Multiplex gene analysis reveals T-cell and antibody-mediated rejection-specific upregulation of complement in renal transplants

In renal transplantation, complement is involved in ischemia reperfusion injury, graft rejection and dysfunction. However, it is still unclear how induction of complement and its activation are initiated. Using allograft biopsies of a well-characterized cohort of 28 renal transplant patients with no rejection (Ctrl), delayed graft function (DGF), acute T-cell-mediated (TCMR) or antibody-mediated rejection (ABMR) we analyzed differences in complement reaction. For that mRNA was isolated from FFPE sections, quantified with a multiplex gene expression panel and correlated with transplant conditions and follow-up of patients. Additionally, inflammatory cells were quantified by multiplex immunohistochemistry. In allograft biopsies with TCMR and ABMR gene expression of C1QB was 2-4 fold elevated compared to Ctrl. In TCMR biopsies, mRNA counts of several complement-related genes including C1S, C3, CFB and complement regulators CFH, CR1 and SERPING1 were significantly increased compared to Ctrl. Interestingly, expression levels of about 75% of the analyzed complement related genes correlated with cold ischemia time (CIT) and markers of inflammation. In conclusion, this study suggest an important role of complement in transplant pathology which seems to be at least in part triggered by CIT. Multiplex mRNA analysis might be a useful method to refine diagnosis and explore new pathways involved in rejection

Can Gene Expression Analysis in Zero-Time Biopsies Predict Kidney Transplant Rejection?

Zero-time biopsies are taken to determine the quality of the donor organ at the time of transplantation. Histological analyses alone have so far not been able to identify parameters that allow the prediction of subsequent rejection episodes or graft survival. This study investigated whether gene expression analyses of zero-time biopsies might support this prediction. Using a well-characterized cohort of 26 zero-time biopsies from renal transplant patients that include 4 living donor (LD) and 22 deceased donor (DD) biopsies that later developed no rejection (Ctrl, n = 7), delayed graft function (DGF, n = 4), cellular (T-cell mediated rejection; TCMR, n = 8), or antibody-mediated rejection (ABMR, n = 7), we analyzed gene expression profiles for different types of subsequent renal transplant complication. To this end, RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) sections and gene expression profiles were quantified. Results were correlated with transplant data and B-cell, and plasma cell infiltration was assessed by immunofluorescence microscopy. Both principal component analysis and clustering analysis of gene expression data revealed marked separation between LDs and DDs. Differential expression analysis identified 185 significant differentially expressed genes (adjusted p < 0.05). The expression of 68% of these genes significantly correlated with cold ischemia time (CIT). Furthermore, immunoglobulins were differentially expressed in zero-time biopsies from transplants later developing rejection (TCMR + ABMR) compared to non-rejected (Ctrl + DGF) transplants. In addition, immunoglobulin expression did not correlate with CIT but was increased in transplants with previous acute renal failure (ARF). In conclusion, gene expression profiles in zero-time biopsies derived from LDs are markedly different from those of DDs. Pre-transplant ARF increased immunoglobulin expression, which might be involved in triggering later rejection events. However, these findings must be confirmed in larger cohorts and the role of early immunoglobulin upregulation in zero-biopsies needs further clarification

Three v-SNAREs and Two t-SNAREs, Present in a Pentameric cis-SNARE Complex on Isolated Vacuoles, Are Essential for Homotypic Fusion

Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit “cis” complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p

Prediction of overall survival for patients with metastatic castration-resistant prostate cancer : development of a prognostic model through a crowdsourced challenge with open clinical trial data

