning styles (Felder & Silverman, 1988), need for affect (Maio & Esses, 2001), need for cognition (Cacioppo & Petty, 1982) and power distance (Hofstede, 1980) on interactions to seek and share information in e-learning are investigated. These investigations were shaped by theory and empirical lessons as briefly mentioned in the next paragraphs. Theoretical support for investigations is derived from the technology acceptance model(TAM) by psychologist Davis (1989) and the hyper-personal model by communication scientist Walther (1996). The TAM was used to describe the influence of learner characteristics on decisions to use e-learning systems (Stantchev et al., 2014). The hyper-personal model described why computer-mediated communication thrives in e-learning (Kaye et al., 2016) and how learners interpret messages exchanged online (Hansen et al., 2015). This theoretical framework was followed by empirical reviews which justified the use of interaction and information seeking-sharing as key components of e-learning as well as the selection of learner characteristics. The reviews provided suggestions for the measurement of variables (Kühl et al., 2014) and the investigation design (Dascalau et al., 2015). Investigations were designed and implemented through surveys and quasi experiments which were used for three preliminary studies and two main studies. Samples were selected from Germany and Ghana with same variables tested in both countries. Hypotheses were tested with interaction and information seeking-sharing as dependent variables while beliefs about the source of knowledge, learning styles, need for affect, need for cognition and power distance were independent variables. Firstly, using analyses of variance, the influence of beliefs about the source of knowledge on interaction choices of learners was supported. Secondly, the role of need for cognition on interaction choices of learners was supported by results from a logistic regression. Thirdly, results from multiple linear regressions backed the influence of need for cognition and power distance on information seeking-sharing behaviour of learners. Fourthly, the relationship between need for affect and need for cognition
was supported. The findings may have implications for media psychology research, theories used in this work, research on e-learning, measurement of learner characteristics and the design of e-learning platforms. The findings suggest that, the beliefs learners have about the source of knowledge, their need for cognition and their power distance can influence decisions to interact and seek or share information. The outlook from reviews and findings in this work predicts more research on learner characteristics and a corresponding intensity in the use of e-learning by individuals. It is suggested that future studies investigate the relationship between learner autonomy and power distance. Studies on inter-cultural similarities amongst e-learners in different populations are also
suggested.
Platelets play an important role in the body, since they are part of the hemostasis
system, preventing and stopping blood loss. Nevertheless, when platelet or
coagulation system function are impaired, uncontrolled bleedings but also irreversible
vessel occlusion followed by ischemic tissue damage can occur. Therefore,
understanding platelet function and activation, mechanisms which are controlled by a
variety of platelet membrane receptors and other factors is important to advance out
knowledge of hemostasis and platelet malfunction. For a complete picture of platelet
function and their modulating behavior it is desired to be able to quantify receptor
distributions and interactions of these densely packed molecular ensembles in the
membrane. This challenges scientists for several reasons. Most importantly, platelets
are microscopically small objects, challenging the spatial resolution of conventional
light microscopy. Moreover, platelet receptors are highly abundant on the membrane
so even super-resolution microscopy struggles with quantitative receptor imaging on
platelets.
With Expansion microscopy (ExM), a new super-resolution technique was introduced,
allowing resolutions to achieve super-resolution without using a super-resolution
microscope, but by combining a conventional confocal microscopy with a highly
processed sample that has been expanded physically. In this doctoral thesis, I
evaluated the potential of this technique for super-resolution platelet imaging by
optimizing the sample preparation process and establishing an imaging and image
processing pipeline for dual-color 3D images of different membrane receptors. The
analysis of receptor colocalization using ExM demonstrated a clear superiority
compared to conventional microscopy. Furthermore, I identified a library of
fluorescently labeled antibodies against different platelet receptors compatible with
ExM and showed the possibility of staining membrane receptors and parts of the
cytoskeleton at the same time.
Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to “free” inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process.
In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions,
To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases.
