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From the article: "To enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement). In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi- organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms. To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish. The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental toxicity screening systems. The results of these studies also provided reassurance that none of the Piperazines tested are likely to have any significant developmental and/or reproductive toxicity issues to humans when used in their commercial applications."

Zebrafish larvae (Danio rerio) are increasingly used to translate findings regarding drug efficacy and safety from in vitro-based assays to vertebrate species, including humans. However, the limited understanding of drug exposure in this species hampers its implementation in translational research. Using paracetamol as a paradigm compound, we present a novel method to characterize pharmacokinetic processes in zebrafish larvae, by combining sensitive bioanalytical methods and nonlinear mixed effects modeling. The developed method allowed quantification of paracetamol and its two major metabolites, paracetamol-sulfate and paracetamol-glucuronide in pooled samples of five lysed zebrafish larvae of 3 days post-fertilization. Paracetamol drug uptake was quantified to be 0.289 pmole/min and paracetamol clearance was quantified to be 1.7% of the total value of the larvae. With an average volume determined to be 0.290 μL, this yields an absolute clearance of 2.96 × 10(7) L/h, which scales reasonably well with clearance rates in higher vertebrates. The developed methodology will improve the success rate of drug screens in zebrafish larvae and the translation potential of findings, by allowing the establishment of accurate exposure profiles and thereby also the establishment of concentration-effect relationships.

Mycobacterium marinum infection in zebrafish has become a well-established model of tuberculosis. Both embryonic and adult zebrafish infection studies have contributed to our knowledge of the development and function of tuberculous granulomas, which are typical of mycobacterial pathogenesis. In this review we discuss how transcriptome profiling studies have helped to characterize this infection process. We illustrate this using new RNA sequencing (RNA-Seq) data that reveals three main phases in the host response to M. marinum during the early stages of granuloma development in zebrafish embryos and larvae. The early phase shows induction of complement and transcription factors, followed by a relatively minor induction of pro-inflammatory cytokines within hours following phagocytosis of M. marinum. A minimal response is observed in the mid-phase, between 6 hours and 1day post infection, when the tissue dissemination of M. marinum begins. During subsequent larval development the granulomas expand and a late-phase response is apparent, which is characterized by progressively increasing induction of complement, transcription factors, pro-inflammatory cytokines, matrix metalloproteinases, and other defense and inflammation-related gene groups. This late-phase response shares common components with the strong and acute host transcriptome response that has previously been reported for Salmonella typhimurium infection in zebrafish embryos. In contrast, the early/mid-phase response to M. marinum infection, characterized by suppressed pro-inflammatory signaling, is strikingly different from the acute response to S. typhimurium infection. Furthermore, M. marinum infection shows a collective and strongly fluctuating regulation of lipoproteins, while S. typhimurium infection has pronounced effects on amino acid metabolism and glycolysis.

A 2-week long theoretical and practical course on innovative microscopy in the field of microbial infection was organized in Pretoria, South Africa. Talks from lecturers from such fields as super-resolution microscopy, fluorescence and bioluminescence imaging, high throughput microscopy assays and image analysis were followed by practicals on cutting-edge microscopes.

Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethal infection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptome level, we have extended and validated previous microarray data by Illumina next-generation sequencing analysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryos using a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcript sequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl transcripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript and an overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165 transcripts was observed with previous microarray data. Based on the combined sequencing-based and microarray-based transcriptome data we compiled an annotated reference set of infection-responsive genes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines and chemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immune response. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafish embryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish we derived a common set of infection-responsive genes. This gene set consists of known and putative innate host defense genes that are expressed both in the absence and presence of a fully developed adaptive immune system and that provide a valuable reference for future studies of host-pathogen interactions using zebrafish infection models.

The rough coat (rc), an autosomal-recessive mutation, arose spontaneously in C57BL/6J mice. Homozygous rc mice develop severe skin and hair abnormalities, including cyclic and progressive hair loss and sebaceous gland hypertrophy. The rc locus was previously mapped to Chromosome 9. To elucidate the genetic basis underlying the rc phenotype development, we carried out positional cloning, and mapped the rc locus to a 246-kb interval. We identified a missense mutation within a novel open reading frame in the rc/rc mice, which is predicted to encode a cell adhesion molecule with the highest homology to myelin protein zero (MPZ) and myelin protein zero-like 2 (MPZL2, also called epithelial V-like antigen). We therefore named this gene Mpzl3 (myelin protein zero-like 3). The mutation in the rc/rc mice occurred at a highly conserved residue within the conserved Ig-like V-type domain, thus likely altering the MPZL3 protein function. Reverse transcriptase-PCR and Western blot analyses revealed expression of the Mpzl3 gene in various adult organs, including the skin. Using indirect immunofluorescence, we detected MPZL3 protein in the keratinocytes and sebocytes in the skin. Results from this study identified a novel gene encoding a predicted adhesion protein whose mutation in the rc/rc mice likely caused the rc phenotype.

