Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear... Show moreNuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-κB translocation dynamics, high-throughput screens that enable the analysis of dynamic NF-κB responses in individual cells in real time are essential. Thus far, only NF-κB downstream signaling responses of whole cell populations at the transcriptional level are in high-throughput mode. In this study, we developed a fully automated image analysis method to determine the time-course of NF-κB translocation in individual cells, suitable for high-throughput screenings in the context of compound screening and functional genomics. Two novel segmentation methods were used for defining the individual nuclear and cytoplasmic regions: watershed masked clustering (WMC) and best-fit ellipse of Voronoi cell (BEVC). The dynamic NFκB oscillatory response at the single cell and population level was coupled to automated extraction of 26 analogue translocation parameters including number of peaks, time to reach each peak, and amplitude of each peak. Our automated image analysis method was validated through a series of statistical tests demonstrating computational efficient and accurate NF-κB translocation dynamics quantification of our algorithm. Both pharmacological inhibition of NF-κB and short interfering RNAs targeting the inhibitor of NFκB, IκBα, demonstrated the ability of our method to identify compounds and genetic players that interfere with the nuclear transition of NF-κB. Show less
Ghotra, V.P.; He, S.; Bont, H.J.G.M. de; Ent, W. van der; Spaink, H.P.; Water, B. van de; ... ; Danen, E.H.J. 2012
A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in... Show moreA quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline. Show less
Janssens, M.; Smeden, J. van; Gooris, G.S.; Bras, W.; Portale, G.; Caspers, P.J.; ... ; Bouwstra, J.A. 2012
The Polyhedral Process Network (PPN) is a suitable parallel model of computation (MoC) used to specify embedded streaming applications in a parallel form facilitating the efficient mapping onto... Show moreThe Polyhedral Process Network (PPN) is a suitable parallel model of computation (MoC) used to specify embedded streaming applications in a parallel form facilitating the efficient mapping onto embedded parallel execution platforms. Unfortunately, specifying an application using a parallel MoC is a very difficult and highly error-prone task. To overcome the associated difficulties, we have developed the pn compiler, which derives PPN specifications from sequential static affine nested loop programs (SANLPs). However, there are many applications that have adaptive and dynamic behavior which cannot be expressed as SANLPs. In order to handle such dynamic applications, in this dissertation we address an important question: whether some of the static restrictions of the SANLPs can be relaxed while keeping the ability to perform compile-time analysis and to derive PPNs in an automated way. Achieving this will significantly extend the range of applications that can be parallelized in an automated way. By studying different dynamic applications we distinguished three relaxations to SANLP programs that would allow one to specify dynamic applications as sequential programs. These relaxations allow dynamic if-conditions, for-loops with dynamic bounds and while-loops in a program. The first relaxation has already been considered. In this dissertation, we consider the other two more difficult relaxations. Show less
Chemokines are signaling proteins that direct the migration and function of many cell types during development and responses of the immune system. The zebrafish embryo model is highly suited to... Show moreChemokines are signaling proteins that direct the migration and function of many cell types during development and responses of the immune system. The zebrafish embryo model is highly suited to investigate cell migration because of its optical transparency and availability of transgenic lines with fluorescently labeled cell types of the innate immune system. In this thesis, we analyzed the phylogenetic relationships between zebrafish and human chemokines and used Salmonella and Mycobacterium infection models to study the function of a zebrafish chemokine receptor gene, cxcr3.2, homologous to human CXCR3. Our data have demonstrated that cxcr3.2 is predominantly expressed in macrophages of the zebrafish embryo and plays an essential role in bacterial-induced macrophage migration and control of local infections. Furthermore, we used in vivo cell migration assays in wild type and cxcr3.2 mutant embryos to discover the putative ligand of the cxcr3.2 receptor. Injection of Cxcl11, an infection-inducible chemokine, resulted in the directional migration of macrophages in a cxcr3.2-dependent manner. As the first ligand-receptor pair with a proposed function in migration of zebrafish macrophages in response to bacterial infection, the identification of Cxcl11-Cxcr3.2 interaction is an important step towards understanding the chemokine signaling network underlying innate immune responses in the zebrafish model. Show less
