Towards single cell proteomics

This thesis focuses on different novel ideas and concepts in the area bioanalytics in order to develop the sensitivity and liquid handling towards the level of single cell proteomics. In biochemical sensors binding events are detected, when target molecules diffuse close enough to interact with specific recognition elements. To develop a fast and sensitive immunosensor, we benefit from short diffusion times and capillarity in microchannels. We fabricated an on-chip immunochemical surface assay which is performed within a microfluidic system. Using such a chip, the concentration of CRP in human blood serum was determined within eleven minutes. We were able to detect less than 1 ng/mL of CRP using only 1 microlitre of sample. To further reduce the sample volume towards single cells, we first structured surfaces with nanometer-sized patterns to separate, handle and culture individual cells. Pillar arrays with a height of 1 micrometer, aspect ratios of 1:5 and a top diameter of 120 nm were fabricated in silicon and were used as a master to produce a PDMS intermediate on which PLLA replicas were casted. At an inter-pillar distance of 200 nm, we could show how individual cells grow along the lines of cones replicated in PLLA. To handle the liquid content of individual cells and to detect single molecules within such heterogeneous analytes, we developed a method to prepare total content sample for electron microscopy. The method combines microfluidic-based in-line negative staining for TEM as well as desalting for mass measurements by STEM. The main advantages are the lossless sample preparation by liquid contact writing of micro-patterns on EM grids and excellent staining at physiological pH. To detect low molecular weight single molecules label-free with a very high specificity, we propose to immobilize arrays of single DARPins on a very flat surface and to discriminate their bound and unbound state by height measurements using AFM. Arrays of immobilization islands for single DARPins were fabricated by EUV-IL, EBL and a newly developed direct immobilization method. By EUV-IL and a glancing angle metal deposition step 20 nm sized gold immobilization islands could be fabricated. By EBL and thermal annealing, arrays of 5 nm sized gold islands have been achieved. These islands are in the size range of single DARPin molecules. To functionalize only the gold islands with DARPins, the surrounding silicon dioxide surface has to be protected against non-specific DARPin adsorption. However PEG molecules for efficient passivation are often to long and the small immobilization islands might be buried by PEG. Therefore we used a photoresist mask and a chemical linker to directly immobilize single DARPins onto silicon dioxide. On the same chip, the pattern size of the mask was varied and besides several mm sized lines full of DARPins, arrays of single immobilized DARPins could be produced. On such arrays, single binding events between DARPins and their corresponding target proteins were detected and bound and unbound DARPins could be discriminated. The developed methodologies and the engineered surfaces are promising tools for the analysis towards single cell proteomics and their further development might result in valuable methods for systems biology. Keywords: microfluidics, pillar arrays, nano-dot array, single cell, cell growth, AFM, TEM, STEM, XIL, EBL, GLAD, thermal annealing, immunoassay, DARPin

Light exposure of roots in aeroponics enhances the accumulation of phytochemicals in aboveground parts of the medicinal plants Artemisia annua and Hypericum perforatum

Light acts as a trigger to enhance the accumulation of secondary compounds in the aboveground part of plants; however, whether a similar triggering effect occurs in roots is unclear. Using an aeroponic setup, we investigated the effect of long term exposure of roots to LED lighting of different wavelengths on the growth andp hytochemical composition of two high-value medicinal plants, Artemisia annua and Hypericum perforatum. In A. annua, root exposure to white, blue, and red light enhanced the accumulation of artemisinin in the shoots by 2.3-, 2.5-, and 1.9-fold, respectively. In H. perforatum, root exposure to white, blue, red, and green light enhanced the accumulation of coumaroylquinic acid in leaves by 89, 65, 84, and 74%, respectively. Root lighting also increased flavonol concentrations. In contrast to its effects in the shoots, root illumination did not change phytochemical composition in the roots or root exudates. Thus, root illumination induces a systemic response, resulting in modulation of the phytochemical composition in distal tissues remote from the light exposure site.publishedVersio