The Role of Attentional Control and Fear Acquisition and Generalization in Social Anxiety Disorder
(2020)
Although Social Anxiety Disorder (SAD) is one of the most prevalent mental disorders, still little is known about its development and maintenance. Cognitive models assume that deviations in attentional as well as associative learning processes play a role in the etiology of SAD. Amongst others, deficits in inhibitory attentional control as well as aberrations during fear generalization, which have already been observed in other anxiety disorders, are two candidate mechanisms that might contribute to the onset and retention of SAD. However, a review of the literature shows that there is a lack of research relating to these topics. Thus, the aim of the present thesis was to examine in which way individuals with SAD differ from healthy controls regarding attentional control and generalization of acquired fear during the processing of social stimuli.
Study 1 tested whether impairment in the inhibitory control of attention is a feature of SAD, and how it might be influenced by emotional expression and gaze direction of an interactional partner. For this purpose, individuals with SAD and healthy controls (HC) participated in an antisaccade task with faces displaying different emotional expressions (angry, neutral and happy) and gaze directions (direct and averted) serving as target stimuli. While the participants performed either pro- or antisaccades in response to the peripherally presented faces, their gaze behavior was recorded via eye-tracking, and ratings of valence and arousal were obtained. Results revealed that both groups showed prolonged latencies and increased error rates in trials with correct anti- compared to prosaccades. However, there were no differences between groups with regard to response latency or error rates, indicating that SAD patients did not exhibit impairment on inhibitory attentional control in comparison to HC during eye-tracking. Possible explanations for this finding could be that reduced inhibitory attentional control in SAD only occurs under certain circumstances, for example, when these individuals currently run the risk of being negatively evaluated by others and not in the mere presence of phobic stimuli, or when the cognitive load of a task is so high that it cannot be unwound by compensatory strategies, such as putting more effort into a task.
As not only deviations in attentional, but also associative learning processes might be pathogenic markers of SAD, these mechanisms were further addressed in the following experiments. Study 2 is the first that attempted to investigate the generalization of conditioned fear in patients with SAD. To this end, patients with SAD and HC were conditioned to two neutral female faces serving as conditioned stimuli (CS+: reinforced; CS-: non-reinforced) and a fearful face paired with a loud scream serving as unconditioned stimulus (US). Fear generalization was tested by presenting morphs of the two faces (GS: generalization stimuli), which varied in their similarity to the original faces. During the whole experiment, self-report ratings, heart rate (HR) and skin conductance responses (SCR) were recorded. Results demonstrated that SAD patients rated all stimuli as less pleasant and more arousing, and overestimated the occurrence of the US compared to HC, indicating a general hyperarousal in individuals with SAD. In addition, ratings and SCR indicated that both groups generalized their acquired fear from the CS+ to intermediate GSs as a function of their similarity to the CS+. However, except for the HR data, which indicated that only SAD patients but not HC displayed a generalization response in this measure, most of the results did not support the hypothesis that SAD is characterized by overgeneralization. A plausible reason for this finding could be that overgeneralization is just a key characteristic of some anxiety disorders and SAD is not one of them. Still, other factors, such as comorbidities in the individuals with SAD, could also have had an influence on the results, which is why overgeneralization was further examined in study 3.
The aim of study 3 was to investigate fear generalization on a neuronal level. Hence, high (HSA) and low socially anxious participants (LSA) underwent a conditioning paradigm, which was an adaption of the experimental design used study 2 for EEG. During the experiment, steady-state visually evoked potentials (ssVEPs) and ratings of valence and arousal were recorded. Analyses revealed significant generalization gradients in all ratings with highest fear responses to the CS+ and a progressive decline of these reactions with increasing similarity to the CS-. In contrast, the generalization gradient on a neuronal level showed highest amplitudes for the CS+ and a reduction in amplitude to the most proximal, but not distal GSs in the ssVEP signal, which might be interpreted as lateral inhibition in the visual cortex. The observed dissociation among explicit and implicit measures points to different functions of behavioral and sensory cortical processes during fear generalization: While the ratings might reflect an individual’s consciously increased readiness to react to threat, the lateral inhibition pattern in the occipital cortex might serve to maximize the contrast among stimuli with and without affective value and thereby improve adaptive behavior. As no group differences could be observed, the finding of study 2 that overgeneralization does not seem to be a marker of SAD is further consolidated.