Protein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these... Show moreProtein-protein interactions play an important role in all cellular processes such as signal transduction, electron transfer, gene regulation, transcription, and translation. Understanding these protein-protein interactions at the molecular level, is an important aim in structural biology. The protein interactions studied in this work are principally involved in the signal transduction and in electron transfer. These protein interactions belong to the family of transient dynamic interactions, meaning they associate and dissociate rapidly. The work presented in chapters two and three gives a detailed description of a study to determine the binding orientation of focal adhesion kinase derived peptide on the Src SH3 domain using paramagnetic NMR spectroscopy. The work presented in chapter four gives detailed information on the cloning, expression and purification of the focal adhesion kinase domain with the SH3 and S H2 binding sequences (32k) using a baculovirus expression system. The results of NMR characterization were presented for the complexes of different Src domains and the 32k. The work presented in the chapters five and six describes the NMR characterization of the interactions between several redox proteins. The aim of the work was to convert the transiently bound weak protein complexes into specific complexes Show less
Data mining tools often only use a single type of information. The method proposed in this thesis allows the user to insert relational information into existing data mining tools that are designed... Show moreData mining tools often only use a single type of information. The method proposed in this thesis allows the user to insert relational information into existing data mining tools that are designed for content-based information. It does so by regarding the contents of the neighborhood of an element. In this way, the content variability of elements is reduced by using the homophily in the network. Show less
We discuss uniform infinite causal triangulations (UICT) and Gibbs causal triangulations which are probabilistic models for the causal dynamical triangulations (CDT) approach to quantum gravity.... Show moreWe discuss uniform infinite causal triangulations (UICT) and Gibbs causal triangulations which are probabilistic models for the causal dynamical triangulations (CDT) approach to quantum gravity. Since there is a bijection between causal triangulations and planar rooted trees we first discuss some aspects of random trees. In particular, we describe new methods to obtain the fractal and spectral dimension for a large class of random tree ensembles which in the thermodynamic limit have the property that they posses a unique infinite spine. The results are applied to obtain the spectral dimension of generic and non-generic trees, as well as a model of randomly grown trees. In the following, we discuss in detail the relation between UICT and size-biased critical Galton-Watson processes. This relation is used to prove convergence of the joint rescaled length-area-process to a diffusion process and to derive from this the quantum Hamiltonian of CDT. In what follows, in an alternative construction to the branching process, we propose a growth process which samples sections of UICT by elementary moves in which a single triangle is added with a certain probability. This construction is used to show that the fractal dimension of UICT is almost surely 2, in an alternative derivation to the branching process picture. Furthermore, we also derive convergence results for the rescaled length-area-process of the grown triangulation to a diffusion process leading to an interesting duality relation and a mathematically rigorous derivation of the so-called peeling procedure. In the final part, we discuss Gibbs causal triangulations and using the transfer matrix formalism we show convergence of the partition function to a limiting measure. Further, we analyse the transfer matrix of the Ising model coupled to (Gibbs) causal triangulations and derive several properties of the latter Show less
Phenomenologically, cosmic inflation is a satisfying and well-tested description of the physics of the very early universe. During this epoch, the universe was dominated by high energy phenomena... Show morePhenomenologically, cosmic inflation is a satisfying and well-tested description of the physics of the very early universe. During this epoch, the universe was dominated by high energy phenomena that can only be truly understood in a quantum gravity theory such as string theory. In this thesis we show that the embedding of inflation in a string theoretic framework is very sensitive to the details of the theory. We consider both the low energy supergravity limit as well as a worldsheet set-up. Moreover, we investigate the constraints imposed by supersymmetry and conformal symmetry. Conformal symmetry is important both in the worldsheet theory as well as in a holographic description of inflation. In the latter case we investigate the imprints of conformal invariance on the (observable) statistical correlations in the cosmic microwave background radiation. Show less