Background Improvements to prognostic models in metastatic castration-resistant prostate cancer have the potential to augment clinical trial design and guide treatment strategies. In partnership with Project Data Sphere, a not-for-profit initiative allowing data from cancer clinical trials to be shared broadly with researchers, we designed an open-data, crowdsourced, DREAM (Dialogue for Reverse Engineering Assessments and Methods) challenge to not only identify a better prognostic model for prediction of survival in patients with metastatic castration-resistant prostate cancer but also engage a community of international data scientists to study this disease. Methods Data from the comparator arms of four phase 3 clinical trials in first-line metastatic castration-resistant prostate cancer were obtained from Project Data Sphere, comprising 476 patients treated with docetaxel and prednisone from the ASCENT2 trial, 526 patients treated with docetaxel, prednisone, and placebo in the MAINSAIL trial, 598 patients treated with docetaxel, prednisone or prednisolone, and placebo in the VENICE trial, and 470 patients treated with docetaxel and placebo in the ENTHUSE 33 trial. Datasets consisting of more than 150 clinical variables were curated centrally, including demographics, laboratory values, medical history, lesion sites, and previous treatments. Data from ASCENT2, MAINSAIL, and VENICE were released publicly to be used as training data to predict the outcome of interest-namely, overall survival. Clinical data were also released for ENTHUSE 33, but data for outcome variables (overall survival and event status) were hidden from the challenge participants so that ENTHUSE 33 could be used for independent validation. Methods were evaluated using the integrated time-dependent area under the curve (iAUC). The reference model, based on eight clinical variables and a penalised Cox proportional-hazards model, was used to compare method performance. Further validation was done using data from a fifth trial-ENTHUSE M1-in which 266 patients with metastatic castration-resistant prostate cancer were treated with placebo alone. Findings 50 independent methods were developed to predict overall survival and were evaluated through the DREAM challenge. The top performer was based on an ensemble of penalised Cox regression models (ePCR), which uniquely identified predictive interaction effects with immune biomarkers and markers of hepatic and renal function. Overall, ePCR outperformed all other methods (iAUC 0.791; Bayes factor >5) and surpassed the reference model (iAUC 0.743; Bayes factor >20). Both the ePCR model and reference models stratified patients in the ENTHUSE 33 trial into high-risk and low-risk groups with significantly different overall survival (ePCR: hazard ratio 3.32, 95% CI 2.39-4.62, p Interpretation Novel prognostic factors were delineated, and the assessment of 50 methods developed by independent international teams establishes a benchmark for development of methods in the future. The results of this effort show that data-sharing, when combined with a crowdsourced challenge, is a robust and powerful framework to develop new prognostic models in advanced prostate cancer.Peer reviewe

Human Practice. Digital Ecologies. Our Future. : 14. Internationale Tagung Wirtschaftsinformatik (WI 2019) : Tagungsband

