Previous investigations on mammalian cells have shown that real microgravity of space and/or simulated microgravity causes severe cellular modifications. One of the most prominent changes occurring under low gravity concerns morphological alterations which involves cytoskeletal reorganization.

The aim of this work was to use Digital Holographic Microscopy (DHM) on the Random Positioning Machine (RPM) to study morphological evolution of cells under simulated microgravity in real time. DHM is a very innovative imaging technique, specially designed to retrieve the topography of objects. It provides quantitative phase images which can be directly related to the optical path length (OPL) induced by the object, and thus it is particularly suitable to investigate cytomorphology.Furthermore real-time DHM images taken on the RPM allow to dynamically follow the processes related to microgravity-induced cellular changes.

Our novel technique was applied on mouse myoblast cells (C2C12), transfected with GFP-tagged actin and exposed to microgravity. Phase images recorded by DHM on living cells were compared to results obtained by fluorescence microscopy on fixed cells. The results have revealed that two major modifications of the cell morphology under microgravity take place: the appearance of disorganized structures in the lamellipodia and a perinuclear accumulation of actin filaments. Our novel setup allows a three-dimensional study of living cells exposed to simulated microgravity. Real-time changes of single cells can be observed during a long time period (several hours). Moreover, the method is non-invasive and no fixation of cells, which may produce undesired effects, is necessary for the investigation. To our knowledge, this is the first study showing time series of three-dimensional images of living cells being exposed to microgravity. The next developmental step is to add a widefield fluorescence channel to the present DHM system, which will vastly increase the scientific output of the studied cells.

Actual constraints as late access to space engines, preparation of samples far away from launch sites or delay between launch and transfer of samples on ISS have or will often lead cell biologists to keep cells at a so-called "pausing" temperature (22-25°C) before starting the experiments. The advantage of this temperature is to maintain cells in a non-proliferative and quiescent state, and is believed to keep them in a "sleeping mode" that would make them insensitive to vibrations and high gravity levels during transportation and launch. We analysed the effect of temperature transition from 25°C to 37°C on a number of genes involved in stress response. WI26 cells were maintained for 1, 2, 3 and 5 days at 25°C followed by a raise of the temperature to 37°C. Proteins and RNA were extracted from cells harvested after 1, 2, 4, 8 and 24 hours at 37°C.

The temperature transition induced phosphorylation of two stress-related kinases, JNK (c-Jun-N-terminal kinase) and p53, coupled with a decreased phosphorylation of the survival protein AKT. The level of HSP70, IL-6, IL-8 and IL-1â mRNA was up-regulated, with different kinetics after rising the temperature. Both the Bcl-XS/Bcl-XL ratio and the expression of a novel VEGF variant (Mineur et al., J. Cell Biol., 2007) VEGF111 showed a time-dependent increase. These results indicate that storage at 25°C and warming up to 37°C induce a cellular stress that regulates their survival, gene expression and pre-mRNA splicing. Moreover, the phosphorylation of p53 and the expression of VEGF111, known to be induced by genotoxic agents, suggest that the temperature transition might result in DNA damages. These effects should be considered before designing space experiments and for data interpretation.

Energetic heavy ions in galactic cosmic rays provide a major contribution to dose equivalent in space, but there is a high uncertainties on their biological effectiveness. It is well known that heavy ions are more effective than sparsely ionizing radiation in the induction of genetic damage, but a large fraction of such lesions are lethal, and cannot eventually contribute to late effects. We have recently concentrated on residual damage in human cells exposed to heavy ions, in an attempt to single out the genetic rearrangements responsible for late effects of cosmic radiation. Human peripheral blood lymphocytes were exposed in vitro to 1 GeV/n Fe-ions produced at the NASA Space Radiation Laboratory at the Brookhaven National Laboratory. Cells were harvested at 144 h from exposure, when the cell population is mostly composed by cells at the 3rd or higher post-irradiation mitosis, i.e. the progeny of the exposed cells.

Chromosomal aberrations were analyzed using arm-specific mFISH. The results demonstrate that the relative biological effectiveness of heavy ions for chromosome damage decreases in the progeny of exposed cells compare to 1st cell-cycle, and is close to 1 for most aberration-type.

However, heavy ions are powerful inducers of chromosomal inter-interchanges, i.e. rearrangements involving both inter- and intra-exchanges. This aberration type could be a fingerprint of exposure to heavy ions.

