Automation and Optimization of Working Speed and Depth in Agricultural Soil Tillage with a Model Predictive Control based on Machine Learning

While facing environmental challenges due to climate change, the need for optimization and automation of agricultural tasks is increasing. Furthermore, costs and the lack of qualified personnel require efficient and highly automated control systems for agricultural machinery. Therefore, this work addresses these challenges by optimizing the working speed of a tractor and soil tillage implement combination to maintain efficient operating points during high power demands. A system was developed that predicts a suitable working speed based on a draft force and traction model in combination with the usage of a neural network for fuel rate prediction. The machine operator is able to customize optimization parameters such as fuel efficiency, performance or total costs depending on the individual needs and situation. These parameters lead to a reward function to value the machines state. Based on these objectives the network is able to predict the system state for various potential target speeds and evaluate their optimization parameters to select the most promising target speed. This target speed gets received by the tractor and leads to a new machine state. The fuel rate prediction network is trained on previously collected training data. Using different methods, for example transfer learning, the network can be adapted easily to different sizes and types of tractors. As the draft force models are based on equations, they can be changed to adapt to turning and no-turning soil tillage. To maintain a sufficient working quality and simplify online parametrization of draft force requirements, the implement working depth is automatically adjusted based on active Lidar measurements. The adjustments take the working conditions and agricultural requirements into account. The system was validated during field measurements on different locations with various customized optimization parameters. The results show a suitable reaction to changing operating conditions

Molekulares Biopatterning technischer Oberflächen und neue Drucktechnologien für innovative Biotransformationssysteme

Im Rahmen dieser Arbeit konnte ein Reaktorsystem entwickelt und eingehend charakterisiert und bewertet werden, wobei es sich als wertvolle Technologie zur Implementierung und spezifischen Regelung von enzymatischen Produktionskaskaden herausstellte

Herausforderungen in der Biokatalyse - Fallstudien zur Expression, Optimierung und Anwendung von Proteinen