We have recently reported a mutation within the conserved immunoglobulin V-type domain of the predicted adhesion protein Mpzl3 (MIM 611707) in rough coat (rc) mice with severe skin abnormalities and progressive cyclic hair loss. In this study, we tested the hypothesis that the human orthologue MPZL3 on chromosome 11q23.3 is a candidate for similar symptoms in humans. The predicted conserved MPZL3 protein has two transmembrane motifs flanking an extracellular Ig-like domain. The R100Q rc mutation is within the Ig-domain recognition loop that has roles in T-cell receptors and cell adhesion. Results of the rc mouse study, 3D structure predictions, homology with Myelin Protein Zero and EVA1, comprehensive database analyses of polymorphisms and mutations within the human MPZL3 gene and its cell, tissue expression and immunostaining pattern indicate that homozygous or compound heterozygous mutations of MPZL3 might be involved in immune-mediated human hereditary disorders with hair loss.

Conventional network protocols and its security mechanisms fail to cope with arising challenges in trust. Well known concepts from the domain of Trusted Computing can be applied to the example of mobile ad-hoc networks (MANETs) in order to establish extended trust capabilities between devices. The approach of such an anchor of trust in MANETs shows interesting possibilities since no central instances such as Access Points are involved in those networks. The communication between directly connected devices of the network is protected by a cryptographic protocol making use of a Trusted Platform Module (TPM) that serves as root-of-trust on each device. Such a hardware chip allows devices to attest the local system state and assess states of remote systems. Building on this, transmission of routing and payload data can be restricted to devices in trustworthy states. The resulting mobile ad-hoc network, by using this protocol, is protected against many of today's security threats. Single malicious devices are automatically recognised and excluded from participation in the network by all devices. Especially the dissemination of misleading routing information, which affects the availability of the whole network, is effectively prevented by the developed protocol. Thus, it is shown that the device itself is secured by a hardware TPM. Also the communication is secured, by verifying the device's state between the counterparts.

As the IP traffic observed on network operator’s backbones keeps increasing year by year, the analysis of NetFlow data metered for this traffic becomes a burden for centralized traffic monitoring solutions. Thus, SCRIPT proposes a decentralized accounting architecture and framework for NetFlow storage and analysis, which is flexible to allow for the development of distributed traffic analysis applications. SCRIPT mechanisms organize multiple PCs or AXP (Application Extension Platform) cards in an analysis network and route NetFlow records according to rules imposed by the analysis application. In turn, the evaluation of the prototype has shown that (a) this approach allows for a linear increase of the number of NetFlow records, which can be processed with the number of nodes in the SCRIPT deployment network, and (b) deploying SCRIPT on router-embedded AXP cards is improving an already existing infrastructure with the capability of storage and processing of NetFlow records.

Network and service performance measurements are essential in IF (Internet Protocol) networks, e.g., for network management, network monitoring, and service quality assurance. In order to measure the service quality received by individual users, a service-specific measurement system is required. Therefore, this paper develops and implements the Network and Service Quality Measurement (NSQM) architecture, which integrates network- and service-specific measurements and can configure all measurement components on demand and according to service signaling in order to setup network-wide measurements in an automated manner. NSQM supports the correlation of measurement data from multiple locations, enabling the determination of link-specific and end-to-end performance characteristics. NSQM allows for measurements at different aggregation levels, including service-class and per-flow measurements. It integrates both active and passive measurements and supports a fine-grained selection of traffic to be measured, which reduces the amount of measurement data to be collected and processed.

The transfer of very large files often faces the problem of performance degradation due to bottlenecks or congestions in a network. Large file transfer is typical in Grid networks [11], where multiple nodes cooperate and run a common application. To improve the performance of large file transfer and to reduce the transfer time, this paper proposes a new file transfer application, called Secure Large File Transfer (SLFT) that supports file transfer over multiple independent network paths. For this purpose, SLFT uses Grid nodes as relays in order to route traffic to the destination. The described problem may also be solved on a lower layer of the OSI model; however, given the high heterogeneity of Grid environments, implementing the required mechanisms on layer 2 or 3 might not be feasible due to the inherent differences between the i volved ISP domains. Taking the approach to the application layer, makes possible to create a generic mechanism able to operate over the different possible underlying communication infrastructure [12]. The SLFT application has been implemented as a part of the GINTONIC toolbox, a set of Grid-specific network enhancements developed under the EC-GIN project [5]. In Grid networks nodes are geographically distributed, several administrative domains may be involved, and the communication is in a potentially hostile environment, the transfer of files involves several security threats which are addressed by the security architecture design of the SLFT application. This paper presents the design and implementation details of the SLFT application, focusing on the necessary security features. Based on practical experiences and evaluation in a test-bed, the performance of the SLFT application has been assessed. Evaluation results show that SLFT can improve the performance thanks to parallel transfer over multiple network paths.