Erschienen bei: universi - Universitätsverlag Siegen. - ISBN: 978-3-96182-063-4Aus dem Inhalt: Track 1: Produktion & Cyber-Physische Systeme Requirements and a Meta Model for Exchanging Additive Manufacturing Capacities Service Systems, Smart Service Systems and Cyber- Physical Systems—What’s the difference? Towards a Unified Terminology Developing an Industrial IoT Platform – Trade-off between Horizontal and Vertical Approaches Machine Learning und Complex Event Processing: Effiziente Echtzeitauswertung am Beispiel Smart Factory Sensor retrofit for a coffee machine as condition monitoring and predictive maintenance use case Stakeholder-Analyse zum Einsatz IIoT-basierter Frischeinformationen in der Lebensmittelindustrie Towards a Framework for Predictive Maintenance Strategies in Mechanical Engineering - A Method-Oriented Literature Analysis Development of a matching platform for the requirement-oriented selection of cyber physical systems for SMEs Track 2: Logistic Analytics An Empirical Study of Customers’ Behavioral Intention to Use Ridepooling Services – An Extension of the Technology Acceptance Model Modeling Delay Propagation and Transmission in Railway Networks What is the impact of company specific adjustments on the acceptance and diffusion of logistic standards? Robust Route Planning in Intermodal Urban Traffic Track 3: Unternehmensmodellierung & Informationssystemgestaltung (Enterprise Modelling & Information Systems Design) Work System Modeling Method with Different Levels of Specificity and Rigor for Different Stakeholder Purposes Resolving Inconsistencies in Declarative Process Models based on Culpability Measurement Strategic Analysis in the Realm of Enterprise Modeling – On the Example of Blockchain-Based Initiatives for the Electricity Sector Zwischenbetriebliche Integration in der Möbelbranche: Konfigurationen und Einflussfaktoren Novices’ Quality Perceptions and the Acceptance of Process Modeling Grammars Entwicklung einer Definition für Social Business Objects (SBO) zur Modellierung von Unternehmensinformationen Designing a Reference Model for Digital Product Configurators Terminology for Evolving Design Artifacts Business Role-Object Specification: A Language for Behavior-aware Structural Modeling of Business Objects Generating Smart Glasses-based Information Systems with BPMN4SGA: A BPMN Extension for Smart Glasses Applications Using Blockchain in Peer-to-Peer Carsharing to Build Trust in the Sharing Economy Testing in Big Data: An Architecture Pattern for a Development Environment for Innovative, Integrated and Robust Applications Track 4: Lern- und Wissensmanagement (e-Learning and Knowledge Management) eGovernment Competences revisited – A Literature Review on necessary Competences in a Digitalized Public Sector Say Hello to Your New Automated Tutor – A Structured Literature Review on Pedagogical Conversational Agents Teaching the Digital Transformation of Business Processes: Design of a Simulation Game for Information Systems Education Conceptualizing Immersion for Individual Learning in Virtual Reality Designing a Flipped Classroom Course – a Process Model The Influence of Risk-Taking on Knowledge Exchange and Combination Gamified Feedback durch Avatare im Mobile Learning Alexa, Can You Help Me Solve That Problem? - Understanding the Value of Smart Personal Assistants as Tutors for Complex Problem Tasks Track 5: Data Science & Business Analytics Matching with Bundle Preferences: Tradeoff between Fairness and Truthfulness Applied image recognition: guidelines for using deep learning models in practice Yield Prognosis for the Agrarian Management of Vineyards using Deep Learning for Object Counting Reading Between the Lines of Qualitative Data – How to Detect Hidden Structure Based on Codes Online Auctions with Dual-Threshold Algorithms: An Experimental Study and Practical Evaluation Design Features of Non-Financial Reward Programs for Online Reviews: Evaluation based on Google Maps Data Topic Embeddings – A New Approach to Classify Very Short Documents Based on Predefined Topics Leveraging Unstructured Image Data for Product Quality Improvement Decision Support for Real Estate Investors: Improving Real Estate Valuation with 3D City Models and Points of Interest Knowledge Discovery from CVs: A Topic Modeling Procedure Online Product Descriptions – Boost for your Sales? Entscheidungsunterstützung durch historienbasierte Dienstreihenfolgeplanung mit Pattern A Semi-Automated Approach for Generating Online Review Templates Machine Learning goes Measure Management: Leveraging Anomaly Detection and Parts Search to Improve Product-Cost Optimization Bedeutung von Predictive Analytics für den theoretischen Erkenntnisgewinn in der IS-Forschung Track 6: Digitale Transformation und Dienstleistungen Heuristic Theorizing in Software Development: Deriving Design Principles for Smart Glasses-based Systems Mirroring E-service for Brick and Mortar Retail: An Assessment and Survey Taxonomy of Digital Platforms: A Platform Architecture Perspective Value of Star Players in the Digital Age Local Shopping Platforms – Harnessing Locational Advantages for the Digital Transformation of Local Retail Outlets: A Content Analysis A Socio-Technical Approach to Manage Analytics-as-a-Service – Results of an Action Design Research Project Characterizing Approaches to Digital Transformation: Development of a Taxonomy of Digital Units Expectations vs. Reality – Benefits of Smart Services in the Field of Tension between Industry and Science Innovation Networks and Digital Innovation: How Organizations Use Innovation Networks in a Digitized Environment Characterising Social