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Exploration of Metagenomic Racemases for Biosynthetic Cascades
Get PDFBiocatalysis becomes more and more appealing to industry due to its inherent ability to address contemporary demands for ecological, stereoselective synthesis from benign and biologically sourced materials, like amino acids. This creates an endless desire for new, diverse enzyme catalysts. One of the advantages of biocatalysis is to inherently provide catalysts for dynamic kinetic resolution like amino acid racemases, which is key to economic stereoselective synthesis. Drawback of most known amino acid racemases is their specificity for only few amino acids. Therefore, this work is dedicated to finding new and unusual amino acid racemases within a library that was created by our industry partner Prozomix Ltd. from their metagenomic database. Besides discovering enzymes with broader, or rare reactivities, the goal was also to demonstrate their synthetic potential in biocatalytic one-pot sequential cascade reactions.
First step was to adapt a spectrophotometric assay to enable efficient probing of the 56 library enzymes for activity on 16 canonical and 14 selected non-canonical amino acids. In addition, the enzymes’ melting points were measured with nanoDSF to assess their thermal stability. Subsequently, biocatalytic reaction cascades were implemented. For the production of the rare sugars 5-deoxy- and 1,5-dideoxyfructose, the detected racemase-hits were combined with D-amino acid oxidase and transketolase, and comparing this oxidase pathway with an alternative transaminase pathway. Valuable 2-keto acids, and D-2-hydroxy acids were produced, employing selected racemases together with oxidase and stereoselective dehydrogenase enzymes. Moreover, new techniques were developed to improve one of these cascades’ critical sub-process, the provision of oxygen to the oxidase at laboratory-scales. Required substrates for screening and sugar-producing cascades were synthesized, optimizing existing protocols and developing new, improved routes and methodologies.
Several racemases with remarkable substrate ranges were discovered, for example enzymes racemising tyrosine, or acting on the dipeptide β-aspartame. Some of the panel enzymes show both, broad substrate range and high thermostability of Tm > 69 °C. Their synthetic potential was successfully demonstrated by producing rare sugars and amino acid derivatives with productivities of up to 15.3 g D-phenyllactic acid per gram of cell-free racemase extract. Thus, the research goal to discover useful, innovative racemases, fit for synthetic application in dynamic kinetic resolutions was achieved and these novel enzymes may even serve as starting points for enzyme engineering
Simulationsbasierter Vergleich von Human Body Modellen und ATD-Dummys in Komfortsitzpositionen hinsichtlich Kinematik und Verletzungsmechanismen in Frontal-Crashs
Get PDFDie Euro NCAP Organisation (europäische Verbraucherschutzorganisation zur Fahrzeugsicherheit) plant zukünftig Simulationsergebnisse im Rahmen des Virtual Testings als festen Bestandteil des Bewertungsverfahrens zu integrieren. Dabei sollen sowohl Menschmodelle, als auch ATD-Dummies, zum Einsatz kommen. Im Rahmen dieser Masterarbeit wird diskutiert, inwieweit sich die Kriterien im bisherigen Testprotokoll des Euro NCAP und die entsprechenden Grenzwerte auf das Menschmodell übertragen lassen. Ebenfalls analysiert wird, welchen Einuss ein vergrößerter Lehnenwinkel (Komfortsitzpositionen) auf die Ergebnisse des Euro NCAP Testprotokolls hat. Im ersten Schritt wird dazu aus den am Markt zur Verfügung stehenden Modellen ein geeignetes Menschmodell ausgewählt. Mit diesem Modell werden in einer Schlittenumgebung (Beschleunigungs-Simulations-Anlage als Fahrzeugersatzumgebung) unterschiedliche Komfortsitzpositionen simuliert. Die Ergebnisse mit Menschmodell werden denen mit Dummy-Modell gegenüber gestellt. Zunächst werden die Unterschiede zwischen Menschmodell und Dummy in ihrem kinematischen Verhalten diskutiert. Daran anschließend erfolgt die Auswertung von ausgewählten Belastungsgrößen sowie die Bewertung nach dem Euro NCAP Testprotokoll. Basierend auf diesen Ergebnissen werden Ideen diskutiert, inwieweit die Modelle zur Beurteilung der Insassensicherheit geeignet sind und warum sich Ergebnisse zwischen den Ersatzmodellen unterscheiden. Dazu werden Ansätze vorgestellt, wie das Euro NCAP Testprotokoll ergänzt und/oder angepasst werden könnte, abhängig davon, ob Menschmodelle oder Dummy-Modelle, beziehungsweise aufrechte Sitzpositionen oder Komfortsitzpositionen bewertet werden sollen
Einfluss variierender Rotorspitzenströmung auf die Turbinenaerodynamik
Get PDFAn moderne Axialturbinen werden hohe technische Ansprüche gestellt. Hohe Flexibilität bei
gleichzeitiger Steigerung der Belastung sind eine interessante und herausfordernde Aufgabenstellung
bei der Auslegung von Maschinen. Daher besteht ein Interesse am Verständnis
von Verlustmechanismen, insbesondere bei Wechselwirkung der Aerodynamik innerhalb zweistufiger
deckbandloser Hochdruckturbinen, was Untersuchungsschwerpunkt dieser Arbeit ist.