In sum, the conducted experiments suggest that individuals with SAD are characterized by a general hyperarousal during the exposition to disorder-relevant stimuli as indicated by enhanced arousal and reduced valence ratings of the stimuli compared to HC. However, the hypotheses that reduced inhibitory attentional control and overgeneralization of conditioned fear are markers of SAD were mostly not confirmed. Further research is required to elucidate whether they only occur under certain circumstances, such as high cognitive load (e.g. handling two tasks simultaneously) or social stress (e.g. before giving a speech), or whether they are not characteristics of SAD at all. With the help of these findings, new interventions for the treatment of SAD can be developed, such as attentional bias modification or discrimination learning.
Assessing particle deposition in a representative in vitro model of the rat respiratory tract
(2014)
The aim of this thesis was to develop an in vitro model (IVR) of the rat lung for the purpose of investigating the deposition of drug particles in the rat airways. The model attempted to account for the affect of drug product characteristics and physiological parameters on deposition in the lungs. In addition, the model outputs were compared with in vivo lung deposition results from live rats and in silico predictions using published computer model of lung deposition in pre-clinical species.
Initial work focussed on developing an aerosol exposure system capable of dosing small rodent to a range of airborne test materials. The system consists of two main parts; a fluidised bed aerosol generator and connection of the generator output to a nose only exposure chamber capable of accommodating 12 small animals in a single layer. In addition, an aerodynamic particle spectrometer (APS) was installed for continuously measuring the size distribution and airborne concentration of aerosol particles generated in the exposure chamber. System validation showed acceptable degree of variation of the test material tested, Fluorescent Microspheres (FMS) throughout the exposure chamber (CV < 15.0%). Particle size (MMAD ± GSD) using the APS was shown to be stable throughout the exposure periods.
The IVR model developed in this project was based on a number of euthanased (n=7), female Sprague-Dawley rats (weight: 372 ± 56 g), which underwent high-resolution micro-CT scans. The physical model consisted of five sub sections; Extra-Thoracic region containing the snout and nasophyarynx, trachea-bronchial region containing the trachea, bronchi, and bronchioles. All sections of the model were attached to one another in numerical order and housed within a containment unit. At the rear end of the cast, a flexible diaphragm was attached in order to collect the fraction of inhaled particles exiting the TB section and possibly reaching the lung, referred to as the Post-TB section.
A study was conducted to assess the influence of inhalation parameters such as the breathing frequency and tidal volume on total and regional dose distribution using FMS as test material. The major finding of this study was the demonstration of the model sensitivity to changes in breathing parameters especially respiratory frequency, where the data showed increased deposition in the peripheral regions of the model with decreased respiratory frequency. Other studies assessed the effect of particle characteristics on deposition on the IVR model, such as particle size, dose increase and formulation changes.
The results assessing particle size effect showed a slightly higher deposition levels for the 4µm sized particles versus 2µm sized particles in the head region; 90.8 ± 3.6% and 88.2 ± 6.6%. However, this difference did not reach statistical significance (P> 0.05) probably due to the polydispersity of aerosolised FMS particles. In addition, the regional deposition analysis showed an increased lung peripheral deposition with the smaller particles. In addition, the model was shown to be sensitive to changes in formulation composition mediated by inclusion of MgSt.
The next stage of work was to validate the model in terms of comparison with lung deposition for in vivo rats. For lung deposition comparison, the absolute amount deposited in the IVR lung model (expressed as µg/kg) was shown to have a reasonably strong correlation with in vivo lung concentration measures (µg/kg); R2= 0.66, P < 0.05. Compounds were predicted well and within 2-folds of the measured lung deposition values. However, knowing the variability in biological systems and the multiple components required to estimate lung doses, predictions within 2-fold of the measured values would seem reasonable
In terms of comparison with in silico model predictions using MPPD, similar deposition levels were noted between the two models, particularly when the data was expressed as percentage of total particles inhaled. The data showed the highest deposition levels were noted in the head region (> 80%) and less than 5.0% deposition for the peripheral lung fractions.