Reading Platforms— A Taxonomy-Based Approach to Structure the Field Less Complex than Expected – What Really Drives IT Consulting Value Modularity Canvas – A Framework for Visualizing Potentials of Service Modularity Towards a Conceptualization of Capabilities for Innovating Business Models in the Industrial Internet of Things A Taxonomy of Barriers to Digital Transformation Ambidexterity in Service Innovation Research: A Systematic Literature Review Design and success factors of an online solution for cross-pillar pension information Track 7: IT-Management und -Strategie A Frugal Support Structure for New Software Implementations in SMEs How to Structure a Company-wide Adoption of Big Data Analytics The Changing Roles of Innovation Actors and Organizational Antecedents in the Digital Age Bewertung des Kundennutzens von Chatbots für den Einsatz im Servicedesk Understanding the Benefits of Agile Software Development in Regulated Environments Are Employees Following the Rules? On the Effectiveness of IT Consumerization Policies Agile and Attached: The Impact of Agile Practices on Agile Team Members’ Affective Organisational Commitment The Complexity Trap – Limits of IT Flexibility for Supporting Organizational Agility in Decentralized Organizations Platform Openness: A Systematic Literature Review and Avenues for Future Research Competence, Fashion and the Case of Blockchain The Digital Platform Otto.de: A Case Study of Growth, Complexity, and Generativity Track 8: eHealth & alternde Gesellschaft Security and Privacy of Personal Health Records in Cloud Computing Environments – An Experimental Exploration of the Impact of Storage Solutions and Data Breaches Patientenintegration durch Pfadsysteme Digitalisierung in der Stressprävention – eine qualitative Interviewstudie zu Nutzenpotenzialen User Dynamics in Mental Health Forums – A Sentiment Analysis Perspective Intent and the Use of Wearables in the Workplace – A Model Development Understanding Patient Pathways in the Context of Integrated Health Care Services - Implications from a Scoping Review Understanding the Habitual Use of Wearable Activity Trackers On the Fit in Fitness Apps: Studying the Interaction of Motivational Affordances and Users’ Goal Orientations in Affecting the Benefits Gained Gamification in Health Behavior Change Support Systems - A Synthesis of Unintended Side Effects Investigating the Influence of Information Incongruity on Trust-Relations within Trilateral Healthcare Settings Track 9: Krisen- und Kontinuitätsmanagement Potentiale von IKT beim Ausfall kritischer Infrastrukturen: Erwartungen, Informationsgewinnung und Mediennutzung der Zivilbevölkerung in Deutschland Fake News Perception in Germany: A Representative Study of People’s Attitudes and Approaches to Counteract Disinformation Analyzing the Potential of Graphical Building Information for Fire Emergency Responses: Findings from a Controlled Experiment Track 10: Human-Computer Interaction Towards a Taxonomy of Platforms for Conversational Agent Design Measuring Service Encounter Satisfaction with Customer Service Chatbots using Sentiment Analysis Self-Tracking and Gamification: Analyzing the Interplay of Motivations, Usage and Motivation Fulfillment Erfolgsfaktoren von Augmented-Reality-Applikationen: Analyse von Nutzerrezensionen mit dem Review-Mining-Verfahren Designing Dynamic Decision Support for Electronic Requirements Negotiations Who is Stressed by Using ICTs? A Qualitative Comparison Analysis with the Big Five Personality Traits to Understand Technostress Walking the Middle Path: How Medium Trade-Off Exposure Leads to Higher Consumer Satisfaction in Recommender Agents Theory-Based Affordances of Utilitarian, Hedonic and Dual-Purposed Technologies: A Literature Review Eliciting Customer Preferences for Shopping Companion Apps: A Service Quality Approach The Role of Early User Participation in Discovering Software – A Case Study from the Context of Smart Glasses The Fluidity of the Self-Concept as a Framework to Explain the Motivation to Play Video Games Heart over Heels? An Empirical Analysis of the Relationship between Emotions and Review Helpfulness for Experience and Credence Goods Track 11: Information Security and Information Privacy Unfolding Concerns about Augmented Reality Technologies: A Qualitative Analysis of User Perceptions To (Psychologically) Own Data is to Protect Data: How Psychological Ownership Determines Protective Behavior in a Work and Private Context Understanding Data Protection Regulations from a Data Management Perspective: A Capability-Based Approach to EU-GDPR On the Difficulties of Incentivizing Online Privacy through Transparency: A Qualitative Survey of the German Health Insurance Market What is Your Selfie Worth? A Field Study on Individuals’ Valuation of Personal Data Justification of Mass Surveillance: A Quantitative Study An Exploratory Study of Risk Perception for Data Disclosure to a Network of Firms Track 12: Umweltinformatik und nachhaltiges Wirtschaften Kommunikationsfäden im Nadelöhr – Fachliche Prozessmodellierung der Nachhaltigkeitskommunikation am Kapitalmarkt Potentiale und Herausforderungen der Materialflusskostenrechnung Computing Incentives for User-Based Relocation in Carsharing Sustainability’s Coming Home: Preliminary Design Principles for the Sustainable Smart District Substitution of hazardous chemical substances using Deep Learning and t-SNE A Hierarchy of DSMLs in Support of Product Life-Cycle Assessment A Survey of Smart Energy Services for Private Households Door-to-Door Mobility Integrators as Keystone Organizations of Smart Ecosystems: Resources and Value Co-Creation – A Literature Review Ein Entscheidungsunterstützungssystem zur ökonomischen Bewertung von Mieterstrom auf Basis der Clusteranalyse Discovering Blockchain for Sustainable Product-Service Systems to enhance the Circular Economy Digitale Rückverfolgbarkeit von Lebensmitteln: Eine verbraucherinformatische Studie Umweltbewusstsein durch audiovisuelles Content Marketing? Eine experimentelle Untersuchung zur Konsumentenbewertung nachhaltiger Smartphones Towards Predictive Energy Management in Information Systems: A Research Proposal A Web Browser-Based Application for Processing and Analyzing Material Flow Models using the MFCA Methodology Track 13: Digital Work - Social, mobile, smart On Conversational Agents in Information Systems Research: Analyzing the Past to Guide Future Work The Potential of Augmented Reality for Improving Occupational First Aid Prevent a Vicious Circle! The Role of Organizational IT-Capability in Attracting IT-affine Applicants Good, Bad, or Both? Conceptualization and Measurement of Ambivalent User Attitudes Towards AI A Case Study on Cross-Hierarchical Communication in Digital Work Environments ‘Show Me Your People Skills’ - Employing CEO Branding for Corporate Reputation Management in Social Media A Multiorganisational Study of the Drivers and Barriers of Enterprise Collaboration Systems-Enabled Change The More the Merrier? The Effect of Size of Core Team Subgroups on Success of Open Source Projects The Impact of Anthropomorphic and Functional Chatbot Design Features in Enterprise Collaboration Systems on User Acceptance Digital Feedback for Digital Work? Affordances and Constraints of a Feedback App at InsurCorp The Effect of Marker-less Augmented Reality on Task and Learning Performance Antecedents for Cyberloafing – A Literature Review Internal Crowd Work as a Source of Empowerment - An Empirical Analysis of the Perception of Employees in a Crowdtesting Project Track 14: Geschäftsmodelle und digitales Unternehmertum Dividing the ICO Jungle: Extracting and Evaluating Design Archetypes Capturing Value from Data: Exploring Factors Influencing Revenue Model Design for Data-Driven Services Understanding the Role of Data for Innovating Business Models: A System Dynamics Perspective Business Model Innovation and Stakeholder: Exploring Mechanisms and Outcomes of Value Creation and Destruction Business Models for Internet of Things Platforms: Empirical Development of a Taxonomy and Archetypes Revitalizing established Industrial Companies: State of the Art and Success Principles of Digital Corporate Incubators When 1+1 is Greater than 2: Concurrence of Additional Digital and Established Business Models within Companies Special Track 1: Student Track Investigating Personalized Price Discrimination of Textile-, Electronics- and General Stores in German Online Retail From Facets to a Universal Definition – An Analysis of IoT Usage in Retail Is the Technostress Creators Inventory Still an Up-To-Date Measurement Instrument? Results of a Large-Scale Interview Study Application of Media Synchronicity Theory to Creative Tasks in Virtual Teams Using the Example of Design Thinking TrustyTweet: An Indicator-based Browser-Plugin to Assist Users in Dealing with Fake News on Twitter Application of Process Mining Techniques to Support Maintenance-Related Objectives How Voice Can Change Customer Satisfaction: A Comparative Analysis between E-Commerce and Voice Commerce Business Process Compliance and Blockchain: How Does the Ethereum Blockchain Address Challenges of Business Process Compliance? Improving Business Model Configuration through a Question-based Approach The Influence of Situational Factors and Gamification on Intrinsic Motivation and Learning Evaluation von ITSM-Tools für Integration und Management von Cloud-Diensten am Beispiel von ServiceNow How Software Promotes the Integration of Sustainability in Business Process Management Criteria Catalog for Industrial IoT Platforms from the Perspective of the Machine Tool Industry Special Track 3: Demos & Prototyping Privacy-friendly User Location Tracking with Smart Devices: The BeaT Prototype Application-oriented robotics in nursing homes Augmented Reality for Set-up Processe Mixed Reality for supporting Remote-Meetings Gamification zur Motivationssteigerung von Werkern bei der Betriebsdatenerfassung Automatically Extracting and Analyzing Customer Needs from Twitter: A “Needmining” Prototype GaNEsHA: Opportunities for Sustainable Transportation in Smart Cities TUCANA: A platform for using local processing power of edge devices for building data-driven services Demonstrator zur Beschreibung und Visualisierung einer kritischen Infrastruktur Entwicklung einer alltagsnahen persuasiven App zur Bewegungsmotivation für ältere Nutzerinnen und Nutzer A browser-based modeling tool for studying the learning of conceptual modeling based on a multi-modal data collection approach Exergames & Dementia: An interactive System for People with Dementia and their Care-Network Workshops Workshop Ethics and Morality in Business Informatics (Workshop Ethik und Moral in der Wirtschaftsinformatik – EMoWI’19) Model-Based Compliance in Information Systems - Foundations, Case Description and Data Set of the MobIS-Challenge for Students and Doctoral Candidates Report of the Workshop on Concepts and Methods of Identifying Digital Potentials in Information Management Control of Systemic Risks in Global Networks - A Grand Challenge to Information Systems Research Die Mitarbeiter von morgen - Kompetenzen künftiger Mitarbeiter im Bereich Business Analytics Digitaler Konsum: Herausforderungen und Chancen der Verbraucherinformati

