EFFECTS OF ALTERED GRAVITY ON INSECTS, PLANTS AND THE HUMAN CARDIOVASCULAR SYSTEM A THESIS SUBMITTED TO THE UNIVERSITY OF PUNE FOR THE AWARD OF THE DEGREE OF
DOCTOR OF PHILOSOPHY IN
PHYSICS BY
SANTOSH BHASKARAN UNDER THE GUIDANCE OF
PROF. P.B.VIDYASAGAR Department of Physics, UNIVERSITY OF PUNE MAY 2008
Certificate This is to certify that the work presented in this thesis entitled, “Effects of Altered
Gravity on Insects, nsects, Plants lants and the Human Cardiovascular System”, submitted by Mr. Santosh Bhaskaran to the University of Pune for the award of Doctor of Philosophy in Physics, was carried out by the candidate under my supervision. Such material obtained from other sources has been duly acknowledged in the thesis.
(P.B. Vidyasagar) Guide
Professor Biophysics Laboratory Department of Physics University of Pune
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Declaration I hereby declare that the present thesis entitled, “Effects of Altered Gravity on
Insects, Plants and the Human Cardiovascular System” is an account of the original work carried out by me. This work or part of the work thereof has not been submitted to any other University or Institution for the award of any degree/ diploma.
(Santosh Bhaskaran)
Candidate
Forwarded through:
(P.B. Vidyasagar) Guide
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Table of Contents Certificate........................................................................................................ii Declaration .................................................................................................... iii List of Abbreviations ....................................................................................vii Abstract ........................................................................................................viii Acknowledgements ........................................................................................ xi Chapter 1: Introduction ............................................................................... 14 1.1
Background ...................................................................................................... 14
1.2
Gravitation ........................................................................................................ 15
1.2.1
Weightlessness and microgravity ...................................................................................... 17
1.2.1.1 1.2.1.2 1.2.1.3
1.3
Weightlessness in an elevator ........................................................................................ 17 Weightlessness in a spacecraft ....................................................................................... 18 Microgravity ..................................................................................................................... 19
Impact of gravity on life ................................................................................. 28
1.3.1
Cells ..................................................................................................................................... 31
1.3.2
Plants ................................................................................................................................... 43
1.3.3
Invertebrates ....................................................................................................................... 47
1.3.4
Vertebrates .......................................................................................................................... 48
1.3.5
Humans ................................................................................................................................ 52
1.4
Thesis Outline .................................................................................................. 56
Chapter 2: Clinostat and Clinostat-Centrifuge......................................... 60 2.1 2.1.1
Introduction ..................................................................................................... 60 The Simulation of Microgravity........................................................................................ 61
2.2
Instrumental Design of Clinostat................................................................... 62
2.3
Testing of clinostat .......................................................................................... 64
iv
2.3.1
Introduction ......................................................................................................................... 64
2.3.2
Materials and Methods ...................................................................................................... 68
2.3.3
Results .................................................................................................................................. 68
2.3.4
Discussion............................................................................................................................. 71
2.4
Instrumental Design of Clinostat-Centrifuge .............................................. 72
2.5
Conclusions ....................................................................................................... 74
Chapter 3: Effects of Altered Gravity Environments on Plants ............... 76 3.1
Introduction ...................................................................................................... 76
3.2
Initial studies on the nature of graviresponse ................................................. 77
3.2.1
Materials and Methods ...................................................................................................... 77
3.2.2
Results .................................................................................................................................. 77
3.2.3
Discussions ........................................................................................................................... 81
3.3
Preliminary experiments on the clinostat ....................................................... 82
3.3.1
Materials and Methods ...................................................................................................... 82
3.3.2
Results .................................................................................................................................. 82
3.3.2.1 3.3.2.2 3.3.2.3
3.3.3
3.4
Direction of roots and shoots............................................................................................ 82 Growth of roots and shoots............................................................................................... 83 UV-Vis Spectra .................................................................................................................. 85
Discussion ............................................................................................................................ 87
Experiments with the clinostat for longer periods ......................................... 87
3.4.1
Materials and Methods ...................................................................................................... 87
3.4.2
Results .................................................................................................................................. 88
3.4.2.1 3.4.2.2 3.4.2.3
3.4.3
3.5
Direction of roots and shoots............................................................................................ 88 Growth of roots and shoots............................................................................................... 88 Chlorophyll content .......................................................................................................... 89
