1,721,020 research outputs found
Engineering of synthetic biological sensors for cell-material interfaces, the presence of aldehydes and for an educational kit
No full textBiosensors are sensors that include a biological component such as enzymes or cells. Analytes vary from metals, toxins, to nucleotides, but also cell-material interfaces can be sensed via mechanosignaling. The latter is especially important for biomaterials when intended for the application in human medicine. They need to undergo rigorous testing to show low immunogenicity and a high biocompatibility. However, how cell-material interactions can influence signaling processes of cells surrounding the materials, have largely been neglected. Especially engineered living materials (ELMs), for example bacterial biofilms, are trending towards applications in biomedicine. To this end, we engineered two E. coli biofilms with different stiffnesses and surface roughness. We demonstrated that HeLa cells show an increased YAP signaling when seeded on biofilms that have a higher stiffness and increased surface roughness compared to softer and smoother biofilms. This shows that before biofilms can be applied as therapeutics with direct contact to surrounding cells, the influence of mechanical properties should be considered and tested beforehand. Furthermore, we also developed a sensor for formaldehyde (FA). Formaldehyde is widely used in cell culture applications for cell fixation. We could show that FA when used in a cell culture setting can diffuse via the gas phase and subsequently activate different signaling processes in cells seeded nearby. We could utilize the reporter cell line based on YAP signaling, to be implemented as a low cost, reusable and sensitive sensor for FA. With this in mind, the handling of FA solutions in cell culture experiments, require careful planning, especially when investigating signaling processes. Finally, we could also implement an in vitro sensor based on CRISPR/Cas12a to be used in high schools. Since CRISPR/Cas is now part of the educational plan in Baden-Württemberg, there is a need for simple experiments that can enhance the understanding of students of this complex topic. By utilizing the side effect of Cas12a, the cleavage of ssDNA once activated, we could show activation of the enzyme by a ssDNA reporter functionalized with a quencher and a fluorophore. Upon cleavage of the reporter, the fluorophore is released and fluorescence can be imaged. In order to make fluorescence visible in high schools, without the need of costly equipment like microscopes, together with the HfG Karlsruhe, we developed a card board box functionalized with acrylic glasses as filters. Testing with students and teachers revealed that this kit is well perceived by both.In summary, this thesis demonstrates the successful implementation of different sensors, cell-based for sensing of aldehydes and cell-material interfaces and in vitro for the development of an educational CRISPR/Cas high school kit
Minimally trans-complementation-dependent HBV reporter vectors
No full textReporter-kodierende Hepatitis-B-Virus-(HBV)-Varianten sollten nach Infektion einen sensitiven und quantitativen Nachweis der Reporterexpression ausgehend von der kovalent geschlossenen, zirkulären (covalently closed circular, ccc)DNA, dem persistierenden viralen Minichromosom, ermöglichen. Frühere Versuche zur Entwicklung von Reporter-(r)HBV-Varianten waren aufgrund des kleinen und komplex aufgebauten Genoms wenig erfolgreich. Das Einbringen transgener Sequenzen führt in der Regel zu multiplen Defekten, die eine Transkomplementierung mit Vollgenom-HBV-Helferplasmiden verlangen.In dieser Arbeit wurden verschiedene Reportervektoren entwickelt, bei denen das HBc-Gen größteneils durch ein Transgen für Gaussia-Dura-Luciferase (Luc) ersetzt wurde. Die Replikation sollte ausschließlich von in trans bereitgestelltem HBc abhängen. Die Translation sowohl des Luc-Reporters wie auch von Pol wurde durch unterschiedliche Kombinationen von Translations-Kontrollelementen ermöglicht, d.h. 2A-Peptide, kleine interne ribosomale Eintrittsstellen (small internal ribosome