Analysis of IP traffic is highly important, since it determines the starting point of many network management operations, such as intrusion detection, network planning, network monitoring, or accounting and billing. One of the most utilized metering data formats in analysis applications are IP (Internet Protocol) flow records. With the increase of IP traffic, such traffic analysis applications need to cope with a constantly increasing number of flow records. Typically, centralized approaches to IP traffic analysis have scalability problems, which are addressed by replacing existing hardware with more powerful CPUs and faster memory. In contrast, this paper developed and implemented SCRIPT (Scalable Real-time IP Flow Record Analysis), which defines a scalable analysis framework that can be used to distribute flow records to multiple nodes performing traffic analysis in order to balance the overall workload among those nodes. Due to its generic design, the framework developed can be extended and used to distribute other metering data, such as packet headers, payloads, or accounting records.

Today’s global food industry represents a huge market of US$ 3,500 billion pa (Wijnands et al. 2006). Nevertheless, food supply chains are characterized by fixed trade relations with long term contracts established between heterogeneous supply chain companies. In addition, consumer demands have undergone a dramatic change during the last four decades. Quality, food safety and uniqueness are the leading factors for buying decisions. Altogether, this causes three main problems companies in the food sector need to be prepared for. First, new ways to coordinate companies in to a supply chain must be installed in order to reach an efficient exploitation of globally distributed capacities. Second, cost-effective mechanisms for collaboration are needed. And third, an integrated tracking and tracing solution is essential to ensure food quality and safety on a global scale. AgroGrid is a Business Experiment addressing above mentioned challenges by providing a Grid-based solution for supply chains in the agricultural industry. AgroGrid implements a Grid-enabled market place that allows companies operating in agriculture food markets to offer and source capacities, to negotiate quality of food to be delivered, to establish contracts, and to create customised dynamic supply chains (Volk et al. 2009b). Thereby, capacities in AgroGrid include any products and services offered by a participant, e.g., food products, transport and/ or storage capacities. AgroGrid also provides facilities to monitor the quality and safety of food products delivered across supply chains.

Novel high-throughput deep sequencing technology has dramatically changed the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of this technology to gain insight into the wide range of transcriptional responses that are associated with an infectious disease process. Using Solexa/Illumina's digital gene expression (DGE) system, a tag-based transcriptome sequencing method, we investigated mycobacterium-induced transcriptome changes in a model vertebrate species, the zebrafish. We obtained a sequencing depth of over 5 million tags per sample with strong correlation between replicates. Tag mapping indicated that healthy and infected adult zebrafish express over 70% of all genes represented in transcript databases. Comparison of the data with a previous multi-platform microarray analysis showed that both types of technologies identified regulation of similar functional groups of genes. However, the unbiased nature of DGE analysis provided insights that microarray analysis could not have achieved. In particular, we show that DGE data sets are instrumental for verification of predicted gene models and allowed us to detect mycobacterium-regulated switching between different transcript isoforms. Moreover, genomic mapping of infection-induced DGE tags revealed novel transcript forms for which any previous EST-based evidence of expression was lacking. In conclusion, our deep sequencing analysis revealed in depth the high degree of transcriptional complexity of the host response to mycobacterial infection and resulted in the discovery and validation of new gene products with induced expression in infected individuals.

Accounting is a key task in commercial networks. With the increasing number of IP-based services and mobility support, accounting needs to evolve towards an integrated, service-oriented accounting approach in a mobile environment. Therefore, this dissertation digest paper presents the Serviceoriented Tailored Accounting Configuration (STACO) architecture that enables a service-oriented accounting configuration management in a mobile, multi-domain networking environment. Additionally, it presents the Diameter flow accounting application as an extension to the Diameter protocol in order to integrate IP flow accounting into any Diameter-based infrastructure and to support an efficient transfer of IP flow records.