Dabei liegt der Fokus auf Interaktionsmechanismen der Rotorspitzenströmung mit dem Stator
der zweiten Stufe. Ziel ist es, ein grundlegendes Verständnis der signifikanten Einflussgrößen
bezüglich der Strömungsverluste des zweiten Stators zu erhalten.
Hierfür wurde von Rolls-Royce Deutschland Ltd. & Co.KG ein Hochdruckstator mit hoher
Belastung entwickelt, welcher die Geometrie einer moderner Triebwerksstatoren abbildet.
Die Variation der Rotorspitzenströmung wird hierbei zum einen durch die Veränderung des
Spaltmaßes zwischen der Spitze einer konventionellen Squealer-Geometrie und dem Gehäuse
erreicht, zum anderen durch ein Neudesign der Spitzengeometrie, welche von der Siemens
Energy AG entwickelt wurde.
Die Untersuchung erfolgte über einen experimentellen (ergänzend) einen numerischen Ansatz.
Simulationen liefern eine gute Zugänglichkeit zum Strömungsfeld, müssen jedoch validiert
werden, da deren Ergebnisse stark vom gewählten Modellierungsansatz und Randbedingungen
abhängen. Daher wurden für letztere experimentelle Daten verwendet, für deren Erzeugung
eine Niedermachzahlturbine an der TU-Darmstadt zur Verfügung stand. Mit Hilfe der numerischen
Ergebnisse konnten die komplexen Strömungsvorgänge besser verstanden werden. Die
quantitative Bestimmung von Einflussgrößen erfolgte jedoch stets auf Basis experimenteller
Daten. Eine deutliche Verbesserung der Datenqualität und Glaubwürdigkeit der Messergebnisse
wurde zum einen durch umfangreiche Änderung in Regelung und Mechanik erreicht und zum
anderen durch eine detaillierte Fehleranalyse der verwendeten Messtechnik. Zusammen mit
aufwendigen numerischen Simulationen entstand so ein valides Bild der Strömungsvorgänge
in der 1, 5-stufigen Modellturbine.
Es konnte gezeigt werden, dass eine Kopplung zwischen den Verlustgebieten der Rotorspitzenströmung
und dem Wirbelsystem des zweiten Stators stattfindet. Hierbei ist nicht der Betrag
des Totaldruckverlustes, sondern des Totaldruckgradienten maßgebend für die Entstehung von
Wirbelpaaren an der Statorvorderkante und Wirbeln in der Passage, wobei die Ausprägung
durch den Inzidenzwinkel bestimmt wird, welcher zum einen die Ausbildung von Wirbelpaaren
an der Vorderkante beeinflusst, zum anderen die Wirbelbildung in der Statorpassage, da der
Inzidenzwinkel die Schaufelbelastung beeinflusst. So wurde festgestellt, dass eine Reduzierung der Spaltströmungsverluste am Rotor, zu erhöhten Verlusten im Stator führen kann, was der
Intuition zunächst widerspricht, mit erarbeiteten Einflussgrößen jedoch erklärbar ist.
Ein umfassendes Verständnis konnte nur durch die Kombination von experimentellen und numerischen
Ergebnissen erzielt werden. Zur Validierung der Numerik standen hierbei Ergebnisse
von dreidimensionalen Strömungsfeldmessungen im Rotor mittels Stereo Particle Velocimetry
(SPIV), Hitzdrahtanemometrie (HDA), Fünflochsonden (5LS) und akkuraten Wirkungsgradmessung
sowie dreidimensionale Daten von Farbanstrichversuchen zur Verfügung.