B2B App Store Governance in Software Platform Ecosystems: Dimensions and Types

The ever-increasing customer demand for use case-specific B2B software puts platform owners into a challenging situation where integrating a B2B app store into their digital platform becomes a necessity to manage the dynamics of software platform ecosystems. However, platform owners face uncertainty and experiment, while platform ecosystem research provides limited guidance for specific B2B app store governance. Closing this gap, we use multiple case studies and develop three taxonomies for architecture, control mechanisms, and demand generation to provide an overview of the solution space for B2B app store governance. We further derive three robust B2B app store governance types: platform play, transaction channel, and community platform. This paper enriches the B2C-driven and core-offering related research on digital platform governance with tangible B2B app store governance dimensions and types. We envision to guide practitioners in identifying and selecting governance characteristics to remain competitive and provide innovation for their B2B app stores

Christian Menn - Reden bei der akademischen Feier aus Anlaß der Verleihung der Ehrendoktorwürde (Dr.-Ing. E.h.) an Prof. Dr. sc. techn. Christian Menn durch die Universität Stuttgart am 2. Februar 1996

Es ist eine alte, sehr schöne Tradition, daß Universitäten die issenschaftlichen Leistungen, die Werke und das Wirken herausragender Persönlichkeiten in esonderer Weise würdigen. Die Universität Stuttgart nimmt diese akademische Tradition sehr ernst und pflegt sie in entsprechend zurückhaltender Weise unter Anlegung strenger Kriterien. So stellt die Verleihung der Würde eines Ehrendoktors ein ganz besonderes Ereignis im akademischen Leben dar. Mit Professor Dr.sc.techn.Christian Menn wird ein Mann geehrt, der einer der kreativsten Brückenbauer unserer Zeit ist, der fundiertes Wissen und Intuition in außergewöhnlicher Form miteinander verknüpft. Verkehrswege verbinden seit jeher die Menschen und ihre Kulturen; Brücken stellen dabei – symbolisch und real – besonders herausragende Elemente dar. Christian Menn hat mit seinen virtuosen Bauwerken unverwechselbaren Charakters einen wesentlichen Beitrag zur Baukultur geleistet und ist daher zu Recht einer der meistzitierten Brückenbauer in der internationalen Fachliteratur. Die Universität Stuttgart ist stolz darauf, diesen großen Ingenieur ehren zu dürfen und über ihn eine weitere Brücke zur ETH Zürich bauen zu können und damit die traditionell engen und guten Beziehungen zu festigen

Iron and Phosphate Deficiency Regulators Concertedly Control Coumarin Profiles in Arabidopsis thaliana Roots During Iron, Phosphate, and Combined Deficiencies

Plants face varying nutrient conditions, to which they have to adapt to. Adaptive responses are nutrient-specific and strategies to ensure supply and homeostasis for one nutrient might be opposite to another one, as shown for phosphate (Pi) and iron (Fe) deficiency responses, where many genes are regulated in an opposing manner. This was also observed on the metabolite levels. Whereas root and exudate levels of catechol-type coumarins, phenylpropanoid-derived 2-benzopyranones, which facilitate Fe acquisition, are elevated after Fe deficiency, they are decreased after Pi deficiency. Exposing plants to combined Pi and Fe deficiency showed that the generation of coumarin profiles in Arabidopsis thaliana roots by Pi deficiency considerably depends on the availability of Fe. Similarly, the effect of Fe deficiency on coumarin profiles is different at low compared to high Pi availability. These findings suggest a fine-tuning of coumarin profiles, which depends on Fe and Pi availability. T-DNA insertion lines exhibiting aberrant expression of genes involved in the regulation of Pi starvation responses (PHO1, PHR1, bHLH32, PHL1, SPX1) and Fe starvation responses (BRUTUS, PYE, bHLH104, FIT) were used to analyze the regulation of the generation of coumarin profiles in Arabidopsis thaliana roots by Pi, Fe, and combined Pi and Fe deficiency. The analysis revealed a role of several Fe-deficiency response regulators in the regulation of Fe and of Pi deficiency-induced coumarin profiles as well as for Pi deficiency response regulators in the regulation of Pi and of Fe deficiency-induced coumarin profiles. Additionally, the regulation of Fe deficiency-induced coumarin profiles by Fe deficiency response regulators is influenced by Pi availability. Conversely, regulation of Pi deficiency-induced coumarin profiles by Pi deficiency response regulators is modified by Fe availability