Introduction to Surface Avatar: the First Heterogeneous Robotic Team to be Commanded with Scalable Autonomy from the ISS

Robotics is vital to the continued development toward Lunar and Martian exploration, in-situ resource utilization, and surface infrastructure construction. Large-scale extra-terrestrial missions will require teams of robots with different, complementary capabilities, together with a powerful, intuitive user interface for effective commanding. We introduce Surface Avatar, the newest ISS-to-Earth telerobotic experiment series, to be conducted in 2022-2024. Spearheaded by DLR, together with ESA, Surface Avatar builds on expertise on commanding robots with different levels of autonomy from our past telerobotic experiments: Kontur-2, Haptics, Interact, SUPVIS Justin, and Analog-1. A team of four heterogeneous robots in a multi-site analog environment at DLR are at the command of a crew member on the ISS. The team has a humanoid robot for dexterous object handling, construction and maintenance; a rover for long traverses and sample acquisition; a quadrupedal robot for scouting and exploring difficult terrains; and a lander with robotic arm for component delivery and sample stowage. The crew's command terminal is multimodal, with an intuitive graphical user interface, 3-DOF joystick, and 7-DOF input device with force-feedback. The autonomy of any robot can be scaled up and down depending on the task and the astronaut's preference: acting as an avatar of the crew in haptically-coupled telepresence, or receiving task-level commands like an intelligent co-worker. Through crew performing collaborative tasks in exploration and construction scenarios, we hope to gain insight into how to optimally command robots in a future space mission. This paper presents findings from the first preliminary session in June 2022, and discusses the way forward in the planned experiment sessions

