Phase

The first phase of space biology was characterized by very simple experiments, naive hypotheses, and rather primitive instrumentation. The most common of such simple but not trivial hypotheses said that "the behavior of a biological system is altered in space and in microgravity in particular". No better arguments were available at that time as there was no history of biological experiments in space. There were, however, old data from experiments performed almost 200 years ago in centrifuges showing that the development of plant seeds was altered at centrifugal forces higher than 1 g. A common speculation was that monocellular organisms like algae, protozoa, and single cells from multicellular organisms, humans included, would change their behavior when exposed to the weightless environment. The rationale for such speculation was that all living beings developed on Earth throughout millions of years under steady gravitational conditions. The lack of proper instrumentation in space and the limited knowledge of cellular signal transduction mechanisms1 allowed only simple experimental approaches at a time when the technologies and the products of genetic engineering were not yet developed. The consequence was that the focus of the experiments was on the determination of so-called end points2 of cellular

' The most important cell functions like mitosis, expression and secretion of specific products are controlled by "biochemical signals", mainly hormones, growth factors, cytokines, that are taken up by receptors located on the cell surface. Once a signal is recognized, its message is transmitted, i.e., transduced, into the cell to organelles. A major effect is a change of genetic expression which is triggered in the cell nucleus and that leads to cell differentiation.

2 The endpoint of signal transduction in a cell is preceded by several intermediate steps that are being identified with the modern techniques of molecular biology, such processes that followed an initial activation step. Examples are cell proliferation assays, light microscopy and electron microscopy observations of morphological changes, biochemical analysis of the secretion, and consumption of metabolites in the culture medium. A major problem was the lack of proper controls like onboard 1-g centrifuges3. A variety of organisms were flown in rather simple devices in batch containers. For example, Yuri Gagarin, the first human in space, carried containers with bacteria on board his space vessel Vostolc in 1961. I remember what one of the U.S. investigators of those days once told me. Containers with E. coli cells were installed inside the upper tip of a rocket to be launched on a cold winter day without any thermal control. To prevent freezing of the cells the wife of the investigator knitted a "pullover" that enveloped the rocket tip to protect the cells from freezing. One exception was the experiment called Woodlawn Wanderer, described in more detail below. This experiment flown on board Skylab in 1973 used a sophisticated fully automated instrument with culture medium exchange, microscope, time-lapse cinematic camera, and sampling device.

In spite of their simplicity, such experiments delivered important information indicating that indeed gravity interacts with critical cellular functions. This was the basis of the research conducted in the following years. Highlights of this period were the flights of two U.S. biosatellites in 1966 and 1967, of several Russian Cosmos-Bion biosatellites between 1974 and 1989, three missions on board Skylab in 1973-1974, and the first flight of Spacelab on board the Space Shuttle Columbia in 1983.

1.4 Phase Two

The second phase started in 1985 with the flight of Biorack on board the Spacelab D-l, a mission organized by the German space agency DFLVR4. Biorack was a multi-user facility developed in Europe by ESA (Figure 4-03).

as the Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and the microarray technologies. The RT-PCR permits to identify qualitatively and quantitatively the genes expressed following a specific signal perception and transduction.

3 A crucial part of every experiment is its control. For instance, it can be argued that effects detected in a space laboratory may be due not only to the exposure to 0 g and/or cosmic radiation but also to other factors of spaceflight like the launch acceleration (3-4 g on the Space Shuttle, 16 g on a sounding rocket), difference of the composition or the incubation temperatures between ground and space laboratory. Therefore, it is important to have controls under artificial gravity conditions at 1 g. This is achieved by means of centrifuges installed in the same incubator in which the static, i.e., the 0-g samples are kept.

4 DFVLR stands for Deutsche Forschungs- und Versuchsanstalt für Luft und Raumfahrt. Today, the name of the Agency is DLR.