With regards to using the IVR model to assess the relationship between dose, particle size and efficacy, an in vivo study using FP with different particle sizes (2.0 and 4.0 µm) but same doses ( 100 and 1000 µg/kg). This study demonstrated that exposure of rat to FP powder resulted in a dose-dependent inhibition of neutrophils in BAL fluids. However, a clear difference in neutrophils suppression was demonstrated for equivalent doses but different particle sizes of FP, where the smaller FP particles (2.0 µm) induced a greater level of neutrophils suppression in comparison with larger FP particles (4.0 µm). In addition, a reasonably good correlation for the relationship between lung deposition in the IVR model and a neutrophils suppression level was demonstrated. Furthermore this data support the hypothesis that regional deposition is an important determinant in efficacy. Therefore, this suggests that the IVR model may be a useful as a tool to describe in vivo efficacy with in vitro data. However, further studies should be conducted to evaluate the validity of this model and relationship.
The IVR model has a number of important limitations. First, the model is based on scans up to generation four of the rat respiratory tract as this represented the limits of the micro-CT scanning technology at the time of this study. Therefore deposition in the deeper region of the lung may not be reflected precisely in the IVR model. Second, the regional deposition data generated using the model tended to show an overestimation of deposition in head region and an underestimation of deposition in the peripheral regions of the lung, in comparison with in vivo lung deposition data. Third, the current model does not take into account lung clearance. However, the amount of the drug present in the in vivo lungs is dependent on numerous physiological processes such as dissolution, passive or active absorption into the systemic circulation, binding to lung tissue and mucociliary clearance. Consequently, the results generated using this IVR model for drug molecules with high lung clearance rate should be treated with some caution.
Future work extending this research could go in a number of directions. In this research, a representative model of the rat respiratory tract was constructed from analysis of imaging data from a number of euthanised Sprague-Dawley rats. This model represented the “average respiratory tract” in terms of dimensions of Sprague-Dawley rats. However, there is considerable variability in the airway dimensions between rats. This variability encompasses a number of factors such as the strains of rats, sex and age, and disease state. Thus, it may be possible to produce a small number of airway models to represent small and large rats and scaled to represent the extrathoracic and peripheral regions based on literature reports of their dimensions in different rat populations. This approach will then enable the effect of intersubject airway dimensions for different rat populations on aerosol deposition to be thoroughly examined.
In addition, due to the limitation of the micro-CT technology used to construct the physical IVR model, detailed morphology only up to generation 4 were captured. However, recent advances in MRI technology, such as the use of in situ-MRI based scanning technology have enabled rat airway morphometry to be extended to 16 airway generation. This coupled with improvements in the resolutions of rapid-prototyping process means it may be possible to construct a rat model that reflects the in vivo lung morphology more accurately, and thus enable greater understanding of the link between aerosol deposition and airway geometry.
In conclusion, a model cast of the rat lung was developed and validated to allow the deposition of inhaled particles in the rat lung to be investigated. The model may be used to estimate the lung concentration in vivo rats in preference to exposure concentration measurements based on filter samples which have been shown to be a poor indicator of the lung concentration immediately after exposure. In addition, the model has the potential to be used along with live rats in an inhalation rig in pulmonary pharmaceutics research and may facilitate in development of inhaled formulations to target specific regions within the lung as well as screening of inhaled drugs in preclinical setting.