Assessing the impact of a combined analysis of four common low-risk genetic variants on autism risk

<p>Abstract</p> <p>Background</p> <p>Autism is a complex disorder characterized by deficits involving communication, social interaction, and repetitive and restrictive patterns of behavior. Twin studies have shown that autism is strongly heritable, suggesting a strong genetic component. In other disease states with a complex etiology, such as type 2 diabetes, cancer and cardiovascular disease, combined analysis of multiple genetic variants in a genetic score has helped to identify individuals at high risk of disease. Genetic scores are designed to test for association of genetic markers with disease.</p> <p>Method</p> <p>The accumulation of multiple risk alleles markedly increases the risk of being affected, and compared with studying polymorphisms individually, it improves the identification of subgroups of individuals at greater risk. In the present study, we show that this approach can be applied to autism by specifically looking at a high-risk population of children who have siblings with autism. A two-sample study design and the generation of a genetic score using multiple independent genes were used to assess the risk of autism in a high-risk population.</p> <p>Results</p> <p>In both samples, odds ratios (ORs) increased significantly as a function of the number of risk alleles, with a genetic score of 8 being associated with an OR of 5.54 (95% confidence interval [CI] 2.45 to 12.49). The sensitivities and specificities for each genetic score were similar in both analyses, and the resultant area under the receiver operating characteristic curves were identical (0.59).</p> <p>Conclusions</p> <p>These results suggest that the accumulation of multiple risk alleles in a genetic score is a useful strategy for assessing the risk of autism in siblings of affected individuals, and may be better than studying single polymorphisms for identifying subgroups of individuals with significantly greater risk.</p

Detection and molecular analysis of Hop latent virus and Hop latent viroid in hop samples from Poland

Die Überwachung von Viruskrankheiten bei Pflanzen ist wichtig für die Durchführung frühzeitiger Kontrollmaßnahmen und die Verhinderung der weiteren Ausbreitung der Erreger. In Polen wurde im Jahr 2004 ein Programm zur Eliminierung von Viren und Viroiden im Hopfen gestartet. In den Jahren 2012/13 wurden in vitro Pflanzen, Proben aus der IUNG-PIB Versuchsstation und aus kommerziellen polnischen Hopfengärten auf das Hop latent virus, Hop latent viroid und Hop stunt viroid getestet. Für die Virus­testung wurden RT-PCR und ELISA eingesetzt. Die Viroide wurden mittels RT-PCR nachgewiesen. Insgesamt war die Nachweishäufigkeit für Viren und Viroide geringer als vor dem Start des Programms. Klonierung und Sequenzierung lassen den Schluss zu, dass das Hop latent virus und das Hop latent viroid aus den polnischen Proben den „type“ Sequenzen und den tschechischen Viren/Viroiden sehr ähnlich sind. DOI: 10.5073/JfK.2014.07.04, https://doi.org/10.5073/JfK.2014.07.04Monitoring the occurrence of virus diseases in plants is important for the implementation of early control measures and prevention of further disease spread. In Poland, in 2004 a health programme for hop was started to eliminate viruses and viroids. In 2012/13, in vitro plants, samples from the IUNG-PIB experimental station and commercial hop gardens in Poland were tested for Hop latent virus (HpLV), and Hop latent and Hop stunt viroids (HpLVd and HpSVd). For virus testing, RT-PCR and ELISA methods were used. In order to detect hop viroids, RT-PCR was employed. The overall incidence of HpLV and hop viroids was lower than reported before the start of the programme. Cloning and sequencing revealed that the HpLV and the HpLVd from Polish sources are very similar to the type sequences and the Czech sources. DOI: 10.5073/JfK.2014.07.04, https://doi.org/10.5073/JfK.2014.07.0

