1. Computational Investigation of Intracellular Networks Centre for Medical Systems Biology CENTRFORINTEGRATIVE BIOINFORMATICSVU E

2. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Agenda Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary

3. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Agenda Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary

4. Gene 1 Gene 2 Phenotype 1 Gene 3 Gene 4Phenotype 1a Phenotype 2 Gene 1 Gene 3

5. Interfaces of Components May Be Damaged during Isolation Intact System Isolation of Components Interfaces Damaged during Isolation

6. Study System at Multiple Scales Molecules Form Pathways Pathways Form Cells Cell Types Form Organs or Ecosystems

7. Widespread RNA transcription found in ENCODE project : Nature, June 2007 "Instead of running errands, RNA appears to be running the whole show," said Isidore Rigoutsos, a lead scientist at IBM's Thomas J. Watson Research Center RNA transcription Do Not Forget the RNA Level

8. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Agenda Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary

9. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Results Rat Bone load vs control C. Reijnders, N. Bravenboer, J van Beek, P. Lips et al.

10. Bone : Differentially expressed genes 6 hours after mechanical loading Gene name Fold change P adj value Biological connection mepe osteoregulin OF45 1.830.054 skeletal development; regulation of bone remodeling; negative regulation of bone mineralization garnl1 tulip 1 1.420.054regulation of transcription creatine kinase, muscle form -2.620.065phosphocreatine biosynthesis and metabolism fibrinogen B beta chain-2.270.074 blood coagulation; wound healing; regulation of blood pressure; positive regulation of cell proliferation putative pheromone receptor V2R2B 1.600.094 related to Ca 2+ -sensing receptor and metabotropic glutamate receptors monoamine oxidase A- 4.000.094 behavior; catecholamine catabolism and metabolism; dopamine metabolism; electron transport; neurotransmitter catabolism troponin-c-3.900.094Ca 2+ -bindingsubunit of the troponin complex QFG-TN1 olfactory receptor 1.460.145 G-protein coupled receptor protein signaling pathway; perception of smell, sensory transduction of chemical stimulus kinesin light chain C-2.060.160unknown 3 significantly down-regulated genes: p21 (c ‑ Ki ‑ ras), amino acid transporter system A (ATA2), and phosphate regulating neutral endopeptidase on the X chromosome (PHEX) Dual Channel Single Channel

11. CENTRFORINTEGRATIVE BIOINFORMATICSVU E PHEXproteases (cathepsin B) MEPE- ASARM ASARM + MEPE Enhances mineralization Inhibits mineralization Role creatine kinase and troponin-C yet unknown Changes after loading a bone Local change in loaded bone Change in all bones

12. Signaling Pathway Map: Epidermal Growth Factor (Kitano) Signaling Pathway Map: Concept Map: MAPK (Van Kampen)

13. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Agenda Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary

14. The Airport Example What do we need to know to determine throughput? Neither the number of airplanes on the ground... … nor the map of the runways... is very helpful

15. ... but the “radar flux” is informative The Airport Example What do we need to know to determine throughput?

16. Carbon Transition Networks (CTNs) Thomas Binsl

17. Simulation of glutamate NMR multiplets due to carbon-13 isotope enrichment during infusion of labeled acetate 4-carbon singlet 4-carbon doublet NMR Spectrum 4-carbon glutamate

18. In Vivo Metabolic Rates Estimated from 13 C NMR Spectrum TCA cycle flux = 7.7 µmol/g/min (n = 60) glutamate content 24.6 µmol/g Transport time 29.8 sec (n=7) 58 % acetyl CoA from infused acetate (n=36) Anaplerosis 16 % of TCA cycle flux (n=19) Transamination 17.4 µmol/g/min (n=9) TCA cycle

19. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Agenda Systems Approach (components and interactions) Bioinformatic and Statistical Analysis (top-down) Metabolomics and Fluxomics (metabolic flux measurements) Mechanistic Modeling : modular and multiscale (bottom-up) Summary

28. Switching from Google Earth, To “Google Human” To “Google Heart” Computational Model of Essential Parts of Molecular System

29. Big Model Many Trees, Forest Visible? Small is Beautiful Well Maintained Garden The K.I.S.S. Principle ("Keep It Simple, Stupid") or ("Keep It Short and Simple") Boehringer Pathways

30. ATPase Pi ATP ADP MM-CK PCrCr Cytosol MOM MIM PCr Mi-CK Cr ATPADP Intermembrane Space Pi ADP ATP OxPhos Model of High Energy Phosphate Group Handling in Energy Metabolism Mitochondrial Matrix ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). Model Elements : two creatine kinase (CK) isoforms diffusion & membrane permeation Communicating Modules: ATP consumption (ATPase) as forcing function ATP production by mitochondria

31. ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). Model Elements : two creatine kinase (CK) isoforms diffusion & membrane permeation Communicating Modules: ATP consumption (ATPase) as forcing function ATP production by mitochondria ATPase Pi ATP ADP MM-CK PCrCr Cytosol MOM MIM PCr Mi-CK Cr ATPADP Intermembrane Space Pi ADP ATP OxPhos Model of High Energy Phosphate Group Handling in Energy Metabolism Mitochondrial Matrix Border of the module