Imprinted genes play important roles in brain development. As the neural developmental capabilities of human parthenogenetic embryonic stem cells (hpESCs) with only a maternal genome were not assessed in great detail, hence here the potential of hpESCs to differentiate into various neural subtypes was determined. In addition DNA methylation and expression of imprinted genes upon neural differentiation was also investigated. The results demonstrated that hpESC-derived neural stem cells (hpNSCs) showed expression of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1), the silencing of pluripotency genes (Oct4, Nanog) and the absence of activation of neural crest (Snai2, FoxD3) and mesodermal (Acta1) markers. Moreover, confocal images of hpNSC cultures exhibited ubiquitous expression of NSC markers Nestin, Sox1, Sox2 and Vimentin. Differentiating hpNSCs for 28 days generated neural subtypes with neural cell type-specific morphology and expression of neuronal and glial markers, including Tuj1, NeuN, Map2, GFAP, O4, Tau, Synapsin1 and GABA. hpNSCs also responded to region-specific differentiation signals and differentiated into regional phenotypes such as midbrain dopaminergic- and motoneuron-type cells. hpESC-derived neurons showed typical neuronal Na+/K+ currents in voltage clamp mode, elicited multiple action potentials with a maximum frequency of 30 Hz. Cell depicted a typical neuron-like current pattern that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels. Furthermore, in hpESCs and hpNSCs the majority of CpGs of the differentially methylated regions (DMRs) KvDMR1 were methylated whereas DMR1 (H19/Igf2 locus) showed partial or complete absence of CpG methylation, which is consistent with a parthenogenetic (PG) origin. Upon differentiation parent-of-origin-specific gene expression was maintained in hpESCs and hpNSCs as demonstrated by imprinted gene expression analyses. Together this shows that despite the lack of a paternal genome, hpNSCs are proficient in differentiating into glial- and neuron-type cells, which exhibit electrical activity similar to newly formed neurons. Moreover, maternal-specific gene expression and imprinting-specific DNA-methylation are largely maintained upon neural differentiation. hpESCs are a means to generate histocompatible and disease allele-free ESCs. Additionally, hpESCs are a unique model to study the influence of imprinting on neurogenesis.
G protein-coupled receptor research looks out for new technologies to elucidate the complex
processes of receptor activation, function and downstream signaling with spatiotemporal
resolution, preferably in living cells and organisms. A thriving approach consists in making use
of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness
and high degree of orthogonality regarding biological processes. This is realized
by the incorporation of molecular photoswitches, which are able to effectively respond to light,
such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic
acetylcholine receptors belong to class A GPCRs and have received special attention in this
regard due to their role as a prototypic pharmacological system and their therapeutic potential.
They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and
thus regulate diverse important biological processes, especially many neurological functions in
our brain.
In this work, the application of photopharmacological tool compounds to muscarinic receptors
is presented, consisting of pharmacophores extended with azobenzene as light-responsive
motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind
concomitantly to the orthosteric and allosteric binding site of the receptor, which is
demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective
photoswitchable ligands, suitable for further ex vivo and in vivo studies.
Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated
extensively with regard to their photochemical behavior and pharmacological profile, outlining
the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho-
fluoro azobenzene. For the first time on a GPCR it was examined, which impact the
different substitution pattern has on both the binding and the activity on the M1 receptor. Results
show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and
F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit
a considerably different biological profile that has to be investigated carefully.
The achievements gained in this study can give important new insights into the binding mode
and time course of activation processes, enabling precise spatial and temporal resolution of the
complex signaling pathway of muscarinic receptors. Due to their role as exemplary model
system, these findings may be useful for the investigation into other therapeutically relevant
GPCRs.