In this thesis we investigate different techniques and formalisms to address complexity introduced by unbounded structures in object-oriented programs. We give a representation of a weakest... Show moreIn this thesis we investigate different techniques and formalisms to address complexity introduced by unbounded structures in object-oriented programs. We give a representation of a weakest precondition calculus for abstract object creation in dynamic logic. Based on this calculus we define symbolic execution including abstract object creation. We investigate the complex behaviour introduced by multi-threading and give a formalism based on the transformation of multi-threaded reentrant call-graphs to thread automata and the application of context free language reachability to decide deadlock freedom of such programs. We give a formalisation of the observable interface behaviour of a concurrent, object-oriented language with futures and promises. The calculus captures the core of the Creol language and allows for a comparison with the concurrency model of thread-based, object-oriented languages like Java or C#. We give a technique to detect deadlock freedom for an Actor-like subset of the Creol language. Show less
Throughout the last decades the water oxidation process has been extensively investigated. However, open questions remain on the reaction mechanism and the possible intermediates in the catalytic... Show moreThroughout the last decades the water oxidation process has been extensively investigated. However, open questions remain on the reaction mechanism and the possible intermediates in the catalytic cycle. Indeed, different catalysts can function through different reaction routes. Some of them work in a proton-coupled electron transfer (PCET) pathway, while other catalysts have a non-PCET behaviour. Some of these problems lie in the very short lifetime of intermediates, which makes it difficult to characterize them experimentally. Within this context, DFT calculations represent a very useful tool. In fact quantum-mechanical computational tools allow exploring and analyzing the possible intermediates and study and compare different reaction pathways in order to find the energetically most likely one. In the same way the thermodynamics of the catalytic cycle and the kinetics of the reaction coordinates can be analyzed. The specific aim of this thesis is the study of different catalysts and reaction pathways to find clues about the mechanism of the reaction and develop guiding principles for the design of efficient water oxidation complexes Show less
We show that weak antilocalization by disorder competes with resonant Andreev reflection from a Majorana zero mode to produce a zero-voltage conductance peak of order e(2)/h in a superconducting... Show moreWe show that weak antilocalization by disorder competes with resonant Andreev reflection from a Majorana zero mode to produce a zero-voltage conductance peak of order e(2)/h in a superconducting nanowire. The phase conjugation needed for quantum interference to survive a disorder average is provided by particle-hole symmetry-in the absence of time-reversal symmetry and without requiring a topologically nontrivial phase. We identify methods of distinguishing the Majorana resonance from the weak antilocalization effect. Show less
Scientific practice is an activity that is data-intensive and widely supported by computerized systems, data repositories included. It is also an activity that is highly creative and, as such, can... Show moreScientific practice is an activity that is data-intensive and widely supported by computerized systems, data repositories included. It is also an activity that is highly creative and, as such, can benefit from a moment of openness, playfulness and exploration. Motivated also by recent developments in the field of Human Computer Interaction regarding play and games, this work investigates playfulness as a desirable attribute of a scientist's interaction with scientific data in repositories. Focus is on data repositories of a specific domain of science, i.e. the life sciences, and of a particular type of data, i.e. image data. Having introduced a new but relevant attribute for interfaces to scientific image repositories, i.e. playfulness, the question we ask is the following: What could playfulness with scientific images amount to and how do we design for it? Via case studies and reviews, we flesh out particular elements of play for exploration and implement artefacts, i.e. interfaces and games, that exemplify instances of playful interaction with image research material in collections. Show less
The exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective... Show moreThe exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective group in the solution-phase synthesis of (phosphorothioate) oligonucleotides. Pioneered by de Koning et al. a Solution-Phase Extraction Method (SPEM) amendable to scale-up was developed for the preparation of DNA and phosphorthioate oligonucleotide fragments. This approach utilizes extractive work-up procedures as the only tool in the isolation of oligonucleotides. Next, the design and synthesis of diamoniod decorated iminosugars as potential inhibitors of glycoside proccesing enzymes are described. Several libraries of natural iminosugars such as deoxynojirimycin and castanospermine decorated with an N-pentyloxy spacer were prepared. These iminosugar derivatives were evaluated as inhibitors of the enzymes involved in glucosylcramide metabolism, namely glucosylceramide synthase, beta-glucocerebrosidase and beta-glucosidase. Show less