Experiments were carried out to identify the effects of spaceflight on Streptomyces lividans, a bacterium that is widely used in gene expression studies. A strain carrying plasmid pIJ702 which includes genes that encode for thiostrepton resistance (tsr+ phenotype) and melanin production (mel+ phenotype) was plated on agar and flown on the Russian satellite Foton-M3 for 16 days. The mel+ phenotype requires an intact tyr gene that is carried on the plasmid. Culture plates were flooded with sufficient water, filtered with glass wool to collect spores from filaments and 50% glycerol added before storing. Bacteria were then spread plated on selective differential media of 0.5 TSA supplemented with CuSO4, L-tyrosine (to promote pigment production) and, with and without, thiostrepton (for thiostrepton sensitivity) will be used. To determine plasmid loss rate, 100ƒÝl of appropriate dilution was spread plated to both TSA and TSA with thiostrepton plates. The colonies yielded in TSA plates will produce total number of spores with or without plasmid and TSA with thiostrepton plates will contain only spores that retained the plasmid needed to survive in medium with thiostrepton added. Aliquots of diluted spore suspension were spread plated for recovery on the selective differential media at a cell density that will permit differentiation of the colony phenotype; brown (melanin producing) vs. white. The percentage loss of plasmid expression in flight samples (6.2% out of 860) was lower than that in ground samples (59.86% out of 437) when both samples were grown in ISP media. When comparing if the difference in media content would cause different percentage in loss of plasmid expression, it was observed that samples in ISP media (59.86% out of 437) have a higher loss of plasmid expression than samples in minimum media (20.21% out of 1052). From the results obtained, we were able to determine that spaceflight cultures when compared to ground controls, microgravity resulted in the increased retention of plasmid pIJ702 by S. lividans. The results for screening of thiostrepton resistant white (tsr+ mel-) mutants show that there are slightly more mutants in the ground samples (0.07%, 4 out of 5807) than the flight samples (0.03%, 1 out of 3347). This result is interesting as we were expecting there to be more mutants in the flight samples but it turned out otherwise. The non-pigmented mel- phenotype that is easily distinguished from the darkly pigmented wild-type on a differential media may result from mutation(s) in the tyr gene on the plasmid. To confirm this, DNA extracted from flight and control non-pigmented mutants is being amplified and the tyr gene sequenced to identify the mutations resulting in loss of pigmentation. By understanding spaceflight effects on the genetic stability of these bacteria, we will be better able to quantify possible spaceflight threats posed to the health of astronauts that are exposed to long periods of spaceflight conditions.

Small fish models, mainly zebrafish (Danio rerio) and medaka (Oryzias latipes), present many advantages for studying development, such as transparency of the embryos, external development, possibility for large scale mutagenesis screening, and rapid development. Similarly, their use is increasingly accepted for applications in toxicology or pharmacology, due to the similarity of many physiological processes to those in mammalians. Further characteristics, e.g. large number of embryos from one single clutch, small size, easy containment in water tanks and utilization of existing instrumentation, are particularly useful for space research. In recent years, many technologies for visualizing and characterizing bones, such as specific staining or fluorescent transgenic animals, have been adapted to these fish and can be routinely performed on large numbers of larvae. Finally, and most importantly, the genome sequencing and annotation is close to completion for these two species.

Due to this huge advancement, we now have access to all the technologies of the genomic age for studying these species. Our aim is to investigate the changes induced by microgravity in small fish species by combining several whole genome approaches, with a special emphasis on bone related genes. We use DNA microarrays to analyze the expression of 16 000 genes deduced from the zebrafish genome annotation, complemented by some 200 genes especially selected for their involvement in bone metabolism. We chose to analyze larvae at 5 days post fertilization, because at this stage the mineralization of bone structures from existing cartilage begins. We compare gene expression at the whole genome level between control zebrafish larvae and larvae treated with bone-destructing or bone-constructing drugs for one day, in order to identify crucial signalling events regulating bone formation. These results are compared to those obtained after submitting larvae for 1 day to on ground microgravity simulation. Clinorotation, random positioning machine (RPM) and rotating wall vessel (RWV) device were used for these simulations. Furthermore, expression of several candidate genes was analyzed in medaka upon drug treatment and clinorotation using quantitative RT-PCR.