The major goal of this thesis is to investigate the effects of the Wnt-signaling pathway on the cytoskeletal rearrangement during the differentiation of human neuronal progenitor cells. In particular, we analyze the dynamic and organization of the microtubule network after inhibition or activation of the wnt-pathway. Quantatitive analyses of the organization of the cytoskeleton in living cells are studied by use of confocal microscopy and GFP-constructs. Simultaneously morphometric analysis are done at a given time of differentiation (0, 12, 24 hrs).In addition, the axonal transport of cytoskeltal proteins and wnt-patway proteins such as β-catenin or dishevelled is characterized. To quantify the transport processes of proteins, involved in Wnt-signaling GFP-transfected cells are subjected to photobleaching (FRAP). High resolution video microscopy in combination with object-tracking is used to investigate the movement of specific organelles and axon growth. The results of the dynamic of single proteins and their effects on the cytoskeleton and axon behavior will provide important data, which can apply to modeling and simulation.

A systems biology approach using mathematical modeling of signal transduction pathways gives insights into the interrelations among the components and thus aims to contribute into the medical research e.g. regenerative medicine. Thereby the application of quantitative measures, have become useful for characterizing the properties of the components, regarding the average time until activation or the average duration of remaining in an activated state. In recent work we applied quantitative measures to analyze the characteristic design of a MAPK pathway consisting of two double phosphorylations. A comparison with a single and triple phosphorylation structure showed, that the design of a MAPK pathway is favorable for pathways which aim to transmit a fast signal which is active only for a short duration. We want to apply those measures to the Wnt pathway in order to confront the following questions: Why is beta catenin phosphorylated several times before degradation? What makes a multiple phosphorylation more favorable compared to a single? Why are there complexes of two kinases and two scaffolding proteins instead of just a single component? What is the advantage of this special design regarding duration and speed of activation? While the quantitative measures defined by Heinrich et al. are restricted to signals that eventually tend to zero after removal of the stimulus, an extension of those measures might be necessary in order to describe signals that tend to a value unequal to zero.

This PhD project deals with mathematical modelling of cellular signal transduction and their analysis. The focus thereby lies on the Wnt-signalling pathway and its kinetic properties. The purpose of modelling and simulation is a better understanding of the biology of signalling cascade and the cellular transport mechanisms that crucially influence the distribution of the proteins. In particular, we focus on the main protagonist and transcriptional co-factor beta-catenin and its antagonists. Additionally, we are using the approach of "sloppy parameters" to investigate the sensitivity of the dynamical behavior of previously published models to experimental errors in the measured values and to rather arbitrarily estimated values used to set the parameters of the model.

This PhD project combines wet-lab experiments as well as in silico experiments. The goal is to analyze the Wnt/beta-catenin pathway. For this a series of wet-lab experiments have been performed. Exploration used techniques like immublotting, infra-red analysis and cell fractionation to analyse the pathway components like LRP6, beta-catenin, GSK3beta and Dishevelled 2 (Dvl2) in cytosol, membrane and nucleus fractions. Observations in the wet-lab have motivated the development of a computational model of the Wnt pathway in which the intra- and intercellular dynamics are analysed. This model is currently validated.

The differentiation of nerve cells from progenitor cells causes a complete restructuring of the cell morphology by activation of receptors and the associated signal cascades. The development of the neural cell extensions, axons and dendrites is mediated by the restructuring of the cytoskeleton and increase of the transportation activity, using especially microtubules and actin filaments for this task. The MAP-1B protein, a very large protein with numerous phosphorylation sites, plays a central role, because of its ability to stabilize microtubules by accumulation. Beside MAP-1B also APC and other factors in their phosphorylation are affected by Wnt/Dvl dependent mechanisms.

In this work the same cell model as in the project R-1 will be used, in order to investigate how MAP-1B is regulated by the Wnt/Fz/Dvl/GSK-3β/β-Catenin signaling pathway.

There are different storage models for XML since they highly depend on the application field and the usage of XML. Other influences are aspects like distribution of document collections, re-use of documents, query and update classes, evolution and adoption of structures (schemas) used. The challenges are the design and deployment of a distributed XML repository and efficiently conceived query and update processing facilities. The retrieval of model components for re-use is one main issue which has to get worked out together with the modelling and simulation group. The retrieval should be based on content or by example, i.e. query by example or query by structure. Therefore, techniques should be employed which are known from the field of structure/schema extraction, structure mining. Additionally, schema matching algorithms are needed as well as query processing techniques for semi-structured data and content based retrieval methods. The combination and selection of the specialized storage and query processing methods rely on knowledge from the application domain. This knowledge can be extracted and derived from existing standards (e.g. SBML markup language), well-documented database applications from system-biology and their schemas. New evolving technologies like ontologies from OBO (Open biomedical ontologies) and other tools from the semantic web should be regarded and employed as well.

In this PhD project the control of the differentiation of neural progenitor cells by Wnt induced changes of the intracellular calcium concentration (non-canonical Wnt calcium Pathway) will be analysed.

The analysis of the mechanisms by inhibitors of the different stages of the Wnt calcium signal pathway, based on the changes of calcium concentration (calcium oscillations), will be cleared up.