Discussion............................................................................................................................. 90
Conclusions....................................................................................................... 91
Chapter 4: Effects of Gravity on the Human Cardiovascular System .. 93 4.1
Introduction ..................................................................................................... 93
4.2
Modelling the Cardiovascular System .......................................................... 94
4.3
Initial Experiments .......................................................................................... 99
4.3.1
Materials and Methods ...................................................................................................... 99
v
4.3.2
4.4
Results and Discussion ..................................................................................................... 100
Later Experiments ......................................................................................... 102
4.4.1
Materials and Methods .................................................................................................... 102
4.4.2
Results and Discussion ..................................................................................................... 103
4.5
Design and Development of Tilt-Table ....................................................... 107
4.6
Experiments on Tilt-Table ........................................................................... 109
4.6.1
Materials and Methods .................................................................................................... 109
4.6.2
Results and Discussion ..................................................................................................... 110
4.7
Program for ECG Analysis .......................................................................... 117
4.7.1
Introduction ....................................................................................................................... 117
4.7.2
Materials and Methods .................................................................................................... 117
4.7.3
Results and Discussion .................................................................................................... 118
4.8
Conclusions..................................................................................................... 123
Chapter 5: Conclusion and Future Scope ................................................ 126 5.1
Summary ......................................................................................................... 126
5.1.1
Studies on insects ............................................................................................................. 126
5.1.2
Studies on plants .............................................................................................................. 127
5.1.3
Studies on humans ........................................................................................................... 128
5.2
Conclusions ..................................................................................................... 134
5.3
Future Scope ................................................................................................... 134
Bibliography .......................................................................................... cxxxvi List of Publications ................................................................................... clviii List of Conferences Attended/Presented................................................... clix
vi
List of Abbreviations ANS
Autonomous Nervous System
BP
Blood Pressure
Chl
Chlorophyll
CNS
Central Nervous System
CO
Cardiac Output
DBP
Diastolic Blood Pressure
ECG
Electrocardiogram
ER
Endoplasmic Reticulum
HR
Heart Rate
SBP
Systolic Blood Pressure
SI
Système International d'unités
SV
Stroke Volume
TPR
Total Peripheral Resistance
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Abstract Millions of years of evolution in an environment containing a constant factor, the unidirectional gravity vector, has led life forms on this planet to acquire characteristics that are not only shaped by this constant factor but are also adapted to utilize it for their benefit. For instance, large marine creatures like whales use their capacious lungs as buoyancy tanks and use their fins and tails to remain afloat or to be mobile. Without these adaptations, they would have quickly sunk to the seabed. Feeding efficiency may be affected by gravity if the animals feed by filtering suspended particles creating currents that carry the particulate food to their mouth opening. In normal gravity, the food particles tend to sink to the bottom and filter feeders must be able to suspend and collect the particles with some apparatus such as ciliary wreaths. In hypergravity, particle sedimentation rate increases thus reducing the animal filtering efficiency while in hypogravity, mainly microgravity, filtering rate will increase. Differently, some bacteriophagous animals do not possess structures to collect the food but commonly live and move into sediment and feed on the bacteria upon encounter. Hypergravity will apply higher pressure on their bodies and could force them to adhere to some surface and to reduce their displacement while hypogravity, mainly microgravity could impede adhesion to the surface and make food item encounters improbable. Thus gravity perturbations may affect animal life – history traits such as survival or fecundity by influencing their feeding efficiency. Organisms live in four dimensional space-time. The time dimension decides the life cycle – an expression of its development of each organism. The cycles of differentiation expressed in the timeframe of evolution has given rise to the biological diversity on Earth. In other words, phylogenesis can be understood as alterations in developmental processes. Thus, species of organisms are groupings of individuals that share developmental processes.
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The process of ontogeny occupies an appropriately central position in gravitational biology. It is thus natural and necessary to study the role of gravity in development. Mammalian development requires an intact, integrated system of mother and offspring. This system develops as a unit based on co-ordinated changes within and between mother and offspring. Any factor that disrupts the mother or the offspring is liable to disrupt the mother-offspring system leading to a likely change in the organising influence of the system on its constituent parts and hence the ontogenic processes within the individuals can be disrupted. Direct effects of gravity on mammalian development are elusive as it involves bidirectional linkages between mother and offspring. A change in one may change the other. Indirect effects of gravity are those that are expressed through avenues of the mammalian system e.g. rat pups grow slowly during spaceflight. The most important physiological changes caused by microgravity include bone demineralization, skeletal muscle atrophy, vestibular problems causing space motion sickness, cardiovascular problems resulting in postflight orthostatic intolerance, and reductions in plasma volume and red cell mass. Pulmonary function is greatly altered but apparently not seriously impaired. Space exploration is a new frontier with long-term missions to the moon and Mars not far away. Understanding the physiological changes caused by long-duration microgravity remains a daunting challenge. Chapter 1 contains literature review related to the field.