Biotechnology is emerging as a key technology in the 21st century. In the past years, scientific and technological advances have established the field of biocatalysis as a competitive alternative to traditional metallo- and organocatalysis in chemical synthesis. Yet many, often basic challenges remain in the key steps of biocatalytic processes, which include the biocatalyst production, characterization, engineering, and application as well as down-stream processing. In the scope of this thesis, three case studies were performed to illustrate solutions for selected, critical challenges in these key steps. The first case study featured dirigent proteins, which are involved in the stereoselective control of the first step of lignan biosynthesis, the phenoxyl radical coupling of coniferyl alcohol radicals to yield optically pure (+)- or (-)-pinoresinol. Up to now, extraction of dirigent proteins from plant material or expression in plant and insect cell culture systems resulted in low yields. The metylotrophic yeast Pichia pastoris was evaluated as a possible host for recombinant protein expression. A reliable high yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg mL-1 of the dirigent protein AtDIR6 representing a more than 250-fold increase compared to other sources was established. Biochemical characterization of AtDIR6 produced with Pichia pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum, validating Pichia pastoris as a suitable expression system for dirigent proteins. Enzymatic deglycosylation of the protein induced conformational changes leading to the loss of function and subsequent protein aggregation. This demonstrated that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein. The second case study focused on a ribokinase from Saccharomyces cerevisiae, which was engineered for an altered substrate spectrum by rational protein design. The enzyme was produced in high yields in a heterologous expression system using Escherichia coli as host and biochemically characterized for the first time. Optimum reaction conditions were investigated regarding the dependency on mono-, di-, and pentavalent ions and the pH. Conditions featuring 100 mM potassium phosphate at pH 6.0 with 2.5 mM magnesium chloride were found to offer the highest catalytic activity. Based on a family alignment and rational design using a homology model of the protein, a small focused library of the ribokinase featuring 72 variants was established. The library was evaluated for alterations in the substrate spectrum with a focus on D-xylose, which is an epimer to the natural substrate D-ribose. More radical alterations such as triple and quadruple mutations abolished catalytic activity towards D-ribose and D-xylose almost completely. Several variants were identified broadening the substrate spectrum to other tetroses, pentoses, and hexoses, albeit with low catalytic efficiency. A single variant exhibited about twice the activity of the wild-type enzyme towards D-xylose (0.77 s-1 versus 0.38 s-1), which can serve as a basis for further studies. In this regard, an attempt to solve a crystal structure for more information about D-xylose binding in the active site was started. While several well diffracting crystals were obtained, molecular replacement did not lead to a solution of the structure so far. The third case study featured a cutinase from the ascomycete Humicola insolens, which was applied as a novel biocatalyst for the synthesis of functionalized acrylic esters by transesterification. The transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system was studied. The enzyme was employed in immobilized form on a microporous, polystyrenic resin to facilitate the usability and stability in a non-aqueous environment and allow simple recovery of the biocatalyst. Besides two minor by-products, 6-mercapothexyl acrylate ester was identified as the main product with the thiol as the functional end group. By optimization of the reaction conditions and critical water content, the transesterification yielded 95.4 ± 0.3% of 6-mercaptohexyl acrylate ester after 6 h at 40°C in the presence of 0.025% (w/w) water without formation of by-products in a solvent free system. When applying methyl methacrylate as an acyl acceptor, transesterification with 6-mercapto-1-hexanol was significantly lower (43.6 ± 0.1%).Die grundlegenden Schritte eines biokatalytischen Prozesses umfassen sowohl die Produktion, die Charakterisierung, die Optimierung, die Anwendung sowie die Wiederverwertung von Biokatalysatoren als auch die Aufarbeitung der Produkte. Im Rahmen der vorliegenden Arbeit wurden ausgewählte, kritische Herausforderungen in diesen Schlüsselschritten in drei Fallstudien näher untersucht. Dirigierende Proteine sind in der stereoselektiven Kontrolle des ersten Schrittes in der Lignan Biosynthese beteiligt. In einem Radikalkupplungsschritt werden zwei Koniferylalkoholradikale unter Beteiligung von dirigierenden Proteinen so verknüpft, dass optisch reines (+)- bzw. (-)-Pinoresinol entsteht. Bisher wurden Dirigierende Proteine in geringen Ausbeuten durch Extraktion aus Pflanzenmaterial oder durch heterologe Expression in pflanzlichen Zellkulturen sowie Insektenzellkulturen gewonnen. Die Hefe Pichia pastoris wurde als möglicher Expressionswirt für die rekombinante Proteinproduktion evaluiert. Mit Pichia pastoris konnte ein zuverlässiger Fed-Batch Fermentationsprozess etabliert werden, welcher in Ausbeuten von 47 mg mL-1 des dirigierenden Proteins AtDIR6 resultierte. Dies entspricht einer ca. 250-fachen Steigerung der Ausbeute verglichen mit den bisherigen Systemen. Die biochemische Charakterisierung von AtDIR6, welches mit Pichia pastoris hergestellt wurde, zeigte eine vergleichbare Proteinstruktur und dirigierende Aktivität sowie eine Glykosylierung an den gleichen N Glykosylierungsstellen im Vergleich zu AtDIR6 aus einem heterologen Pflanzenzellsystem (Solanum peruvianum). Diese Ergebnisse bestätigten die Verwendung von Pichia pastoris als geeigneter Expressionswirt für die Herstellung dirigierender Proteine. Eine enzymatische Deglykosylierung des Proteins induzierte strukturelle Veränderungen, welche zu einem Verlust der Funktion und anschließender Proteinaggregation führten. Dadurch konnte die essentielle Bedeutung der Glykanstrukturen von AtDIR6 für die Proteinstruktur sowie die Löslichkeit und Funktion des Proteins demonstriert werden. In der zweiten Fallstudie wurde eine das Substratspektrum einer Ribokinase aus Saccharomyces cerevisiae durch rationales Proteindesign verändert. Das Enzym konnte zum ersten Mal in hoher Ausbeute mit Escherichia coli als heterologer Expressionswirt hergestellt und biochemisch charakterisiert werden. Die optimalen Reaktionsbedingungen bezüglich der Abhangigkeit des Enzyms von mono-, di- und pentavalenten Ionen als auch dem pH Wert wurden ermittelt. Dabei zeigte das Wildtyp Enzym die höchste katalytische Aktivität in 100 mM Kaliumphosphatpuffer bei pH 6.0 mit 2,5 mM Magnesiumchlorid. Basierend auf einem Sequenzvergleich von 200 Ribokinase Sequenzen aus der Familie der Ribokinasen und rationalem Proteindesign mit einem Homologiemodell des Proteins konnte eine kleine, fokussierte Mutantenbibliothek erstellt werden. Die fokussierte Bibliothek wurde in Bezug auf Veränderungen im Substratspektrum evaluiert. Dabei lag der Fokus auf D-Xylose, welches ein Epimer zu dem natürlichen Substrat D-Ribose ist. Starke Veränderungen der Substratbindetasche durch dreifach und vierfach Mutationen führten zu einem nahezu vollständigen Verlust der katalytischen Aktivität gegenüber D-Ribose und D-Xylose. Mehrere Varianten zeigten ein erweitertes Substratspektrum mit geringen Aktivitäten gegenüber Tetrosen, Pentosen und Hexosen. Eine einzelne Variante wies eine etwa verdoppelte Aktivität im Vergleich zu dem Wildtyp Enzym gegenüber D-Xylose auf (0.77 s-1 gegenüber 0.38 s-1), was als Basis für weitere Studien verwendet werden kann. In der dritten Fallstudie wurde eine Cutinase aus dem Schlauchpilz Humicola insolens als neuartiger Biokatalysator für die Synthese von funktionalisierten Acrylestern eingesetzt. Dabei wurde als Modellreaktion die Transesterifizierung von Methylacrylat mit 6-Mercapto-1-hexanol in einem lösungsmittelfreien System untersucht. Das Enzym wurde auf mikroporösem Trägermaterial aus Polystyren immobilisiert um die Verwendbarkeit und Stabilität in einer nicht-wässrigen Umgebung sowie das einfache Wiederverwenden des Biokatalysators zu ermöglichen. Als Hauptprodukt wurde ausschließlich 6 Mercaptohexylacrylatester mit der freien Thiogruppe als funktionelle Endgruppe identifiziert. Die Bildung zweier Nebenprodukte, welche durch Michael-Additionsreaktionen entstanden waren, konnte durch die Optimierung der Reaktionsbedingungen verhindert werden. Die optimierte Transesterifizierung erreichte den Umsatz von 95,4 ± 0,3 % zu dem Hauptprodukt 6-Mercaptoacrylatester in 6 h bei 40°C in der Anwesenheit von 0,025 % (w/w) Wasser in einem lösungsmittelfreien System. Die Verwendung von Methylmethacrylat als Acyl-Akzeptor in einer Transesterifizierung mit 6-Mercapto-1-hexanol lieferte einen deutlich geringeren Umsatz zum entsprechenden Hauptprodukt (43,6 ± 0,1%)