Streptococcus pneumoniae (pneumococci) are Gram-positive bacteria and commensals of the nasopharyngeal cavity. Besides colonization, pneumococci are responsible for severe local infections such as otitis media, sinusitis and life-threatening invasive diseases, including pneumonia, sepsis and meningitis. The surface of pneumococci is decorated with proteins that are covalently or non-covalently anchored to the cell wall. The most unique group of cell wall associated proteins in pneumococci are the choline-binding proteins (CBPs). PspC, also known as SpsA or CbpA, is a multifunctional choline-binding protein that plays an essential role in pneumococcal pathogenesis by functioning as an adhesin. PspC promotes adherence of pneumococci to mucosal epithelial cells by interacting in a human specific manner with the free secretory component (SC) or to SC as part of the secretory IgA (SIgA) or polymeric immunoglobulin receptor (pIgR). PspC also interacts specifically with the soluble complement Factor H. Apparently, PspC uses two different epitopes for binding the soluble host protein Factor H and SC of pIgR. However, the mechanism by which these independent interactions facilitate pneumococcal infections under physiological and host specific conditions have not yet been completely elucidated. This study aims to explore the impact of the PspC interaction with human pIgR (hpIgR) or complement regulator Factor H on pneumococcal virulence. Here the cellular and molecular basis of PspC-mediated adherence to and invasion of host epithelial and endothelial cells was demonstrated. The genetic approach, specific pharmacological inhibitors and immunoblot analysis demonstrated the complexity of the induced signal transduction pathways during PspC-hpIgR mediated pneumococcal uptake by host cells. Inhibition studies with specific inhibitors of actin cytoskeleton and microtubules demonstrated that the dynamics of host cell cytoskeleton are essential for pneumococcal uptake by mucosal epithelial cells. Moreover, this study reports for the first time that the small GTPase Cdc42 is essential for pneumococcal internalization into epithelial cells via the PspC-hpIgR mechanism. In addition, in infection experiments performed in presence of specific inhibitors of PI3-kinase/Akt and protein tyrosine kinase (PTKs), hpIgR-mediated pneumococcal uptake by host cells was significantly blocked. Amongst PTKs the Src kinase pathway, ERK1/2 and JNK pathways were implicated during pneumococcal ingestion by hpIgR expressing cells. In addition, inhibition experiments performed in the presence of individual inhibitors or with a combination of inhibitors suggested the independent activation of PI3-kinase/Akt and Src kinase pathways during pneumococcal infections of hpIgR expressing cells. By employing specific inhibitors and siRNA in cell culture infection experiments it was further demonstrated that pneumococcal endocytosis by host epithelial cells via the PspC-hpIgR mechanism depends on clathrin and dynamin. PspC recruits also Factor H to the pneumococcal cell surface. Consequently, the impact of pneumococcal cell surface bound Factor H on adherence to host cells and the molecular mechanism facilitating the uptake of Factor H bound pneumococci by epithelial cells was investigated. Flow cytometry and immunoblots revealed that S. pneumoniae has evolved the ability to recruit both purified Factor H as well as Factor H from human plasma or serum. Moreover, it was demonstrated that the recruitment of Factor H is independent of the PspC-subtypes and that capsular polysaccharide (CPS) interferes with its recruitment. Factor H bound to pneumococci significantly increased bacterial attachment to and invasion of host epithelial cells including nasopharyngeal cells (Detroit562), lung epithelial cells (A549), and human brain-derived endothelial cells (HBMEC). Blocking experiments demonstrated that bacteria bound Factor H interacts via the heparin binding sites on Factor H with eukaryotic cell surface glycosaminoglycans and that this interaction promotes pneumococcal adherence to host cells. In addition, inhibition studies with mAbs recognizing specifically different short consensus repeats (SCR) of Factor H suggested that SCR 19-20 of Factor H are essential for the pneumococcal interaction with host epithelial cells via Factor H. In the presence of Factor H, attachment of pneumococci to human polymorphonuclear leukocytes (PMNs) is enhanced. The integrin CD11b/CD18 was identified as the cellular receptor on PMNs. By using pharmacological inhibitors the impact of host cell cytoskeleton and signalling molecules, such as PTKs and PI3-kinase, for Factor H-mediated pneumococcal internalization into eukaryotic cells was shown. Taken together, the results revealed that Factor-H mediated pneumococcal infection requires a concerted role of host epithelial cell surface glycosaminoglycans, integrins and host cell signalling pathways.