Durch die Analyse ergab sich auf Basis der identifizierten Einflussgrößen ein Verbesserungspotential
des isentropen Stufenwirkungsgrades von 0,5pp
Engineering a toolbox of synthetic dual-input hybrid riboswitches
Get PDFThe design of genetic regulators for the precise control of genes in complex genetic circuits remains a challenging task. Systems that rely on protein-based regulation to precisely control individual genes can suffer from the increased metabolic burden on the host cell organism that results from the additional gene expression required to produce the transcription factors. The introduction of longer genetic sequences required for the implementation of additional transcription factors (and their respective promoters/operators) can also be time consuming and difficult to adapt to a different host cell organism strain (codon optimization, difficulties during the cloning process of longer sequences, etc.). Synthetic riboswitches are a type of genetic element that can be used as an alternative to traditional protein-based regulation. Riboswitches are RNA sequences that form complex tertiary structures with the ability to specifically bind a designated target molecule (ligand) and regulate gene expression through a variety of different mechanisms. They are composed entirely of RNA, are compact in size (typically less than 100 nucleotides) and do not require additional factors to regulate gene expression. For these reasons, synthetic riboswitches have a low metabolic footprint, are strain independent, and can be easily adapted to different genetic circuits. While riboswitches were originally discovered in bacterial mRNAs, synthetic riboswitches have been engineered to function in all domains of life. Riboswitches are highly affine for their specific ligand, reaching picomolar binding affinities. A method called SELEX (Synthetic Evolution of Ligands by EXponential enrichment) can be used to discover new binding domains for any (water-soluble) target molecule of choice. An interesting type of riboswitch-based regulation in yeast is the roadblock mechanism. Regulation relies solely on a riboswitch, placed in the 5' UTR of a gene of interest, to stabilize its structure upon ligand binding to inhibit translation initiation by serving as a physical "roadblock" to the scanning ribosome. Since no sequestration of a regulatory sequence motif (e.g., ribosome binding site) is required, insertion of the riboswitch sequence into any 5' UTR is sufficient to enable regulation. A disadvantage of this type of regulation is that riboswitches exist in a pre-structured state even in the absence of ligand. As a result, the expression of the regulated gene is inhibited to some extent by the riboswitch even in the unbound state (basal expression). While previous studies have shown that the dynamic range (ratio of expression levels between the OFF and ON states) of these riboswitches can be increased by inserting multiple constructs in close spatial proximity (tandem riboswitches), the simultaneous increase of basal expression levels and dynamic range remains a challenge. A new type of dual-input riboswitch architecture, termed hybrid riboswitches, aims to solve this problem by incorporating two different ligand binding pockets into a single continuous structure. Hybrid riboswitches can rely on fewer scaffold sequences that would inhibit gene expression in the unbound state to provide stability, because the presence of the second binding pocket provides additional stabilization in the ligand-bound state. The idea was that hybrid riboswitches would retain the advantages of single-input riboswitches as compact and low-footprint regulators of gene expression, while providing the improved regulation of tandem riboswitches. This study aimed to create a variety of hybrid constructs from the combination of different parental riboswitches using both rational design and screening approaches. The goal was to create a toolbox of new highly efficient genetic regulators for use in circuit design, as well as to investigate how hybrid riboswitches can be efficiently designed. Several hybrid riboswitch constructs were generated that were able to outperform their parental riboswitches, with the best constructs achieving more than 50-60% basal expression and more than 20-fold dynamic range in the presence of both ligands. The best regulatory active construct achieved over 36-fold dynamic range in the presence of both ligands. In addition, it was investigated if the dual input nature of hybrid riboswitches could be used to design genetic regulators capable of emulating Boolean logic gate behavior. In collaboration with Heinz Koeppl's group, a screening pipeline was developed to discover a hybrid riboswitch responsive to neomycin and tetracycline that regulates gene expression only in the simultaneous presence of both ligands, thereby acting as a Boolean NAND gate
Closing the loop: establishing an autonomous test-learn cycle to optimize induction of bacterial systems using a robotic platform
No full textOne goal of synthetic biology is to provide well-characterised biological parts that behave predictably in genetic assemblies. To achieve this, each part must be characterised in a time-resolved manner under relevant conditions. Robotic platforms can be used to automate this task and provide sufficiently large and reproducible data sets including provenance. Although robotics can significantly speed up the data collection process, the collation and analysis of the resulting data, needed to reprogram and refine workflows for future iterations, is often a manual process. As a result, even in times of rapidly advancing artificial intelligence, the common design-build-test-learn (DBTL) cycle is still not circular without human intervention. To move towards fully automated DBTL cycles, we developed a software framework to enable a robotic platform to autonomously adjust test parameters. This interdisciplinary work between computer science and biology thus transforms a static robotic