Untersuchungen zur optimalen spektralen Strahlungsverteilung für die Erzielung einer hohen Farbwiedergabegüte mit LEDs

This diploma thesis discusses the colour rendering qualities of LED-spectra. It has been one intent to initially determine the influences on colour rendering of the constitutive parameters of particularly couloured LEDs. With this information optimal configurations of components have been established regarding to different colour-sets and rendering-indices. This has been reached by using Gaussian functions as approximation functions to the spectral power distribution of single LEDs involved in the spectrum, whose colour rendering qualities have been optimized by different algorithms. Further on those virtual power distributions have been transduced into spectra that could be realized by available LEDs. Their colour rendering capacities have been verified with subjective surveys at colour temperatures of 3000K and 6000K. The obtained distances in colour space and the derived Subjective Rendering Index [SBI] have been compared with those of the prediction models for colour rendering CIE-UVW, CIELAB and CIECAM02. A significant correlation between a) the theoretically best spectra and their factual colour rendering quality and b) the distances in colour space and rendering indices of the colour difference models and those of the SBI.Die Diplomarbeit beschäftigt sich mit der Farbwiedergabegüte von LED-Spektren. Ein Ziel der Arbeit ist, zunächst die Einflüsse der konstituierenden Parameter einzelner farbiger LEDs auf die Farbwiedergabe festzustellen, um dann optimale Zusammensetzungen für verschiedene Sätze von Farbproben und Bewertungsindices zu erzeugen. Dies wurde mit Hilfe von Gaußfunktionen als mathematische Näherungsfunktionen an die Strahlungsverteilungen einzelner LEDs erreicht, deren Farbwiedergabeindex dann mit verschiedenen Algorithmen optimiert wurde. Weiterhin wurden diese virtuellen Strahlungsverteilungen in durch vorhandene LEDs realisierbare Spektren überführt. Deren Farbwiedergabeeigenschaften wurden an Hand subjektiver Untersuchungen für die Farbtemperaturen 3000K und 6000K geprüft. Die daraus entstehenden Farbabstände und der abgeleitete subjektive Bewertungsindex [SBI] wurden mit jenen der Vorhersagemodelle für Farbwiedergabe CIE-UVW, CIELAB und CIECAM02 verglichen. Damit konnte eine hohe Korrelation zwischen a) den theoretisch besten Spektren und deren tatsächlichen Farbwiedergabeeigenschaften und b) den Farbabständen und Farbwiedergabewerten der Farbmodelle und denen des SBI festgestellt werden.Ilmenau, Techn. Univ., Diplomarbeit, 200

Construction of Escherichia coli producer strains for the fermentative production of succinate from glycerol