Synthetic enzyme supercomplexes: Co-immobilization of enzyme cascades

A sustainable alternative to traditional chemical synthesis is the use of enzymes as biocatalysts. Using enzymes, different advantages such as mild reaction conditions and high turnover rates are combined. However, the approach of using soluble enzymes suffers from the fact that enzymes have to be separated from the product post-synthesis and can be inactivated by this process. Therefore, enzymes are often immobilized to solid carriers to enable easy separation from the product as well as stabilization of the enzyme structure. In order to mimic the metabolic pathways of living cells and thus to create more complex bioproducts in a cell-free manner, a series of consecutive reactions can be realized by applying whole enzyme cascades. As enzymes from different host organisms can be combined, this offers enormous opportunities for creating advanced metabolic pathways that do not occur in nature. When immobilizing this enzyme cascades in a co-localized pattern a further advantage emerges: as the product of the previous enzyme is directly transferred to its co-immobilized subsequent catalyst, the overall performance of the cascade can be enhanced. Furthermore when enzymes are in close proximity to each other, the generation of by-products is reduced and obstructive effects like product inhibition and unfavorable kinetics can be disabled. This review gives an overview of the current state of the art in the application of enzyme cascades in immobilized forms. Furthermore it focuses on different immobilization techniques for structured immobilizates and the use of enzyme cascade in specially designed (microfluidic) reactor devices

Optimization of enzyme immobilization on magnetic microparticles using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as a crosslinking agent