Chapter 2 is
dedicated to the design and development of a 1-D clinostat and a clinostatcentrifuge for studying effects of altered gravity on plants and insects. The clinostat and the clinostat-centrifuge were designed and developed in-house. It also includes the testing of the clinostat by studying effects of simulated weightlessness on insects. The eggs of the harquelian fly (Chironomus ramosus) were exposed to different durations of clinorotation and their development studied. Studies show faster development of the eggs leading to faster hatching. The effects of altered gravity on plants are discussed in Chapter 3. It also includes the preliminary studies carried out on the gravitropism in plants. Rice
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seeds were exposed to clinorotation for different durations. Growth and development of rice seedlings is promoted by simulated microgravity. However, the chlorophyll content isolated from shoots showed a decrease for clinorotated plants. The effects of gravity and posture on the cardiovascular system have been described in Chapter 4. It also includes the design and development of a tilt-table which can be rotated through 360° in steps. It was found that regular practice of Sheersasana, a Yogic posture, is possibly effective in tuning the cardiovascular system to the pooling of blood in the head as in early days of spaceflight. A Java based program developed for ECG analysis has also been included in this chapter. It can be concluded that the regular practice of Sheersasana possibly tunes the cardiovascular system to the pooling of blood in the head as in early days of spaceflight. Thus it is possible that astronauts with a regular practice of Sheersasana might adapt sooner to the microgravity environment in space than without. Simulated microgravity enhances the developmental processes in plants and insects. These conclusions are summarised in Chapter 5. The relevant references used in the work are enlisted in the Bibliography section at the end of the thesis.
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Acknowledgements It is my great privilege and honour to work under the guidance of Prof. P.B. Vidyasagar, Department of Physics, University of Pune for this thesis. He provided stimulus and various ideas for pursuing this endeavour and taught me various aspects of biophysics. Without his constant moral support, inspiration and encouragement, it would not have been possible for me to complete this work. My gratitude towards him cannot be expressed in words. My parents, especially my mother, have been a constant source of support and encouragement throughout my study. I am highly indebted to them. I am also very much thankful to all my friends mainly Mr. Anup Dhirghangi, Mr. Saytavinayak Mule, Mr. Sahil Wagh, Mr. Sandip Patil, Mr. Subhadip Das, Mrs. Yogeeta Apte and Ms. Sashauna Mitchell for all the support and encouragement they have given. I am thankful to the Head, Department of Physics, University of Pune for granting me permission and providing me with facilities to carry out my Ph.D. work. I would also like to thank Mr. A.G. Bhosale, Mr. V.L. Jadhav, Mr. A.F. Sheikh, Mr. K.A. Deshpande, Mr. J.J. Lokhande and Mr. D.B. Potdar, University Workshop, University of Pune for their help in designing the clinostat and clinostat-centrifuge. Special thanks to Mr. J.J. Lokhande for his discussion on various technical aspects of the designing of the clinostat and clinostat-centrifuge. I would like to thank Dr. B.B. Nath, Reader, Department of Zoology, University of Pune, for allowing me to use Chironomus as a model system and for his valuable suggestions. I also would like to thank my friend and colleague, Mr. Anand Babrekar, Ph.D. student, Department of Zoology, University of Pune for his collaboration on studying the effects of altered gravity on Chironomus. He also has given me moral support. I would also like to thank Prof. S.S. Bhargava, Department of Botany, University of Pune for giving us knowledge on the various media for plant growth.