entry sites, sIRESs) oder "Slip"-Sequenzen zur Induktion einer programmierten ribosomalen Leserasterverschiebung (programmed ribosomal frameshifting, PRF). Alle Vektoren wurden auf Reporterexpression, Replikation und Bildung der funktionellen Genomkonformation, der relaxierten zirkulären (relaxed circular, rc)DNA, untersucht. Ein Reporterkonstrukt, in dem Luc in-frame mit N-terminalen HBc-Resten kotranslatiert und die Pol-Transation über ein Slip-Motiv gesteuert wird, wurde weiter optimiert. Dies beinhaltete die Verkürzung der verbliebenen HBc-Restsequenz sowie die Deletion nicht-essentieller Luc-interner Sequenzen. Infektiosität und infektionsabhängige Reporterexpression wurden durch Inokulation von HepG2-NTCP-Zellen mit rHBV untersucht. Eindeutige Ergebnisse wurden zunächst durch in den rHBV-Inokula enthaltenes, während der Virusproduktion von rHBV-Plasmid transfizierten Zellen exprimiertes Luc Protein erschwert. Aus mehreren Methoden zur Entfernung oder Inaktivierung dieses Luc Proteins erwies sich ein GluC-Protease-Verdau der Inokula als am effizientesten, wobei die rHBV-Infektiosität vollständig erhalten blieb.Ansätze zur Steigerung der Virustiter beinhalteten die rHBV-Produktion durch Transduktion von Hepatomzellen mit rekombinanten Adeno-assoziiertes Virus-(rAAV)-rHBV-Partikeln sowie die lentivirale Integration einer rHBV-kodierenden TetON-Expressionskassette in das Genom von HepG2-HBc-Zellen, die konstitutiv HBc exprimieren. Schlussendlich wurden die besten Ergebnisse jedoch mittels konventioneller Kotransfektion unter Anwendung eines optimierten Protokolls erzielt.Andere Quellen als cccDNA für die Luc-Expression wurden experimentell durch Mutationsanalysen ausgeschlossen, einschließlich durch rHBV-Mutanten mit Defekten in der Bildung von rcDNA, dem authentischen cccDNA-Vorläufer. Die Eignung von rHBV als wt HBV-Surrogat wurde durch den Nachweis der antiviralen Wirkung zweier Kapsid-Assemblierungs-Modulatoren (capsid assembly modulators, CAMs) demonstriert, wobei die Lumineszenz vergleichbare Ergebnisse wie die Expression des HBV-Oberflächenantigens (surface, HBs) und des e-Antigens (HBe) nach einer wt HBV-Infektion zeigte. Darüber hinaus produzierte die Transkomplementation von rHBV mit HBc-Varianten mit Mutationen der hydrophoben Tasche, L60G und L60W, nackte Kapside, aber keine umhüllte Virionen, analog zu früheren Ergebnissen mit wt HBV. Eine Inokulation mit rHBV L60G oder rHBV L60W erzeugte dementsprechend keine Lumineszenz.Mit rHBV als Werkzeug zur Identifizierung neuer antiviraler Substanzen wurde die Wirkung des zyklischen Decapeptids Antamanid aus dem Pilz Amanita phalloides auf die HepG2-NTCP-Infektionanalysiert, wobei mikromolare Konzentrationen dosisabhängig die Reporteraktivität verringerten.Schließlich wurden in einem knockdown-Ansatz zelluläre Wirtsfaktoren, darunter bereits beschriebene HBV-Interaktionspartner wie auch neue Kandidaten, durch lentivirale Expression von shRNAs in HepG2-NTCP Zellen gezielt herabreguliert, um ihren Einfluss auf die Bildung von cccDNA zu untersuchen. Die Inokulation dieser Zellpools identifizierte u.a. die Flap-Endonuklease 1 (FEN1) als cccDNA-relevanten Wirtsfaktur, in Einklang mit früheren Studien. Somit ist rHBV auch zur Charakterisierung von HBV-Wirt-Interaktionen geeignet.Zusammengefasst wurde in dieser Arbeit ein neues HBV-Reportersystem entwickelt, welches eine sensitive und quantitative Detektion der cccDNA-Bildung nach Infektion ermöglicht. Validierung und Optimierung erfolgten im HepG2-NTCP-Zellkultursystem, dem derzeit primären Infektionsmodell. Dabei reproduzierte rHBV diverse, zuvor für wt HBV beschriebene Ergebnisse und demonstrierte damit sein Potenzial als wt HBV Surrogat. Schließlich erlaubte rHBV die Ermittlung der unbekannten antiviralen Kapazität von Antamanid. Künftig sollte dieses neue rHBV-System dazu beitragen, die komplexen Wechselwirkungen zwischen HBV und Wirt aufzuklären und ein besseres Verständnis der cccDNA-Bildung zu ermöglichen sowie die Entwicklung neuer therapeutischer Ansätze zu fördern
AptaSwift - Development of a novel aptamer selection method based on copied DNA microarrays
