RAMIROS

1. Philippe Baert, Geert Meesen, Dr. Patrick Van Oostveldt, Dr. Andre Poffijn Ghent University Dept. of Molecular Biotechnology Coupure Links 653, 9000 Gent Dept. of Subatomic & Radiation Physics Proeftuinstraat 86, 9000 Gent RAMIROS

2. Experiment name Experiment Team/University/Sponsoring Agency Scientific Background Proposed Experiment Experiment Hardware Experiment Protocol Benefits to Science/Mankind Lesson outline / Contents

3. “RAMIROS” Radiation And Microgravity Related Oxidative Stress Experiment Name

4. Dr. P. Van Oostveldt, Ph. Baert Dept. of Molecular BiotechnologyCoupure Links 653B-9000 GentTel: +32 9 264 59 69Fax: +32 9 264 62 19E-Mail: Patrick.VanOostveldt@rug.ac.be, Philippe.Baert@rug.ac.be Dr. A. Poffijn, G. Meesen Dept. of Subatomic and Radiation Physics (Radiation and Environmental Physics group)Proeftuinstraat 86B-9000 GentTel: +32 9 264 65 40Fax: +32 9 264 66 97E-Mail : Andre.Poffijn@rug.ac.be, Geert.Meesen@rug.ac.be Contract grant sponsor: ESA Experiment Team/University/Sponsoring Agency

5. Cosmic radiation Primary radiation : Trapped particle radiation (TR): protons and electrons Galactic cosmic radiation (GCR):85% protons14% He 1% HZE’s (Charged Particles with Atomic Number > 2; peak for 26Fe) Solar particle events (SPE)protons, electrons Secondary radiation : Brehmstralung radiation Neutrons Scientific Background

6. Very little human radio-epidemiology data on bio-effects of high-energy charged particle radiation Radiation induces DNA damage through direct ionisation or indirectly through the generation of reactive oxygen species Acute and long term effects of radiation; DNA damage is a starting point for: senescence, ageing cancer Scientific Background

7. AIM: To analyse the biological effects of heavy particle (HZE) radiation on primary mammalian tissue in space, in order to understand how single cells and their environment deal with HZE impact, with a contribution to radiation safety guidelines for human space activities in mind. Proposed Experiment

8. Confluent murine bone marrow culture on top of HZE track detector stack Co-localisation of tracks and hit cells Analysis of tracks (HZE characterisation) with corresponding effect/response both at DNA and protein level in the single cell level and its microenvironment Proposed Experiment

9. Use of high contrast two photon/confocal fluorescence microscopy enabling 3D imaging of bothtrack detector and biology Irradiation Etching Damage trail (Latent track) SSNTD HZE particle Proposed Experiment - Physics

10. Length and direction of tracks by confocal sectioning : information about particle type and which cell is hit. Proposed Experiment - Physics

11. Use of high contrast two photon/confocal fluorescence microscopy enabling 3D imaging of both track detector and biology DNA adducts and localization/expression of protein targets Immunochemical methods for detecting: DNA single and double strand breaks, modified DNA bases, DNA-repair machinery Proposed Experiment - Biology

13. Proposed Experiment - Biology • Irradiation induced DNA lesions • single or double strand breaks • base modifications • destruction of sugars • base dimer formation • need for efficient DNA repair, if not: • less efficient gene reading, senescence (ageing) features • DNA mutations • Cancerous transformation (late effect)

14. Track position linked with hit cells and immediate surroundings called bystander effects To be linked with information of HZE (SSNTD’s) and bulk radiation (TLD’s) ~25 HZE hits/day randomly distributed throughout the 800 mm² cell culture plane Proposed Experiment - Biology

15. Semi automated image acquisition and processing co-localization of tracks and cells image enhancement (deconvolution) DNA damage and protein expression profiles track parameter extraction : length direction Proposed Experiment Dedicated software routines

16. Experiment Hardware • CCM based concept for plunger activation • wet and dry compartment for biology and • physics respectively • 3 plungers/plungerbox • 2 fluids: -MEM, 0.5% Formaldehyde

17. Experiment Hardware

18. 1st level of containment: plunger module 2nd level of containment: Type II/E or CIS container (flushed with CO2) Scenario: 4 plungerbox units in 2 Type II/E containers (excluding electrical activation unit) - mass: 200 g x 2 x 2 = 0.8 kg - volume: 595 ml x 2 = 1.190 dm³ 3th level of containment? Experiment Hardware

19. Prior to operations: passive (if ambient T° is close to 20°C) or Aquarius CTA (< 27 W) Transfer Aquarius CTA from Soyuz to ISS At ISS (T0): cosmonaut should activate the time line for plunger activation and set 37°C condition for Aquarius (<27 W) first plunger activation: T0 +2.5 days second (fixative) and third plunger (detector shift) activation: T0 +5 days set 5°C Experiment Protocol

20. = Crew activity Experiment Protocol t0 =start of exp. timeline Set CTA temperature to +37°C Activate 4 plungers Fixation and Detector shift (2x4 plungers) Transfer CTA from Soyuz to ISS 1 2 3 4 22°C 0xg 22°C >> 37°C, 60 h 37°C 60-84 h RAMIROS 1 RAMIROS 2 CTR CTR 2 EC’s Type II/E stored at +22°C in CTA for 3 days (tbc) Activator unit Activator unit 1 Chemicals used inside the plunger boxes and Type II/E containers: 1) Activation: αMEM medium 2) Fixation: 0.5% Formaldehyde

21. Post-operations: transfer 2 type II/E containers to CTR Transfer CTR from ISS to Soyuz Experiment Protocol

22. This research will help : 1) to refine the knowledge about the space radiation environment 2) to better understand fundamental biological processes such as the occurrence of different DNA lesions (hallmarks of cancer and ageing) under particular conditions 3) to better understand the relation between radiation and biological damage (long duration space flights, heavy particle radiation therapy) Benefits to Science/Mankind

23. Single cell analysis procedure for HZE impact Experiment could be self-supporting Summary