Glycerin fällt in großen Mengen bei der Biodieselproduktion an und kann als billiger, nachwachsender Rohstoff betrachtet werden, aus dem sich biotechnologisch viele industriell relevante Fein- oder Bulkchemikalien herstellen lassen. Eine der Bulkchemikalien ist Succinat, das Anion der Bernsteinsäure, welche in Studien des Department of Energy der USA (DOE) in den Jahren 2004 und 2010 als wichtige Plattformchemikalie bezeichnet wurde. Plattformchemikalien bzw. chemische Bausteine können kostengünstig in eine Vielzahl von weiteren, höherpreisigen Materialien umgewandelt werden. In dieser Arbeit wurden Escherichia coli Produktionsstämme für Succinat konstruiert. Die Bildung größerer Zellmassen erfolgte vor der Produktionsphase in einer aeroben Wachstumsphase in Minimalmedium mit Glycerin als C-Quelle. In der mikroaeroben oder anaeroben Produktionsphase wurde dann wachstumsentkoppelt Succinat aus Glycerin und Kohlenstoffdioxid bzw. Hydrogencarbonat gebildet. Die Entstehung von 1 mol Succinat aus 1 mol Glycerin unter Fixierung von 1 mol CO2 ist redoxneutral und stellt die theoretisch maximale Succinatausbeute dar. Zuerst wurde eine Methode entwickelt, um die Succinatproduktion verschiedener Stämme schnell und einfach miteinander vergleichen zu können. Diese Biotransformationen im Kleinmaßstab wurden in 1,5 ml Plastikgefäßen durchgeführt. Zuvor wurden diese mit 1,2 ml einer Zellsuspension von 0,5 OD600/ml in M9-Minimalmedium gefüllt, welches definierte Mengen Glycerin und Hydrogencarbonat enthielt. Anschließend wurde der zeitliche Verlauf der Zelldichte und der exkretierten Metabolite gemessen. Nach einem anfänglichen, kleinen Anstieg der Zelldichte auf durchschnittlich 0,7 OD600/ml blieb diese während des Produktionszeitraumes bis zu 15 Tage konstant. In einem Experiment wurde der Einbau von 13C-markiertem Hydrogencarbonat in das gebildete Succinat per Massenspektrometrie bestätigt. Verschiedene Stoffwechselwege führen zum Succinat. Aus diesen Wegen wurden drei Strategien zur Succinatproduktion abgeleitet, wobei jede Strategie eine Kombination mehrerer Wege beinhaltete. Der metabolische Fluß über bestimmte Wege wurde durch die Deletion von Genen mit der &#955;Red-Rekombinationsmethode oder durch Plasmid-basierte Expression von Genen, die für auf den jeweiligen Routen benötigte Enzyme codieren, begünstigt. Außerdem sollten die Deletionen die Entstehung von Nebenprodukten wie beispielsweise Acetat verhindern. Nach der ersten Strategie sollte Succinat durch PEP-Carboxylierung und über den Glyoxylatzyklus produziert werden. Dem Stamm ss328 (&#916;ackA-pta &#916;mgsA &#916;gldA &#916;tdcE &#916;ldhA &#916;poxB &#916;pflB) fehlt der Großteil der Pyruvat-verbrauchenden Enzyme. Zusätzlich wurde die Acetatentstehung verhindert. Stamm ss328 verbrauchte bei einer Biotransformation mit 0,5 OD600/ml Zellen innerhalb von 6 Tagen 43,2 ± 5,7 mM Glycerin und bildete 28,1 ± 3,3 mM Succinat. Dies entsprach einer molaren Succinatausbeute von 65,1 ± 1,4 %. Nach der zweiten Strategie sollte Succinat neben PEP-Carboxylierung vor allem durch Pyruvat-Carboxylierung gebildet werden. Stamm ss331 (&#916;lipA &#916;ldhA &#916;poxB &#916;pflB) kann viele Pyruvat-verbrauchende Enzyme nicht mehr synthetisieren. Außerdem ist wegen &#916;lipA der Pyruvat-Dehydrogenase-Komplex inaktiv, falls keine Liponsäure zugegeben wird. ss331 wurde mit Plasmid pSS84.4 transformiert, welches das Gen für das Malic-Enzym 2 von Arabidopsis thaliana trug. Der resultierende Stamm verbrauchte bei einer Biotransformation mit 0,5 OD600/ml Zellen innerhalb von 6 Tagen 37,6 ± 1,4 mM Glycerin und bildete 26,6 ± 0,7 mM Succinat. Dies kam einer molaren Succinatausbeute von 70,9 ± 0,7 % gleich. Nach der dritten Strategie sollte Succinat ausschließlich durch PEP-Carboxylierung entstehen, weswegen die Gene pykA und pykF, die für Pyruvatkinasen codieren, deletiert wurden. Die Pyruvatkinasen katalysieren die direkte Umwandlung von PEP in Pyruvat. Die erhaltenen Stämme konnten auf Glycerin kaum wachsen, da sie nur ungenügende Mengen Pyruvat bilden konnten. Erst nach einer Selektion auf schnelleres Wachstum in Minimalmedium mit Glycerin zeigten die erhaltenen Mutanten Wachstumsraten im Bereich von 0,3 h-1. Pyruvat wurde in diesen Stämmen vermutlich verstärkt über die POMP-Umleitung gebildet, welche folgende Schritte beinhaltet: PEP-Carboxylierung zum Oxalacetat, Umwandlung von Oxalacetat in Malat und Decarboxylierung von Malat zum Pyruvat. Mit dem Stamm ss279 (&#916;gldA &#916;tdcE &#916;pykA &#916;pykF &#916;ldhA &#916;poxB &#916;pflB) wurde in Biotransformationen mit 0,5 OD600/ml Zellen innerhalb von 6 Tagen 57,8 ± 2,8 mM Glycerin verbraucht und 47,1 ± 1,1 mM Succinat gebildet. Dies entsprach einer molaren Succinatausbeute von 81,5 ± 2,0 %. Berücksichtigt man nur den reinen Produktionszeitraum ohne Zellmassebildung ab dem zweiten Tag, so wurden noch höhere Ausbeuten von 89,5 ± 3,7 % erhalten.Glycerol is produced in large quantities in the process of biodiesel production. It is a cheap, renewable resource that can be biotechnologically transformed into many industrially relevant fine or bulk chemicals. One such bulk chemical is succinate, the anion of succinic acid, which was described in studies of the U.S. Department of Energy (DOE) in 2004 and 2010 as an important platform chemical. Platform chemicals or chemical building blocks can be economically converted into a variety of other, higher-priced materials. In this work succinate production strains of Escherichia coli were constructed. The generation of substantial cell mass occurred prior to the production phase during an aerobic growth phase in minimal medium with glycerol as sole carbon source. In the following microaerobic or anaerobic production phase succinate was produced from glycerol and carbon dioxide or bicarbonate under non-growth conditions. The formation of 1 mol of succinate from 1 mol of glycerol with concomitant fixation of 1 mol of CO2 is redox neutral, and represents the theoretical maximum yield. First, a protocol was developed to compare the succinate production of different strains easily and quickly. 1.5 ml plastic vials were used for this small-scale biotransformation assay. They were filled beforehand with 1.2 ml cell suspension of 0.5 OD600/ml in M9 minimal medium, which contained defined amounts of glycerol and bicarbonate. Then the time course of cell density and excreted metabolites was analyzed. After an initial, small increase in cell density to 0.7 OD600/ml on average it remained constant up to 15 days during the the production period. In one experiment, the incorporation of 13C-labeled bicarbonate into the produced succinate was confirmed by mass spectrometry. Several metabolic pathways lead to succinate. Three strategies for the production of succinic acid were derived from these pathways. Each strategy is comprised of a combination of several routes. The metabolic flux through the desired routes was favored by deletion of genes of competing pathways with the &#955;Red-recombination method or by plasmid-based expression of genes that code for enzymes needed for the particular route. In addition, the deletions prevented the formation of by-products such as acetate. Production strains following the first strategy should produce succinate by PEP carboxylation and the glyoxylate shunt. Strain ss328 (&#916;ackA-pta &#916;mgsA &#916;gldA &#916;tdcE &#916;ldhA &#916;poxB &#916;pflB) amongst others lacks the majority of pyruvate-consuming enzymes. Moreover the formation of acetate was prevented. It consumed 43.2 ± 5.7 mM glycerol and produced 28.1 ± 3.3 mM succinate during a biotransformation with 0.5 OD600/ml cells within 6 days. This corresponded to a molar yield of 65.1 ± 1.4 %. Strains that were constructed according to the second strategy could form succinate by PEP carboxylation and especially by pyruvate carboxylation. The strain ss331 (&#916;lipA &#916;ldhA &#916;poxB &#916;pflB) lacks many of the pyruvate-consuming enzymes. In addition, the pyruvate dehydrogenase complex is inactive if no lipoic acid is added to the medium. Strain ss331 transformed with plasmid pSS84.4 carrying the malic enzyme 2 from Arabidopsis thaliana was used in a biotransformation with 0.5 OD600/ml cells. Within 6 days 37.6 ± 1.4 mM glycerol was used and 26.6 ± 0.7 mM succinate was formed. This represented a molar yield of 70.9 ± 0.7 %. The third strategy involved succinic acid production exclusively by PEP carboxylation, which is why the genes pykF and pykA, encoding pyruvate kinases have been deleted. The pyruvate kinases catalyze the direct conversion of PEP into pyruvate. The resulting strains could barely grow on glycerol. Only after a selection for faster growth in minimal medium with glycerol, the resulting mutants showed growth rates on glycerol in the range of 0.3 h-1. Pyruvate was formed in these strains through the POMP-pathway, a local rerouting of metabolism which includes the following steps: PEP carboxylation to oxaloacetate, conversion of oxaloacetate to malate, and decarboxylation of malate to pyruvate. Strain ss279 (&#916;gldA &#916;tdcE &#916;pykA &#916;pykF &#916;ldhA &#916;poxB &#916;pflB) was used in biotransformations with 0.5 OD600/ml cells. In 6 days 57.8 ± 2.8 mM glycerol was consumed and 47.1 ± 1.1 mM succinate was produced. This corresponded to a molar yield of 81.5 ± 2.0 %. When only the production period from the second day on is considered an even higher yield of 89.5 ± 3.7 % was obtained