32. ATPase Pi ATP ADP MM-CK PCrCr Cytosol MOM MIM PCr Mi-CK Cr ATPADP Intermembrane Space Pi ADP ATP OxPhos Mitochondrial Matrix ? ? Border of the module ADP and inorganic phosphate (Pi) enter intermembrane space and stimulate oxidative phosphorylation (OxPhos). Model Elements : two creatine kinase (CK) isoforms diffusion & membrane permeation Communicating Modules: ATP consumption (ATPase) as forcing function ATP production by mitochondria Model of High Energy Phosphate Group Handling in Energy Metabolism

34. Ingredients for this Simulation Dynamics of response O 2 uptake at whole heart level Transport time blood vessels and diffusion Decrease total O 2 amount in whole heart Increase O 2 uptake heart NMR determined diffusion coefficients Mitochondrial Membrane Permeability Steady state responses isolated mitochondria Enzyme kinetics of two enzymes = Outer Membrane Permeability for ADP = 21  m/s

35. Our model analysis implies that in rabbit heart at 487 μM/sec ATP hydrolysis, the diffusion flux carried by PCr is 154 μM/sec. This suggests that the PCr shuttle is of minor importance. But what is the function of the two creatine kinase isoforms?

36. Effect of creatine kinase expression level on metabolic oscillations and on average concentrations

37. Pictorial Summary Modular Modeling, Using Information from Multiple Scales, makes Systems Accessible to Deep and Wide Analysis Using stable isotopes to trace networks

39. Networks of Correlation Clish, Van der Greef, Naylor OMICS Vol. 8, 2004

40. Acknowledgments Thomas Binsl Kate Mullen Olav Kongas Mark Noble Frits Prinzen Joli Bussemaker David Alders Johan Groeneveld Lori Gustafson Koert Zuurbier Jan Bart Hak Glenn Harrison Marcel Eijgelshoven Bas de Groot Jaap Heringa Ivo van Stokkum Hans van Beek

41. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Threshold False Discovery Rate (Benjamini-Hochberg Linear Stepup) Rank Order P value Linear Stepup Threshold FDR = 20%

42. Time [min] FluxSimulator (Simulation Results) Isotopomer Fraction Time (sec) 0100200 0 1 0 1 Metabolite B Metabolite D

43. Oxygen Consumption by Blood Gas (μmol / g dry /min) Oxygen Consumption by NMR Method (μmol / g dry /min) COMPARING THE NEW NMR METHOD WITH OXYGEN CONSUMPTION MEASURED BY ‘GOLD STANDARD’ (BLOOD GAS) n = 42 pigs

44. ATP ADP PCr MitochondriaMyofibril creatine kinase Module : set of molecular processes performing a cellular function Functions of the adenine nucleotide-creatine-phosphate module: 1.transfer high-energy phosphate bonds from mitochondria to cytosolic ATPases 2. dynamic adaptation of ATP synthesis to time-varying hydrolysis 3.emergency buffer system Adenine Nucleotide – Creatine - Phosphate Module

45. Predicted oscillation of ADP in cytosol for low permeability of mitochondrial outer membrane Membrane Permeability 0.16  m/s (Vendelin, Saks et al. 2000) ADP levels much too high Response much too slow

46. Isolating the Module under Study Experimentally Response measured of venous oxygen in mouse heart Activation time of oxidative phosporylation 3.7 s … and Measuring the Response of the System as a Whole

47. The response is simplified by removing pulsatility from the ATP hydrolysis forcing function 5 1 3 4 2 Membrane Permeability 1 =   m/s (for sake argument) 2 = 85  m/s (Beard 2006) 3 = 21  m/s (optimised t mito = 3.7 s) 4 = 3.5  m/s (Saks 2003) 5 = 0.16  m/s (Saks 2000)

48. Partitioning the Contributions of Mitochondrial and Muscle CK Isoforms experiment: normal CK experiment: both CK isoforms inhibited MM-CK changes Mi-CK changes Both CKs change

49. Time varying load on the mitochondria at two levels: First level: ATP hydrolysis in beating heart muscle pulsates during each heartbeat ATP Hydrolysis (μM/s) ATP Synthesis (μM/s) Time ( s ) ATP Hydrolysis (μM/s) Time ( s )

50. At t=0 paced heart rate is stepped from 135 to 220 beats/min Time varying load on the mitochondria at two levels: Second level: Heart rate is variable

51. Metabolites in the Same Biochemical Reaction Often Correlate Poorly ATP Level (mM) Phosphocreatine (mM) Attack Stay in Pack

52. J. van der Greef (CMSB) False discovery rate of edges potentially VERY high Centre for Medical Systems Biology

53. Simulation of glutamate NMR multiplets due to carbon-13 isotope enrichment during infusion of labeled acetate 4-carbon singlet 4-carbon doublet time amplitude TCA cycle

54. Systems Consist of Components

55. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Results for Bone : load vs control

56. ATPase Pi ATP ADP MM-CK PCrCr Cytosol MOM MIM PCr Mi-CK Cr ATPADP Intermembrane Space Pi ADP ATP OxPhos Mitochondrial Matrix Outer Membrane Permeability for ADP 21  m/s (tmito optimised to 3.7 s) Diffusion in Cytosol characteristic diffusion time about 0.35 millisec Outer Membrane Permeability Determined from Functional Response In Vivo Estimation !

57. File 3 FluxSimulator (Network Specification)

58. CENTRFORINTEGRATIVE BIOINFORMATICSVU E Organism 1 Organism 2 Environmental State 1 Organism 4 Environmental State 1a Environmental State 2 Organism 1 Organism 3 Ecological System