1. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Vitamin D R. Bouillon - G. Carmeliet Legendo K.U. Leuven Herestraat 49, 3000 Leuven tel: 32-16-34.60.23 e-mail : geert.carmeliet@med.kuleuven.ac.be

2. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Introduction: normal bone metabolism osteoblastosteoclast mineralized matrix osteoid bone resorptionbone formation equilibrium osteocyte

3. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Introduction: spaceflight induces bone loss 0 -2 -4 -6 % difference from control resorption bone mass formation 123 weeks

4. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Introduction: spaceflight decreases bone formation proliferation bone marrow stromal cell preosteoblast   + osteoclast mineralized matrix   osteoid UNLOADING

5. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Introduction: spaceflight induces changes in osteoblast characteristics extracellular matrix growth factors growth factor receptors ?  change in gravity change in adhesion change in gene expression

6. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity AIM of microgravity project characterization of effect of microgravity on gene expression by osteoblasts identification of effect of microgravity on intracellular pathways

7. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Model: cellular mechanisms previous MG63 cells: human osteosarcoma cell line stimulus: vitamin D + Transforming Growth Factor  endpoints:change in matrix proteins, growth factors presentdifferent species, correlation with transgenic mice MC3T3-E1 cells:mouse osteoblastic cells stimulus:vitamin D endpoints:matrix proteins, growth factors, metabolism

8. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Model: molecular mechanisms VDR GENE TRANSCRIPTION NUCLEUS VDR interaction of vitamin D receptor (VDR) and DNA: stable cell line with reporter construct Vitamin D DNA

9. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Technical outline : cell culture unit M M L LS M MC3T3-E1 cells on thermanox slide M: medium S: stimulus L: lysis

10. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Technical outline: manipulations and timeline 1 1 = Crew activity Transfer CTA from Soyuz to ISS VITA 1 VITA 2 22°C 0xg 2 Set CTA temperature to +37°C 3 Activate 4 plungers 4 Medium Exchange (4 plungers) 22°C >> 37°C37°C 48 h Activator unit 37°C 72 h 5 Cell lysis (4 plungers) Activator unit CTR Chemicals used inside the plunger boxes and Type II/E containers: 1) Activation: αMEM medium + 10% FCS 2) Medium exchange: αMEM medium + 10% FCS + 1,25- dihydroxyvitamin D3 10 -8 M 3) Termination: Buffer (hypotonic) t 0 = start of exp. timeline 2 EC’s Type II/E stored at +22°C in CTA for 3 days (tbc)

11. e Insert Experiment Name Belgian Taxi Flight - Life Science Background Training B.USOC, Brussels11. - 12.03.2002 Directorate of Manned Spaceflight and Microgravity Benefits insight in the effects of spaceflight on osteoblast function better prevention of bone loss in astronauts insight in fundamental process of normal (cell) physiology: mechanisms by which bone responds to mechanical (un)loading influence treatment of other etiologies of osteoporosis