No full textAptamers are short single-stranded DNA or RNA oligonucleotides with the ability to bind a molecular target with high affinity and specificity. Other positive properties are their high thermal and chemical stability and that their denaturation is reversible, so they can be easily reused. Therefore, they have the potential to replace antibodies in many diagnostic and therapeutic applications in the future.However, a major problem in aptamer research is to find the right aptamer sequence with the desired properties. The current selection method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) consists of alternating selection and amplification cycles, with the aim of evolutionary enriching the “best/fittest” aptamers. In reality, however, SELEX is severely limited due to many biases. Sequences that perform best in PCR amplification have the greatest selection advantage, but often displace better binders that amplify worse. In addition, SELEX is very time-consuming and cost-intensive, as the best of all sequences in the final pool have to be characterised individually.Therefore, a completely novel selection method called AptaSwift was developed, and a first proof-of-concept was partly realised within this dissertation. AptaSwift aims to decouple selection and amplification by limiting each amplification and also providing optimal monitoring at each amplification step. This is enabled by the combination of a novel digital solid-phase PCR method which limits the amount of generated DNA per sequence with a label-free measurement system called SCORE (Single COlor REflectometry), enabling to monitor the binding properties of each sequence in parallel. In the context of this thesis, the digital solid-phase PCR and SCORE analysing step is tested, as well as additionally combined with a photocleavage process, in order to enable a later release of DNA sequences according to demand. Using the SCORE system, all potential aptamers on a microarray generated by digital solid-phase PCR can be analysed and characterised simultaneously. Subsequently, the best binders are regained in the photocleavage process by cleaving the photocleavable groups of the solid-phase primer using a microscope and short-wave light.In this thesis, microarrays were generated from a pool of three aptamer species via the solid-phase PCR process using two different approaches, and the aptamers were successfully analysed for their binding affinity to thrombin using SCORE. In addition, the photocleavage of a thrombin aptamer was demonstrated with sequencing data. To evaluate the kinetic analytical power of the SCORE system, aptamer screenings were performed with synthetic aptamer arrays binding thrombin and ICAM-1 (intercellular adhesion molecule 1), and DNA binders against Min proteins. A systematic investigation of thrombin aptamer sequences could be performed, showing especially that the height of the binding signal nearly linearly depends on the number of Thymine (T-) spacer between aptamer and surface.5In contrast to thrombin aptamers, DNA interactions with ICAM-1 were found to be nearly completely non-specific, as positive and negative controls also showed binding. Also contrary to current models and theories, the investigated Min proteins bind much better to single-stranded than to double-stranded DNA. The validation of the SCORE system was concluded with a comparative study of six different biosensing systems. In addition, the influence of the analysis software and the user on the determination of the binding constants was investigated. It could be shown that the software has only minor impact, but the quite often manual selection of the fitting windows can impact the KD value by a factor of up to 10. But this selection is already impacted by the definition of the assay itself, as, for example, the off-kinetic time may be determined to be 5 min instead of 10 min, which can have an impact of an order of magnitude for the KD analysis. Therefore “the human factor” may be a major impact to many kinetic analysis publications
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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