Malaria still persists as one of the deadliest infectious disease in addition to AIDS and tuberculosis. lt is a leading cause of high mortality and morbidity rates in the developing world despite of groundbreaking research on global eradication of the disease initiated by WHO, about half a century ago. Lack of a commercially available vaccine and rapid spread of drug resistance have hampered the attempts of extinguishing malaria, which still leads to an annual death toll of about one million people. Resistance to anti-malarial compounds thus renders search for new target proteins imperative. The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Several reports till date have suggested involvement of parasite kinases in the human host and as well as in the mosquito vector. Kinases essential for life cycle stages of the parasite represent promising targets for anti-malarial compounds thus, provoking characterization of additional malarial kinases. Despite extensive research on most plasmodial enzymes, very little information is available regarding the four identified members of the cyclin dependent kinase like kinase (CLK) family. Thus, the present thesis dealt with the functional characterization of four members of the PfCLK kinase family of the parasite denoted as PfCLK-1/Lammer, PfCLK-2, PfCLK-3 and PfCLK-4 with a special focus on the first two kinases. Additionally, one Ca2+/Calmodulin dependent putative kinase-related protein, PfPKRP, presumed to be involved in sexual stage development of the parasite, was investigated for its expression in the life cycle of the parasite. In other eukaryotes, CLK kinases regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Transcription analysis revealed abundance of PfCLK kinase genes throughout the asexual blood stages and in gametocytes. By reverse genetics approach it was demonstrated that all four kinases are essential for completion of the asexual replication cycle of P. falciparum. PfCLK 1/Lammer possesses two nuclear localization signals and PfCLK-2 possesses one of these signals upstream of the C-terminal catalytic domains. Protein level expression and sub-cellular localization of the two kinases was determined by generation of antiserum directed against the kinase domains of the respective kinase. Indirect immunofluorescence, Western blot and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and in vitro assays show that both enzymes are associated with phosphorylation activity. Finally, mass spectrometric analysis of co immunoprecipitated proteins shows interactions of the two PfCLK kinases with proteins, which have putative nuclease, phosphatase or helicase functions. PfPKRP on the other hand is predominantly expressed during gametocyte differentiation as identified from transcriptional analysis. Antiserum directed against the catalytic domain of PfPKRP detected the protein expression profile in both asexual and gametocyte parasite lysates. Via immunofluorescence assay, the kinase was localized in the parasite cytoplasm in a punctuated manner, mostly in the gametocyte stages. Reverse genetics resulted in the generation of PfPKRP gene-disruptant parasites, thus demonstrating that unlike CLK kinases, PfPKRP is dispensable for asexual parasite survival and hence might have crucial role in sexual development of the parasite. On one hand, characterization of PfCLK kinases exemplified the kinases involved in parasite replication cycle. Successful gene-disruption and protein expression of PfPKRP kinase on the other hand, demonstrated a role of the kinase in sexual stage development of the parasite. Both kinase families therefore, represent potential candidates for anti-plasmodial compounds.
The aim of the present study was to design different dosage forms as carrier systems to deliver sorafenib to the lung of BXB-23 transgenic mice using different routes of administration. Three dosage forms were used one of them was an oil-in-water emulsion and the oral route was chosen for this experiment. The other delivery system was a liposome preparation for intratracheal instillation. In this case the oral route was considered as a control experiment. The last dosage form was PLGA microspheres. Before sorafenib administration it was important to develop a HPLC method to assess sorafenib absorption after its administration and to determine its concentrations in mouse serum. The HPLC method allowed sorafenib quantification in small volumes (30 µl) of mouse serum and tissues. The developed HPLC method was validated resulting in satisfactory selectivity, good linearity, good accuracy and precision over the concentration range examined. Sorafenib was successfully incorporated in a fat emulsion (o/w) using a traditional method resulting in a white homogenous emulsion and no particle aggregation was observed. Sorafenib exhibited antitumor activity on the lung adenoma in BXB-23 transgenic mice when administered orally (2 mg sorafenib per mouse) in the emulsion preparation. The determined effect was an approximately 29 % reduction in the tumor area of the adenoma foci and a proliferation reduction. In order to improve the pharmacological effects of sorafenib on the lung adenoma in BXB-23 mice, the targeting of sorafenib directly to the site of action (the lung) was an attractive concept. For this purpose the intratracheal route was used. Since sorafenib administration by instillation required incorporation of sorafenib in a dosage form suitable for its lipophilic nature, a liposome suspension was the second dosage form used. A lyophilization method was employed for sorafenib