The collected data will be used in an integrative model of the Wnt signal paths into neural progenitor cells together with the project R1.

Apoptosis is known to be an essential mechanism for eliminating redundant cells during CNS development. The proliferation and differentiation of neuronal progenitor cells are accompanied by massive apoptotic cell death and the canonical Wnt signaling pathway driven differentiation of neuronal progenitor cells is presumed to interfere with apoptosis pathways as well.

However, the molecular interlock between the canonical Wnt pathway and apoptotic pathways is still unknown. The description of the expression pattern of apoptosis-related proteins over time after targeted manipulation of the canonical Wnt pathway shall help to elucidate the molecular interference of the canonical Wnt and apoptosis pathways in neuronal progenitor cells. Computer based modelling of the data will increase the understanding of the molecular bases of the cross-talk between Wnt pathway, apoptosis and neuronal differentiation.

Aim of this project is the clarification of the function of the canonical Wnt/Fz/Dsh/GSK-3ß/ß- Catenin- signalling pathways for the differentiation of human neural progenitor cells. The analyses will help to understand to which extend this signalling pathway induces or fastens the differentiation. The necessity of the pathway for differentiation as well as possible interactions with non-canonical Wnt-pathways will be tested by the selective perturbation on different stages. Data for the mathematical modelling of the pathway will be based on gene expression and proteome analysis. The identification of new target genes of the canonical pathway is anticipated, which will of high importance for the neuronal differentiation.

The aim of this project is to investigate the effects of Wnt/calcium-pathway on the differentiation of ReNcell VM cells by physiological state or upon the stimulation with purified Wnt proteins or in down-regulation of endogenous Wnts by specific small interfering RNA (siRNA) transfection. The quantities of cellular free calcium concentration, calcium sensitive enzymes like protein kinase C (PKC), calcium-calmodulin dependent kinase II (CamKII), calcineurin (CaCN) and other proteins, e.g., nuclear factor of activated T cells (NFAT) will be analysed during cell differentiation at different time points by real-time PCR and quantitative Western blot. The quantitatively collected data will be used to create an integrative model of Wnt signal pathway for neural progenitor cells.

This project focuses on the Wnt-dependent control and dynamics of Wnt receptor molecules Frizzled (Fz) and Low Density Lipoprotein Receptor-related Protein (LRP) and their binding partners, as well as on possible interactions with growth factors. The results should shed light on how receptor distribution, internalization and interaction relate to the Wnt signaling pathway:

• How does receptor distribution change during differentiation? (confocal microscopy and image analysis (IMARIS) for quantification of co-localization of different proteins)
• How stable are the receptor molecules in the membrane? (expression of GFP-fusion proteins (LRP, Fz) for photobleaching (FRAP) and quantification of recovery)
• Which influence does the activation of receptors have on their half-life in the membrane (receptor recycling)? (TIRF for the measurement of membrane activities in living cells, 3D-microscopy; a combined TIRF/Confocal Live Cell Analysis Station has recently been acquired)
• Which correlations exist between the signaling pathways of Growth Factors and Wnt in proliferating and differentiating cells?

The aim is the modeling of the dynamic processes on the membrane level – also with the aid of the already existing cytoplasm/nucleus-shuttling models (such as [MJM+09]), the modeling formalisms already developed or to-be-developed at the college ([JLNU08] and Uhrm A2) as well as the evaluation methods [LU10] and Uhrm K2.

The Wnt proteins are members of a highly conserved family of signaling molecules that play a crucial role in development, by controlling proliferation and differentiation of neural progenitor cells in a stage-specific and cellular-dependent manner. However, the molecular events mediating cell differentiation, especially in human neural progenitor cells, remain poorly understood. We could show that Wnt-3a in contrast to stabilized beta-catenin increases the neuronal population in human neuronal progenitor cells (ReNCell VM) which suggests a beta-catenin independent mechanism. The Wnt pathway is also known to interact with a plurality number of different signaling pathways e.g. Notch. Preliminary results reveal a regulation of Notch target genes HES1 and HES5 by Wnt-3a, but not stabilized beta-catenin Since HES1 and HES5 are helix-loop-helix (HLH) transcription factors which are known to regulate the fate of neural stem cells, this cross-talk between Wnt and Notch and its impact on neuronal differentiation of human NPCs will be investigated further.

The thesis deals with the evaluation of novel small molecules as potential GSK-3β inhibitors within canonical Wnt signaling in human neural progenitor cells. The newly synthesised compounds are provided by Prof. Dr. Matthias Beller of the Leibniz Institute for Catalysis in Rostock and are analysed in multi-screening step. The first screening is an ELISA specific for β-catenin, the key component of canonical Wnt signaling. The second screen investigates the TCF-activity driven by the potential new inhibitors. Finally, the impact on the transcription of Wnt-specific target genes are examined. Follow-up studies deal with the possible influence of new GSK-inhibitors (their activation of canonical Wnt signaling) on neuronal differentiation of human neural progenitor cells. Follow-up studies deal with proliferation and neuronal differentiation of human neural progenitor which are linked to the activation of canonical wnt signaling and how they can be manipulated by novel GSK-3β inhibitors.