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I wish to thank Mr. Dhananjay, my friend and colleague, for his encouragement and technical help in the design of the clinostat during his M.Sc. project which he did under me. I would also like to thank my other colleagues viz., Mr. Salil for his help in working on human subjects and also making models for studying effects of gravity on the cardiovascular system, Mr. Vikas for his help in experiments on effects of clinorotation on Chironomus, Mr. Vijay and Mr. Vikram for their help in clinorotation on plants and Mr. Sayan for his help on the experiments on the tilt-table with human subjects. Special thanks to Dr. Selma Nunes, Dr. Ujwal Yeole, Physiotherapy College, Sancheti Hospital, Pune for their help in the tilt-table experiment by measuring BP using cuff method and also the students therein, for volunteering for the experiment. Thanks also to Hemangi Aero Gym for providing us tilt-table facility for carrying out preliminary studies on the effects of gravity and posture on the human cardiovascular system. I would like to thank all the volunteers especially to Mr. Ajit , Mr. Pradip and Mr. Arpan for giving their valuable time and cooperating to volunteer for this project. My sincere thanks to my labmates viz., Dr. Kaveh S. Haghparvar, Dr. Ni Nyoman Rupiasih, Mr. A. Balraj and Mr. Sagar Jagtap and Dr. Pratip Shil, Biophysics Laboratory, Department of Physics, University of Pune for all the support and help, they have given during the project. I also thank my senior colleague, Dr. Jyoti Gaikwad for her moral support mainly in the early stages of this project. Special thanks to my labmate, Dr. Kaveh S. Haghparvar for his help related to medicine. Thanks to Mr. Rahul, Bioinformatics Center, University of Pune, my friend Mr. Anirban and the Java forums on the Sun Microsystems site for helping me in debugging many parts of my program developed for ECG analysis. I also acknowledge the Council of Scientific & Industrial Research (CSIR) for providing me fellowship without which it would have been difficult to do this research.
Santosh Bhaskaran
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CHAPTER 1
Chapter 1: Introduction 1.1 Background
All living beings on earth experience the gravitational field of the earth. Gravity has been constant throughout the evolution of life on Earth. Gravity is a vector, i.e. a force that has magnitude and direction at each point in space. Millions of years of evolution in an environment containing this constant factor have led life forms on this planet to acquire characteristics that are not only shaped by it but are also adapted to utilize it for their benefit. For instance: large marine creatures like whales use their capacious lungs as buoyancy tanks and use their fins and tails to remain afloat or to be mobile. Without these adaptations, they would have quickly sunk to the seabed. Protozoa seem to utilize gravity depending on the preferred living conditions (1). Paramecium which feeds on aerobic bacteria, shows a negative gravitaxis, i.e. the cell population swims mainly upwards, against the gravity vector, thus reaching oxygen saturated layers. Loxodes, which prefers low oxygen concentrations, shows a positive or a bimodal gravitaxis: alternately moving upwards or downwards increases the chance of finding optimal conditions of light and oxygen concentrations which change with the depth of the water column in an aquatic ecosystem. Travel in space was a dream till Yuri Gagarin, the first Russian astronaut, successfully scaled the space on April 12, 1961 aboard the spaceship Vostok 1 and experienced the effects of gravitation. Since then the situations have changed to a great extent. Space travel has now become a routine affair. Space shuttles are being operated on a regular basis. Astronauts have spent about six months at a stretch in space. Man has put his foot on the moon’s surface. These missions have provided opportunities for researchers to observe the physiological changes when astronauts are exposed to altered gravity conditions. In the pre-space flight era it was believed that any changes in the magnitude of the gravitational vector would affect the organisms only on a macro scale, physiological level (changes in blood flow volume, changes in inter-cranial fluid
14
pressure, vestibular dysfunction, etc.). Mechanical forces important to pulmonary structure and function are produced by gradients in gravity, motion, osmotic forces, and interactions between cells and/or cell matrix. But subsequent observations have shown that gravity or the absence of it has a direct effect at the cellular level where cellular organelles or components transduce the mechanical gravistimulus to an electrochemical signal. How this transduction occurs remains a mystery and is still driving a lot of frenetic research. Exciting research on the interaction of the cell cytoskeleton with membrane components and the extracellular matrix is attempting to explain all the different phenomena associated with the behaviour of cells in microgravity as well as hypergravity. Manned missions into space and significant problems in developmental and evolutionary biology in zero and low gravity conditions demand a concentrated research effort in space-medicine, physiology and on a larger scale - gravitational biophysics. Research in these areas would also provide us with fascinating insights into how gravity has shaped our evolution on this planet and how it still governs some of the basic life processes. Yet experiments carried out aboard space shuttles and in bioreactors have shown that gravity changes are sensed at the cellular level in cell cultures (2, 3) while global changes in neural circuitry like change in synapse density occur during experiments done on live animals aboard International Space Station (4).