Food supply chains today are characterized by fixed trade relations with long term contracts established between heterogeneous supply chain companies. Production and logistics capacities of these companies are often utilized in an economically inefficient manner only. In addition, increased consumer awareness in food safety issues renders supply chain management even more challenging, since integrated tracking and tracing along the whole food supply chain is needed. Facing these issues of supply chain management complexity and completely documented product quality, this paper proposes a full lifecycle solution for dynamic capacity markets based on concepts used in the field of Grid [1], like management of Virtual Organization (VO) combined with Service Level Agreement (SLA). The solution enables the cost-efficient utilization of real world capacities (e.g., production capacities or logistics facilities) by using a simple, browser-based portal. Users are able to enter into product-specific negotiations with buyers and suppliers of a food supply chain, and to obtain real-time access to product information including SLA evaluation reports. Thus, business opportunities in wider market access, process innovation, and trustworthy food products are offered for participating supply chain companies.

Monitoring of Service Level Objectives (SLOs) determines an essential part of Service Level Agreement (SLA) management, since customers are to be reimbursed, if a provider fails to fulfil them. By automating this process, a timely detection of a violation is possible. The compliance approach must be flexible to adapt to potential changes, must be scalable with respect to the amount of data, and has to support multi-domain environments. This paper determines a Hosted Streaming Services scenario and defines relevant SLOs. Key requirements are derived, the respective architecture is designed, and the approach is implemented prototypically based on a generic auditing framework. Further-more, a new scheme is proposed that considers the degree and duration of SLO violations in calculating reimbursements.

Flow accounting in IP networks is used by network operators for various purposes, such as network management, traffic management, or traffic analysis. In order to integrate flow accounting into an Authentication, Authorization, and Accounting (AAA) infrastructure, this work designs and evaluates an accounting extension to the Diameter protocol - termed Diameter IP Flow Accounting (IPFA) application - in support of the efficient transfer of IP flow records. The new Diameter IPFA application has been implemented as a prototype and its evaluation shows that it achieves a better performance for the transfer of IP flow records than the traditional Diameter accounting approach.

Charging for IP-based communications determines the overall term for metering or monitoring, accounting, pricing, charge calculation, and billing. These five actions are detailed in this chapter to provide a clear view on their interdependencies as well as their relations to distributed computing. Since an ubiquitous computing approach does require communication means between all entities involved, the provisioning of these communication channels is supported typically by commercial service providers— covering network, transport, and value-added services. Thus, the legal and contractual relationships between customers and providers as well as technological choices of protocols, mechanisms, and parameters define the area of interest here.

Grid networks aim to build a future architecture for efficient resource sharing and distributed service provisioning in a multi-provider environment. However, mobility, QoS support, and commercial service provisioning -all essential issues in future networks -pose new challenges to grid networks, both from a technical and economic point of view. Therefore, the Akogrimo project aims at developing an integrated service architecture for commercial mobile grid networks. This paper presents the Akogrimo architecture and its key characteristics, integrating mobility and network layer QoS support in a commercial grid environment.

Accounting for resource and service usage in communication networks forms the basis of network operators' business, and is therefore essential. Within a heterogeneous, multidomain mobile environment of emerging new access technologies, the demand for a distributed accounting infrastructure becomes more relevant. Driven by the analysis of current accounting solutions in Universal Mobile Telecommunications System (UMTS) and Wireless Local Area Networks (WLAN) networks, the newly developed approach presents an integrated accounting architecture based on the Diameter protocol, forming a flexible basis for charging UMTS and WLAN communication services. The system developed is described, discussed and evaluated by a prototypical implementation.

Accounting of service usage is one of the main tasks of service providers in their operation and management processes, providing the necessary information for the subsequent charging process. Because of the increasing number and diversity of services in IP-based networks, accounting has to provide flexible and extensible means supporting different service specific accounting attributes in order to meet the providers' requirements. Furthermore, users typically access services of different providers, resulting in a service composition in a multi-provider environment. After identifying key players in this scenario, this paper presents a service model, classifying different service types, and proposes an architecture (SMArch) for a common accounting scheme, which is implemented based on the IPDR framework and reference code.

Accounting performed by network and service providers covers the tasks of determining, collecting, and evaluating information on the service usage of their customers. This information forms the basis of the subsequent charging process. For performing these tasks in case of heterogeneous mobile networks a generic configuration management, specifically tailored at the provisioning of various Internet services is needed. This work defines a role model covering all participating entities of a distributed service provisioning environment for mobile networks together with key requirements. In addition, it outlines different accounting scenarios and proposes an architecture for a generic, technology- and service-independent configuration management of accounting in a distributed, mobile, and multi-provider environment. In turn, the approach specifies required configuration and state information in network components. Finally, a use case with an inter-domain scenario provides an example for the proposed accounting configuration management process.