Enzyme immobilization is a versatile tool in biotransformation processes to enhance enzyme activity and to secure an easy separation of catalysts and products and the reusability of enzymes. A simple and commonly used method for crosslinking enzymes to a solid support is the zero-length crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). This work shows the optimization of the EDC-crosslinking protocol for two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), to functionalized magnetic microparticles. For GOx the optimization of the immobilization parameters pH-value and the enzyme to particle ratio results in activity yields of up to 36%, which is in the usual range for undirected enzyme immobilisations. In contrast, for HRP the activity yield does not exceed 6% even after optimization of the protocols. The main reasons for this unusually low activity yield are the presence of multiple HRP isoforms in the enzyme solution used for immobilisation and the observed tendency of HRP to be inactive even in the case of simple physisorption to the particle surface. © 2015 The Royal Society of Chemistry

Matrix stiffness controls lymphatic vessel formation through regulation of a GATA2-dependent transcriptional program

Tissue and vessel wall stiffening alters endothelial cell properties and contributes to vascular dysfunction. However, whether extracellular matrix (ECM) stiffness impacts vascular development is not known. Here we show that matrix stiffness controls lymphatic vascular morphogenesis. Atomic force microscopy measurements in mouse embryos reveal that venous lymphatic endothelial cell (LEC) progenitors experience a decrease in substrate stiffness upon migration out of the cardinal vein, which induces a GATA2-dependent transcriptional program required to form the first lymphatic vessels. Transcriptome analysis shows that LECs grown on a soft matrix exhibit increased GATA2 expression and a GATA2-dependent upregulation of genes involved in cell migration and lymphangiogenesis, including VEGFR3. Analyses of mouse models demonstrate a cell-autonomous function of GATA2 in regulating LEC responsiveness to VEGF-C and in controlling LEC migration and sprouting in vivo. Our study thus uncovers a mechanism by which ECM stiffness dictates the migratory behavior of LECs during early lymphatic development.Peer reviewe

MicroRNA regulation of endothelial homeostasis and commitment—implications for vascular regeneration strategies using stem cell therapies

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration

Factors Affecting European Farmers’Participation in Biodiversity Policies

This article reports the major findings from an interdisciplinary research project that synthesises key insights into farmers’ willingness and ability to co-operate with biodiversity policies. The results of the study are based on an assessment of about 160 publications and research reports from six EU member states and from international comparative research.We developed a conceptual framework to systematically review the existent literature relevant for our purposes. This framework provides a common structure for analysing farmers’ perspectives regarding the introduction into farming practices of measures relevant to biodiversity. The analysis is coupled and contrasted with a survey of experts. The results presented above suggest that it is important to view support for practices oriented towards biodiversity protection not in a static sense – as a situation determined by one or several influencing factors – but rather as a process marked by interaction. Financial compensation and incentives function as a necessary, though clearly not sufficient condition in this process

Inhibition of GATA2 restrains cell proliferation and enhances apoptosis and chemotherapy mediated apoptosis in human GATA2 overexpressing AML cells

GATA2, a zinc finger transcription factor predominantly expressed in hematopoietic cells, acts as an essential regulator of hematopoietic stem cell generation, survival and functionality. Loss and gain of GATA2 expression has been implicated in myelodysplastic syndrome and acute myeloid leukemia (AML) yet the precise biological impact of GATA2 expression on human AML cell fate decisions remains ambiguous. Herein, we performed large-scale bioinformatics that demonstrated relatively frequent GATA2 overexpression in AML patients as well as select human AML (or AML-like) cell lines. By using shRNAi to target GATA2 in these AML cell lines, and an AML cell line expressing normal levels of GATA2, we found that inhibition of GATA2 caused attenuated cell proliferation and enhanced apoptosis exclusively in AML cell lines that overexpress GATA2. We proceeded to pharmacologically inhibit GATA2 in concert with AML chemotherapeutics and found this augmented cell killing in AML cell lines that overexpress GATA2, but not in an AML cell line expressing normal levels of GATA2. These data indicate that inhibition of GATA2 enhances chemotherapy-mediated apoptosis in human AML cells overexpressing GATA2. Thus, we define novel insights into the oncogenic role of GATA2 in human AML cells and suggest the potential utilization of transient GATA2 therapeutic targeting in AML