liposome preparation utilizing dilauroylphosphatidylcholine (DLPC) which is safe and tolerable for the lung. Incorporation of sorafenib in the liposomes did not influence the particle size and its distribution. The sorafenib liposomes showed high encapsulation efficiency, good stability at 4 °C for one month and satisfactory in vitro release properties and inhibited Raf-1 mediated activation of ERK in cell culture assay. In a pharmacokinetic experiment sorafenib loaded liposomes were instilled directly into the lung. The results revealed that a significant level of sorafenib was achieved in the lung tissues after 2 hours and then reduced after 48 h and remained nearly constant for one week. On the other hand, only traces of sorafenib were found in the mice serum up to 48 h. Subsequently, the pharmacological activity of sorafenib (1 mg per mouse) was studied when delivered in a liposomal suspension intratracheally to treat the lung adenoma of BXB-23 mice. The data of this experiment demonstrated that sorafenib intratracheal instillation resulted in a reduction of tumor area of adenoma foci (67 %) and an elevation of the percent of apoptotic cells. In contrast, prolongation of the treatment period did not further enhance sorafenib activity on the lung adenoma. This previous finding suggested a development of multidrug resistance (MDR) by the adenoma foci cells against sorafenib instillation, which was examined by immunohistochemistry staining. The percent of MDR positive cells was higher after two and three weeks sorafenib liposome instillation treatment than that after one week treatment. The last dosage form used for sorafenib was microspheres, which were prepared by emulsion-diffusion-evaporation method using biodegradable PLGA 50:50 resulting in a white lyophilized powder. The system was characterized physicochemically and revealed a good microspheres yield, high encapsulation efficiency, a homogenous particle size distribution and slow in vitro release of sorafenib. The other strategy studied in the present research project was gene delivery to target the lung bearing tumor of BXB-23 mice using a non-viral vector (polyethylenimine). Polyethylenimine (PEI) was used to investigate its efficiency in transfecting lung bearing tumor of BXB-23 mice model and its ability to transfect the adenoma foci cells. LacZ, which encodes Beta-galactosidase was used in the present study as a reporter gene and was complexed with PEI before delivered intravenously. A high LacZ expression in the alveolar region with some expression in the adenoma foci was observed. On contrary, a low LacZ expression in the alveoli and in the adenoma foci was achieved after instillation of the same polyplex intratracheally.
The high failure rate of new drug candidates in preclinical or clinical studies due to hepatotoxicity represents a considerable problem in the drug development. Hence, there is an urgent need to develop new approaches for early and reliable prediction of drug-induced hepatotoxicity that enables a better identification of drug candidates with high potential for toxicity at early stages of drug development. Therefore, the aim of this work was to improve the prediction of drug-induced liver injury in preclinical studies through evaluation of more reliable and sensitive biomarkers of hepatotoxicity and a better understanding of the underlying mechanistic basis for drug-induced toxicity. First, the ability of a set of potential markers (NGAL, thiostatin, clusterin, PON1) to detect early signs of liver injury was assessed in rats treated with drug candidates that were dropped from further development, in part due to toxic adverse effects in the liver. In summary, PON1 and clusterin were not consistently altered in response to liver injury and thus provide no additive information to the traditional liver enzymes in detecting drug-induced hepatotoxicity. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, such as ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment. In the second part of this work, combined application of genomics profiling technology and RNAi to inhibit the pharmacological target of a drug candidate BAY16, a glucagon receptor (GCGR) antagonist, was used to determine if interference with the pharmacological target plays a role in the toxic response to BAY16, and to narrow down those molecular changes that are associated with toxicity, and not the pharmacological action of BAY16. In contrast to Bay 16, which was found to be cytotoxic at concentrations of 75 µM, silencing of the glucagon receptor did not affect cell viability in primary rat hepatocytes. Thus, it can be concluded that hepatotoxicity of Bay 16 was not related to the drugs inhibitory effect on the glucagon receptor in vitro and in vivo. These findings were supported by the fact that most of BAY16-induced changes in gene expression occurred independently of the pharmacological modulation of GCGR. These off-target effects include altered xenobiotic metabolism, oxidative stress, increased fatty acid synthesis, and alterations in cholesterol and bile acid metabolic processes. Although it was not possible to draw a final conclusion about the mechanism of BAY16 hepatotoxicity, changes in these molecular mechanisms appear contribute to progression of hepatic injury. With regard to drug safety assessment in preclinical studies, the utilization of siRNA technology in vitro represents a new approach to improve mechanistic understanding of the nature of drug’s toxicity, being either chemically mediated or due to primary or secondary pharmacological mode of action.