Special Topic: Identifying effective learning environments -Part I Guest Editorial

Researchers and laypeople alike continue to argue over the relative importance of genes versus environment in explaining superior accomplishments in art, science, sport and education. Although this debate shows no signs of abatement, many researchers in the fields of giftedness and expertise have focused on how the environment influences the attainment of achievement. Obviously, environmental influences can be wide-ranging; studies in this area vary from understanding differences between systems of development (e.g., between nations) and global policies regarding instruction to the manipulation of specific variables in training/practice environments to determine their effectiveness to maximize learning effects. Leading models of expertise development and giftedness (e.g.

Regelsystem zur Einstellung des optimalen Schaltzeitpunktes bei Schaltflankensynchronisation

Durch den vermehrten Einsatz von neuartigen schnellschaltenden Leistungstransistoren auf Basis von Halbleitermaterialien mit großer Bandlücke und der Miniaturisierung von leistungselektronischen Schaltungen werden Probleme, verursacht durch EMV-Störungen, immer größer. Herkömmlich werden hierfür EMV-Filter eingesetzt, welche jedoch in ihrer Größe und ihres Gewichts steigen. Deshalb wird versucht, diese herkömmlichen EMV-Filter zu vermeiden. Eine Möglichkeit dabei ist der Einsatz von aktiven EMV-Filtern. Hierbei werden dem Störsignal entgegengesetzte Signale erzeugt und so aufgeschaltet, dass sie das Störsignal vermindern [1– 3]. Eine weitere Möglichkeit die Störsignale zu vermindern ist die Synchronisation entgegengesetzter Schaltflanken [4–10]. Hierbei werden jeweils eine steigende und fallende Schaltflanke miteinander synchronisiert, sodass sich die erzeugten Gleichtaktstörungen gegenseitig kompensieren. Entscheidend für die Kompensation sind gleich große Schaltgeschwindigkeiten, ein symmetrischer Ausbreitungsweg der Störungen und möglichst gleichzeitig auftretende Schaltzeitpunkte. Durch Laufzeitunterschiede im Gatepfad, wie beispielsweise die des Gatetreibers, treten die Schaltzeitpunkte zweier Transistoren trotz gleichzeitigem Schaltbefehl aus dem Ansteuersystem nicht gleichzeitig auf. Diese Unterschiede sollen im Regelsystem dieses Beitrages automatisch kompensiert werden und die Schaltbefehle so aufbereitet werden, dass die Schaltzeitpunkte gleichzeitig auftreten. Für ein Regelsystem sind im allgemeinen folgende Komponenten nötig. Zunächst muss eine Größe erfasst werden, welche den Zustand des Systems beschreibt. Mit dieser Größe muss es möglich sein, eine ausreichend große Abhängigkeit der Störungen vom Schaltzeitpunkt zu messen. Anhand dieser Messgröße kann ein Regelalgorithmus den optimalen Schaltzeitpunkt, welcher über ein geeignetes Stellglied einstellbar sein muss, bestimmen. [11] beschreibt ein solches Regelsystem, welches die Schaltflanken aufeinander abstimmt. Dieser Beitrag unterscheidet sich jedoch deutlich in der Erfassung und Bewertung des Systemzustandes. Weiterhin wird in [11] eine Einstellung des Schaltzeitpunktes in deutlich gröberen Zeitschritten vorgestellt. Die Schrittweite der einzelnen Schaltzeitpunkte ist in [11] durch die Modulation bedingt im Bereich mehrerer Nanosekunden. Dieser Beitrag präsentiert ein Regelverfahren zur Einstellung eines optimierten Schaltzeitpunktes mit einer Auflösung von 100 ps