This facility was exclusively dedicated to the study of small organisms like bacteria, slime moulds, fungi, small plants and animals, as well as single plant and animal cells. The main feature of Biorack was that nearly all experiments had inflight 1 -g controls in a centrifuge installed within the same incubator as the 0-g samples. Basic biology became a priority and it was the privilege of European scientists to be the first users of Biorack. After D-l, Biorack flew on board the Spacelab International Microgravity Laboratory missions (IML-1 in 1992, IML-2 in 1994) and three times on board SpaceHab in 1996-1997. Biorack was followed by other biological multi-user facilities like Biolabor flown in Spacelab D-2 in 1993, NIZEMI5, a slow rotating microscope flown in Spacelab IML-2, and Biobox, an automated incubation facility installed on Russian biosatellites.

This second phase began with more systematic investigations and is characterized by the transition from tests of naive hypotheses to investigations of molecular mechanisms at the cellular level. It is accompanied by studies on signal transduction on board sounding rockets delivering few minutes of microgravity. Large amounts of data were collected showing that even single cells undergo dramatic changes in microgravity. The discovery of dramatic

5 NIZEMI, for Niedergeschwindigkeit Zentrifuge Mikroskop, consists of a rotating microscope on which samples could be observed at 20-400x magnifications at accelerations from 0 g (static) to 200 g.

Figure 4-03. Biorack as installed in the Spacelab: two incubators (22°C and 37°C) above and below the glovebox provided the thermal environment and 1-g reference centrifuges for the experiments. A freezer accommodated frozen samples. A laptop computer was used as an interface for facility and experiment data. A camcorder allowed on-line video observation or recordings of the glovebox activities. Source ESA.

gravitational effects in mammalian cells, the progress of techniques like the reverse transcriptase polymerase chain reaction and of fluorocytometry with fluorescent monoclonal antibodies as markers, gradually directed the focus of the research towards the intermediate steps of intracellular signal transduction. Examples are the studies of the genetic expression of early oncogenes and the activation of the G-proteins-inositol-triphosphate or the protein kinase C pathways. A prominent task is the discrimination between direct and indirect effects of gravity, as we will review it below.

1.5 Phase Three

The third phase consists of experiments selected by the major national and international space agencies according the criteria established by international peers of scientists and based on the most actual trends and findings of basic research and applied technology. It started in 1996-1997 with three flights of Biorack during three Shuttle missions to the Russian space station Mir, followed by the Spacelab mission Neurolab in 1998.

Hopefully, this phase will continue in the next decade with investigations on sounding rockets and with the use of Biolab on board the ISS. There, the potential benefits of bioprocessing in space will be investigated in addition to basic research. The gap between the last flight of Biorack and the first flight of Biolab should have been bridged by Biopack (see Figure 3-10), a small and flexible instrument that was installed on board the Columbia STS-107 mission, which ended catastrophically on February 1st, 2003 (Figure 4-01).

A comprehensive review of the most important biological experiments of the first three phases has been presented in Moore and Cogoli (1996).

1.6 Phase Four

The fourth phase is beginning now and it is driven by two main factors. One is the technological and scientific knowledge achieved in thirty years of gravitational cell biology research in space and in machines providing vectorless gravity. The other is given by the costs of the space activities. In fact, the average price of an experiment on board a manned space laboratory is of the order of magnitude of ten million dollars. In order to justify such sums in front of the taxpayer, the space agencies are supporting the development of commercial applications on board the ISS. Such programs shall involve scientists from universities and non-aerospace industries interested in medical, biotechnological, and material applications.

One example is the Microgravity Application Program (MAP) of ESA. Several projects have been approved that confirm the industrial and scientific interest to make use microgravity as a new tool for research and development.

Ly so some

Endoplasmic reticulum

Goigi complex

Nucleus :

Nuclear envelope —

Chromatin

Micro tub ui es

Mitochondrion

Plasma membrane

Centrioies

Figure 4-04. A cell is a complex living system in which organelles of different densities perform specific vital functions.

Ly so some

Micro tub ui es

Mitochondrion

Endoplasmic reticulum

Plasma membrane

Goigi complex

Nucleus :

Nuclear envelope —

Chromatin

Centrioies

Figure 4-04. A cell is a complex living system in which organelles of different densities perform specific vital functions.

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