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1	A recombineering pipeline for functional genomics applied to Caenorhabditis elegans Sarov, Mihail 19 February 2007 (has links) (PDF) Genome sequencing and annotation projects define the complete sets of RNA and protein components for living systems. They also present the challenge to generate functional information for thousands of previously uncharacterized genes. Protein tagging with fluorescent or affinity tags provides a generic way to describe protein expression and localization patterns and protein-protein interactions. The genome wide application of this approach in Saccharomyces cerevisiae has resulted in a comprehensive picture of the core proteome of a simple, well-studied model system. Extending these studies to more complex, multicellular model organisms, would allow us to place protein function onto a 4 dimensional space-time map, and will improve our understanding of the complex processes of development and differentiation. This will require efficient protein tagging methods and new high performance tags. Here we present a generic protein tagging approach for the model nematode Caenorhabditis elegans. The method is based on recombination mediated DNA engineering of genomic BAC clones into tagged transgenes for integrative transformation. C.elegans offers unique advantages for function discovery through protein tagging: compact and a well annotated genome, combined with a simple and well-understood anatomy and pattern of development. However, the methods for protein tagging in C.elegans have so far been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. In contrast, our approach combines the advantages of authentic regulation with a new application of recombineering, which is simple, fast and efficient. For the first time we apply liquid culture cloning for multiple recombineering steps. This is particularly important when high throughput applications are considered, as it offers significant advantages in scale up and automation. We show that the BAC derived transgenes can be used for stable, integrative transformation in C. elegans. We show that the tagged transgene can take over the function of its endogenous counterpart. Using florescent reporter, we reproduce known and document new expression patterns. The second part of the thesis describes a project that we undertook to develop improved double affinity cassettes for protein purification. We evaluated the performance of 5 new double tag combinations in vitro and in mammalian culture cells. All of the new cassettes performed well and present a valuable tool for protein interaction studies in higher model systems. functional genomics recombineering high throughput DNA engineering protein tagging funktionale Genomik recombineering Hochdurchsatz DNS manipulation Proteinmarkierung ddc:570 rvk:WD 5360 Genomik DNS Rekombination Proteine Markierung Caenorhabditis elegans
2	A recombineering pipeline for functional genomics applied to Caenorhabditis elegans Sarov, Mihail 11 December 2006 (has links) Genome sequencing and annotation projects define the complete sets of RNA and protein components for living systems. They also present the challenge to generate functional information for thousands of previously uncharacterized genes. Protein tagging with fluorescent or affinity tags provides a generic way to describe protein expression and localization patterns and protein-protein interactions. The genome wide application of this approach in Saccharomyces cerevisiae has resulted in a comprehensive picture of the core proteome of a simple, well-studied model system. Extending these studies to more complex, multicellular model organisms, would allow us to place protein function onto a 4 dimensional space-time map, and will improve our understanding of the complex processes of development and differentiation. This will require efficient protein tagging methods and new high performance tags. Here we present a generic protein tagging approach for the model nematode Caenorhabditis elegans. The method is based on recombination mediated DNA engineering of genomic BAC clones into tagged transgenes for integrative transformation. C.elegans offers unique advantages for function discovery through protein tagging: compact and a well annotated genome, combined with a simple and well-understood anatomy and pattern of development. However, the methods for protein tagging in C.elegans have so far been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. In contrast, our approach combines the advantages of authentic regulation with a new application of recombineering, which is simple, fast and efficient. For the first time we apply liquid culture cloning for multiple recombineering steps. This is particularly important when high throughput applications are considered, as it offers significant advantages in scale up and automation. We show that the BAC derived transgenes can be used for stable, integrative transformation in C. elegans. We show that the tagged transgene can take over the function of its endogenous counterpart. Using florescent reporter, we reproduce known and document new expression patterns. The second part of the thesis describes a project that we undertook to develop improved double affinity cassettes for protein purification. We evaluated the performance of 5 new double tag combinations in vitro and in mammalian culture cells. All of the new cassettes performed well and present a valuable tool for protein interaction studies in higher model systems. info:eu-repo/classification/ddc/570 ddc:570
3	Internet censorship in the European Union Ververis, Vasilis 30 August 2023 (has links) Diese Arbeit befasst sich mit Internetzensur innnerhalb der EU, und hier insbesondere mit der technischen Umsetzung, das heißt mit den angewandten Sperrmethoden und Filterinfrastrukturen, in verschiedenen EU-Ländern. Neben einer Darstellung einiger Methoden und Infrastrukturen wird deren Nutzung zur Informationskontrolle und die Sperrung des Zugangs zu Websites und anderen im Internet verfügbaren Netzdiensten untersucht. Die Arbeit ist in drei Teile gegliedert. Zunächst werden Fälle von Internetzensur in verschiedenen EU-Ländern untersucht, insbesondere in Griechenland, Zypern und Spanien. Anschließend wird eine neue Testmethodik zur Ermittlung der Zensur mittels einiger Anwendungen, welche in mobilen Stores erhältlich sind, vorgestellt. Darüber hinaus werden alle 27 EU-Länder anhand historischer Netzwerkmessungen, die von freiwilligen Nutzern von OONI aus der ganzen Welt gesammelt wurden, öffentlich zugänglichen Blocklisten der EU-Mitgliedstaaten und Berichten von Netzwerkregulierungsbehörden im jeweiligen Land analysiert. / This is a thesis on Internet censorship in the European Union (EU), specifically regarding the technical implementation of blocking methodologies and filtering infrastructure in various EU countries. The analysis examines the use of this infrastructure for information controls and the blocking of access to websites and other network services available on the Internet. The thesis follows a three-part structure. Firstly, it examines the cases of Internet censorship in various EU countries, specifically Greece, Cyprus, and Spain. Subsequently, this paper presents a new testing methodology for determining censorship of mobile store applications. Additionally, it analyzes all 27 EU countries using historical network measurements collected by Open Observatory of Network Interference (OONI) volunteers from around the world, publicly available blocklists used by EU member states, and reports issued by network regulators in each country. EU DPI Blocklisten Netzstörung Informationskontrolle Informationsfilterung Zugangskontrolle Internet-Zensur DNS-Hijacking DNS-Sperrung DNS-Manipulation Deep-Paket-Inspektion Netzmessungen Regulierungsbehörde Europäische Union Deep Packet Inspection European Union Offenes Internet Internet censorship EU DNS hijacking DPI blocklists national regulator authority DNS blockling DNS manipulation network interference network measurements information controls information filtering access control open Internet 940 Geschichte Europas 004 Informatik QR 760 ddc:000 ddc:940 ddc:004

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