1.2 Gravitation In 1665, Sir Isaac Newton proposed the law of gravitation. According to this law every object in the universe attracts every other object with a force directed along the line of centres for the two objects and is proportional to the product of their masses and inversely proportional to the square of the separation between the two objects. If m1 and m2 are the masses of the two bodies which are separated by a distance r then the magnitude of the gravitational force of attraction F is given by 1.1
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where G is the universal gravitational constant and r$ is the unit vector along r. This force acts along the line joining the centres of the two bodies. The value of G is 6.673 x 10-11 Nm2/kg2. The small value of G compared to other forces of nature explains the reason why the forces of a gravitational nature are almost imperceptible in case of small objects and even massive structures and buildings. They are perceptible only in a planetary scale, where masses are in the order of 1030 kg or more. If we consider one of the bodies as a planet whose mass is M and radius is R, then the weight
uur W of an object on the surface of the planet is, 1.2
or, 1.3
As the earth is not exactly spherical but oblate spheroid in shape, the value of g is not same on all points on the surface of the earth. Hence the value of g at the poles is greater than that at the equator. g has a value of 9.83 m/s2 at the poles while it has a value of 9.78 m/s2 at the equator. The taken value of g is 9.81 m/s2. This is also known as earth gravity or 1 g. If the value of g happened to be substantially lesser, then one would be hopping instead of walking on the earth’s surface. And if it had been substantially greater, then the creatures would appear to be short and bulky. The g-values for different bodies in the Solar System are calculated and tabulated in Table 1.1. If the value of g is lower than 1 then it is termed hypogravity and if it is greater than 1 then it is called hypergravity.
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Astronomical
Mass M
Radius R
g
g
Body
1023Kg
106m
m/s2
units
Sun
19890000
695
275
27.99
Hypergravity
Mercury
3.3
2.44
3.7
0.38
Hypogravity
Venus
48.69
6.0518
8.87
0.91
Hypogravity
Earth
59.72
6.37815
9.81
1
Earth gravity
Moon
0.735
1.738
1.62
0.17
Hypogravity
Mars
6.4219
3.397
3.71
0.38
Hypogravity
Jupiter
19000
71.492
24.8
2.52
Hypergravity
Europa
0.48
1.569
1.3
0.13
Hypogravity
Ganymede
1.48
2.631
1.43
0.15
Hypogravity
Saturn
5680
60.268
10.4
1.07
Hypergravity
Titan
1.35
2.575
1.36
0.14
Hypogravity
Uranus
868.3
25.559
8.87
0.91
Hypogravity
Ariel
0.0127
0.579
0.25
0.03
Hypogravity
Neptune
1024.7
24.766
11.1
1.14
Hypergravity
Triton
0.214
1.35
0.78
0.08
Hypogravity
Pluto
0.127
1.137
0.66
0.07
Hypogravity
Charon
0.019
0.606
0.35
0.04
Hypogravity
Table 1.1: g values on the surface of various astronomical bodies in the solar system. Courtesy (http://www.nineplanets.org/)
Gravitational force plays an important role in the motion of satellites. It makes the satellites orbit around the earth. The satellites tend to move away from earth but when they reach a particular distance in their respective orbits, they curve back inwards towards the earth. Hence they have circular or elliptical orbits around the earth.
