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ID: 545807.0, MPI für Dynamik komplexer technischer Systeme / Bioprocess Engineering
WPT7: Model Validation, Parameter Analysis and Experimental Design.

Part I: Parameter estimation and model invalidation using uncertain data.
Part II: Redox Signaling in the facultative photosynthetic bacterium Rhodospirillum rubrum.
Part III: Sensitive cycling assays for measuring key metabolic enyzme activities in MDCK cells.
Authors:Streif, S.; Findeisen, R.; Zeiger, L.; Grammel, H.; Janke, R.; Genzel, Y.; Reichl, U.
Name of Conference/Meeting:FORSYS/FORSYS Partner Status Seminar
Place of Conference/Meeting:Heidelberg, Germany
(Start) Date of Event 
End Date of Conference/Meeting 
Audience:Experts Only
Intended Educational Use:No
Abstract / Description:Model validation, parameter estimation and design of experiments are central, yet challenging problems that must be addressed in systems biology. This project consists of three sub-projects aiming to tackle these challenging problems from a theoretical and application side. It combines the expertise of three interdisciplinary research groups, the method and theory oriented group (Findeisen), a bioengineering focused group (Reichl), and a mainly biologically focused group (Grammel). Special focus is put on the model organisms Rhodospirillum rubrum (group Grammel) and MDCK cells (group Reichl), since one of these is a typical representative for a rather simple, yet not completely understood and modeled organism (Rhodospirillum rubrum) and the other is a complex organism (MDCK cells), often being very difficult to model and understand.
Theoretical developments: On the theoretical side new parameter estimation, model invalidation, and experimental design methods are developed, that provide conclusive/mathematically provable. The methods are set-based and employ suitable relaxation strategies [1,2]. They allow to cope with: the often unavoidable nonlinearities in the mathematical models; the appearing uncertainties and biological variability’s in the measurement; the fact that typically, even under stationary conditions, not all states, e.g. concentrations, of the considered biological system/model are directly experimentally accessible; the fact that some of the variables/inputs/stimuli’s appearing might be discrete by nature, such as genetic switches, possible genetic modification in experiments, discrete valued stimuli applied to the process.

Rhodospirillum rubrum: In previous studies we found evidence that the formation of photosynthetic membranes in R. rubrum is controlled by a highly cooperative mechanism in response to redox states of electron transport chain components [1,2]. However, direct experimental demonstration of cooperative (or bistable) photosynthetic gene expression is still lacking [3,4]. During the present project, by monitoring gene expression at the transcriptional level, the signal-response functions of key genes are being determined. For reliably adjusting specific redox conditions (aerobic, microaerobic) in bioreactor cultivations, an unstructured redox process model was developed based on batch experiments and used for parameter estimation.

Madin-Darby canine kidney (MDCK) cells: Adherent MDCK cells have been shown to produce large amounts of lactate and ammonium. A promising approach to reduce this inhibiting by-product formation in mammalian cell cultures is to replace glutamine in the medium by pyruvate [5]. In glutamine-free medium with pyruvate as carbon source, MDCK cells not only released no ammonia during cell growth but glucose consumption and lactate production was also reduced significantly. However, concerning the interpretation of experimental data and corresponding flux distributions, still some open questions remain. To address these questions an enzyme platform has been established that uses four sensitive enzymatic cycling assays to determine 28 key enzyme activities of central carbon metabolism in mammalian cells [6,7]. Cell extracts could therefore be highly diluted, which (i) minimized interferences from other components (enzymes or inhibitors) in the extract, (ii) minimized over- and underestimates of actual enzyme activities as substrate concentration could be maintained at a near constant level (which, in addition, minimized product inhibition), and (iii) allowed to detect low enzyme levels.

[1] P. Rumschinski, S. Borchers, S. Bosio, R. Weismantel, and R. Findeisen. Set-based dynamical parameter estimation and model invalidation for biochemical reaction networks. BMC systems biology, 4:69, 2010.
[2] S. Borchers, S. Bosio, R. Findeisen, U. Haus, P. Rumschinski, and R. Weismantel. Graph problems arising from parameter identification of discrete dynamical systems. Mathematical Methods of Operations Research, 2011.
[3] Grammel, H. and R. Ghosh. 2008. Redox state dynamics of ubiquinone-10 imply cooperative regulation of photosynthetic membrane expression in /Rhodospirillum rubrum/. J. Bacteriol. 190 (14), 4912-4921.
[4] Klamt, S., H. Grammel, R. Straube, R. Ghosh, and E.D. Gilles. 2008. Modeling the electron transport chain of purple non-sulfur bacteria. Mol. Syst. Biol. 4:156
[5] Genzel, Y., Ritter, J.B., König, S., Alt, R., Reichl, U., (2005). Substitution of glutamine by pyruvate to reduce ammonia formation and growth inhibition of mammalian cells. Biotechnol Prog 21, 58-69.
[6] Janke, R., Genzel, Y. Wahl, A. & Reichl, U. (2010). Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate-based assays. Biotechnology and Bioengineering, Vol. 107, No. 3, 566-581.
[7] Janke, R., Genzel, Y., Freund, S., Wolff, MW., Grammel, H., Rühmkorf, C., Seidemann, J., Wahl, A., Reichl, U. (2010). Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells. Journal of Biotechnology 150, 396–403.
Document Type:Poster
Communicated by:Udo Reichl
Affiliations:MPI für Dynamik komplexer technischer Systeme/Systems Biology
MPI für Dynamik komplexer technischer Systeme/Bioprocess Engineering
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