platform into a dynamic one. The software framework includes software components such as an importer that retrieves measurement data from the platform’s devices and writes it to a database. This is followed by an optimizer that selects the next measurement points based on a balance between exploration and exploitation. The platform is shown to be able to automatically and autonomously optimize the inducer concentration for a Bacillus subtilis system and the combination of inducer and feed release for a Escherichia coli system. As a target product the readily measurable green fluorescent reporter protein (GFP) is produced over multiple, consecutive iterations of testing. An evaluation of chosen (learning) algorithms for single and dual factor optimization was performed. In this article, we share the lessons learned from the development, implementation and execution of this automated design-build-test-learn cycles on a robotic platform
When Service Robots Enter the Workplace: Providing Information to Humanoid and Android Robots
Get PDFService robots are on the rise and are increasingly being used by companies in the workplace and at the customer interface. Recent developments in AI are enhancing the capabilities of service robots. In the workplace, service robots can be used by knowledge workers, i.e., employees who use knowledge as a powerful resource to perform tasks. At the customer interface, service robots are especially on the rise in retail, health care, food service, and hospitality. However, it is unclear how knowledge workers and external customers provide personal information to service robots, especially in the light of growing privacy concerns. Therefore, the overall goal of this dissertation is to investigate privacy concerns and patterns of information disclosure in customer-robot interactions. A particular focus is on android service robots, which look very much like human service providers. The behavior of knowledge workers and external customers towards these android robots is compared to both humanoid robots and human employees. Thus, this dissertation contributes to scientific research at the interface between marketing management and human-robot interaction.
With both types of robots, experimental studies were conducted in the workplace, where the service robots performed autonomous tasks in the HR department. Study 1 describes the preparation of these service robots for autonomous customer-robot interactions in the workplace from a design science perspective. Study 2 contributes to the overall goal of this dissertation by examining the influence of privacy concerns on the reluctance to provide personal information in a field experiment. The results show that privacy concerns do not prevent knowledge workers from working with service robots for long periods of time, nor do they mirror actual information reluctance. As the task complexity of customer-robot interactions increases, privacy concerns increase, but information reluctance decreases. There are also differences in the behavior of knowledge workers when providing personal information to humanoid robots versus android robots, depending on task complexity.
Study 3 contributes to the overall goal of this dissertation by examining the conversational flow in the interactions with the service robots using video analysis of the field experiment. It can be seen that knowledge workers show more variation in verbal and nonverbal forms of expression when talking to android robots than when talking to humanoid robots.
In addition to the field experiment in the workplace, further studies have been conducted with the service robots. Study 4 deals with the effect of social influence on information disclosure when interacting with service robots. It turns out that customers are more willing to provide personal information to android robots when they have previously observed this behavior from peers.
Study 5 contributes to the overall goal of this dissertation by introducing a new theoretical concept to research on service robots and information disclosure, the so-called "Blurring Paradox". Based on political speeches in which politicians talk a lot but say little, it is suggested that customers may also tend to talk a lot but say little when interacting with a service robot.
In addition to introducing this theoretical concept, this dissertation integrates various theoretical perspectives from different research disciplines, such as the CASA paradigm, task-technology-fit theory, communication accommodation theory, and social impact theory. In addition, this dissertation provides methodological contributions by introducing a scale for measuring the conversational flow when interacting with service robots. From a content-related perspective, the different studies show that employees and external customers adapt the way they provide information depending on whether they are interacting with a service robot or a human service provider. Thus, this dissertation contributes to scientific research and managerial practice in several important ways
DFG-Abschlussbericht: Dynamische datenbasierte Schätzung des technischen Missionsrisikos unbemannter Flugsysteme
Get PDFIn diesem Projekt wird die Risikobewertung des Betriebs komplexer technischer Systeme mit datenbasierten Methoden untersucht. Diese erfordert die Betrachtung der Degradationszustände verschiedener Subsysteme sowie deren Interaktionen. Beispielhaft wird die Überwachung des Missionsrisikos unbemannter Flugsysteme (Unmanned Aerial Vehicles, UAVs) betrachtet. Am Beispiel einer hybriden Quad-Plane Konfiguration, welche die Vorteile eines Multicopters mit denen eines Starrflüglers vereint, werden die Subsysteme hinsichtlich ihrer Kritikalität analysiert und die Aktoren (Antriebs- und Stellsysteme) sowie die Batterie als zu überwachende Subsysteme ausgewählt. Für diese werden anhand von Prüfstandsdaten, Simulationsmodellen und öffentlichen Datensätzen Diagnose- und Prognoseansätze zur Überwachung ihres Gesundheitszustandes entwickelt.
Zur Betrachtung des Gesamtsystems wird ein Flugsimulationsmodell eingesetzt. Dieses ermöglicht, den Einfluss von Funktionseinbußen mehrerer Aktoren auf die Flugstabilität zu analysieren. Zur Simulation der Degradation wird eine zeitlich abnehmende Effizienz der Antriebsmotoren, eine zunehmende Varianz der Stellmotorsteuerung sowie eine sich verringernde Batteriekapazität modelliert.
Mehrere UAV-Missionssequenzen werden simuliert, wobei die Missionsprofile zufällig generiert und durch eine Wegplanung unter Berücksichtigung von Luftraumbeschränkungen ergänzt werden. Dabei wird auch der Einfluss von Wind und verschiedenen Flugmodi berücksichtigt, darunter Start, Landung und Übergangsphasen zwischen Starrflügel- und Multicopter-Flug.
Zur Risikobewertung wird ein Verfahren entwickelt, das den aktuellen Systemzustand mithilfe von Hidden-Semi-Markov-Modellen (HSMM) abschätzt. Simulierte Flugdaten werden für das Training in Segmente bis hin zu fehlgeschlagenen Missionen unterteilt, und mithilfe von K-Means- oder Entscheidungsbaum-Algorithmen geclustert. Die trainierten HSMMs sowie Entscheidungsbäume ermöglichen dann eine Analyse der Wahrscheinlichkeit eines Missionsfehlschlags
NFDI4Ing Conference 2022: Unifying the Understanding of Research Data Management in Engineering Science : Editorial Letter for the NFDI4Ing Conference 2022 Special Issue
No full textWith the title “Unifying the Understanding of Research Data Management (RDM) in Engineering Science”, NFDI4Ing welcomed you online on October 26th and 27th, 2022 to its annual conference, hosted by the RWTH Aachen University. As the NFDI4Ing speaker and conference host, I am personally highly delighted about the numerous and multifaceted contributions at the conference. In this special issue of ing.grid, we present publications based on some of the presentations given in the conference
Generating systems for modular forms for the Weil representation and Hecke operators for orthogonal modular forms
Get PDFThe transformation behaviour of the vector-valued theta function of a positive-definite even lattice under the metaplectic group Mp2(Z) is described by the Weil representation. In the first part of this thesis we study modular forms for the Weil representation. This is divided into three projects:
For an isotropic subgroup H of a discriminant form D there exists a lift from modular forms for the Weil representation of the discriminant form H^⊥/H to modular forms for the Weil representation of D. We determine a set of discriminant forms such that all modular forms for any discriminant form are induced from the discriminant forms in this set. Furthermore, for any discriminant form in this set there exist modular forms that are not induced from smaller discriminant forms.
Next we investigate the invariants of the Weil representation. In particular, we show that they are induced from 5 fundamental invariants.
In the third project we show that the space of cusp forms for the Weil representation is generated by theta series. This gives a positive answer to Eichler's basis problem in this case. As application we derive Waldspurger's result on the basis problem for scalar-valued modular forms.
The second part of this thesis is about orthogonal modular forms. First, we give a new proof of the surjectivity of the multiplicative Borcherds lift based on the analysis of local Picard groups that follows immediately from the basis problem. Then we study orthogonal Hecke operators, in particular, we compute the Hecke eigenvalues of Borcherds' Phi12
Synthesis of Methoxy-X04 Derivatives and Their Evaluation in Alzheimer's Disease Pathology
Get PDFBackground: Alzheimer's disease is characterized by two notorious protein aggregates in the brain: extracellular senile plaques mainly consisting of amyloid-β peptides and tau-protein-derived intracellular paired helical filaments. The diagnosis of Alzheimer's disease is impaired by insufficient sensitivity and specificity of diagnostic methods to visualize these pathological hallmarks over all disease stages.
Objective: The established fluorescence marker methoxy-X04 stains plaques, tau tangles and amyloid-derived angiopathies with good specificity, yet it is limited by slow elimination in vivo. Since the need for new markers is high, we prepared methoxy-X04 derivatives and evaluated their potential as imaging agents in Alzheimer's disease pathology.
Methods and Results: In this study, we describe an improved synthesis for methoxy-X04 and its derivatives and their affinity determination for the respective protein targets by immunohistology and a displacement assay.
Conclusion: This resulted in the identification of new derivatives of methoxy-X04 with improved binding affinity