1.2.1 Weightlessness and microgravity 1.2.1.1 Weightlessness in an elevator Consider a man of mass m standing in a stationary lift as shown in Figure 1.1. He exerts a force equal to his weight mg on the floor of the lift. At the same time, the floor exerts an equal and opposite force of reaction on him. If the lift starts
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moving up with acceleration a, then the apparent weight of the man is the normal reaction acting on him which is mg + ma = m (g +a). Now if the lift is accelerating downwards then his apparent weight would be mg – ma = m (g – a). Illustration of weightlessness during free fall
Figure 1.1: Man standing in an elevator in various conditions of acceleration
Now, consider the case when the cable by which the lift is suspended, suddenly breaks. Then the lift and the man standing in it experience free fall i.e. both will move down with an acceleration g. Therefore, apparent weight of the man = m (g – mg) = 0 i.e. the man experiences a complete feeling of weightlessness. Now the question that arises is has the force of gravity exerted by the Earth on the man ceased to act. The answer is no since the Earth exerts the same force of attraction mg on him as before. However, when the lift was stationary, the floor of the lift exerted an equal and opposite reaction on him. And when the lift started falling, the floor of the lift moved down with same acceleration as the man and therefore it did not exert any reaction on him and hence the feeling of weightlessness. 1.2.1.2 Weightlessness in a spacecraft Imagine an astronaut standing on the surface of the Earth. His weight acts in the downward direction i.e. he exerts a force (equal to his weight) on the
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ground. At the same time, the ground exerts equal and opposite force of reaction on the astronaut. Due to this force of reaction, the astronaut has a feeling of weight. Now consider the same astronaut on the floor of a spacecraft orbiting the Earth. His weight is still equal to the gravitational force mg acting on him towards the centre of the Earth. This gravitational force provides the centripetal acceleration for the circular motion around Earth. Therefore the astronaut is acted upon by two equal and opposite forces viz., the gravitational force acting towards the centre of the Earth and the centrifugal force acting away from the centre of the Earth. Hence the astronaut does not exert any force on the floor of the satellite. As a result, the floor does not exert any force of reaction on him. Consequently, the astronaut has a feeling of weightlessness. Weightlessness condition is called in popular language as zero gravity condition. However, it must be remembered that the force of gravity acting on the astronaut is far from zero. It is the force of normal reaction acting on the astronaut which is zero giving rise to the feeling of weightlessness. 1.2.1.3 Microgravity Perfect weightlessness is difficult to achieve within a spacecraft since in practice, the spacecraft follows an elliptical path and not a circular path around the Earth. Hence, there exists some residual force. Also, atmospheric drag, solar wind, light pressure, etc. also cause additional forces which would be non-existent in a perfect weightless environment. Therefore, acceleration of the order of 10-4-10-6 g is more typically experienced in today’s spacecraft and is usually referred to as microgravity. Thus, one can consider the second constraint in studying the effects of gravity on a spacecraft to be the determination of whether or not nearweightless environment results in sufficiently small force acting on the experimental system so as to be deemed statistically negligible. There are different methods used to achieve altered gravity environments on Earth. These include:
Drop-towers are high-tech versions of the elevator analogy mentioned above. NASA’s Lewis Research Centre has 145 m drop tower facility that begins on the surface and descends into Earth like a mine shaft. The test section of the facility is
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6.1 m in diameter and 132 m deep which can create microgravity environment for about 5-6 seconds. Below the test section, is a catch basin filled with polystyrene beads. The pressure is maintained below 10-2 Torr so that microgravity of the order of 10-5 g is achieved.
Figure 1.2: 100 Meter Drop Tube at the NASA Marshall Space Flight Center (5)
The NASA Marshall Space Flight Centre has a smaller facility for the same purpose. It is a 100 m high, 25.4 cm diameter evacuated drop tube that can achieve microgravity for upto 4.5 seconds. Japan has a 490 m deep drop tower facility which can create microgravity environment of the order of 10-5 g for upto 10 seconds. A Free-Fall Machine (FFM) has been invented and patented by Mesland at ESA (6). As its name suggests, it works on the principle of free fall. Biological samples fixed to a carriage sliding along a vertical 1 m guiding bar fall freely. At the bottom of the bar, a thrust of compressed air (8 Bar) bounces the carriage to the top of the rod. The assumption is that the approximately 20 times shorter bounce of 15-40 g that alternates ~ 800 ms microgravity phase is not sensed by the
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biological samples under study. FFM has been found to be a good simulator of weightlessness for unicellular green algae (6). However it was found that human lymphocytes possibly are able to register the bounces and hence FFM is not suitable for simulated microgravity experiments on these cells (7). Although microgravity of the order of 10-5 g is achieved, this technology has few drawbacks. The duration of microgravity is too short for studying the microgravity effects on physiological systems. Also experimenters cannot ride with their experiments.
Parabolic flights using aircrafts can achieve microgravity for about 25 seconds. The NASA Johnson Space centre employs a commercial-sized aircraft with most of its passenger seats removed. The people inside are protected by padded walls.
Figure 1.3: Parabolic flight characteristics (5)
A typical flight lasts for 2-3 hours and carries experiments and crew members to a beginning altitude of about 7 km above sea level. The aircraft climbs rapidly at a 45º angle (pull up), traces a parabola (pushover) and finally descends at a 45 degree angle (pull out). During the pull up and pull out segments, crew and experiments experience hypergravity of the order of 2 - 2.5 g. During the parabola, at altitudes ranging from 7.3-10.4 km, microgravity of the order of 10-2 g is achieved
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for more than 15 seconds. On a typical flight, the aircraft flies 40 parabolic trajectories. Aircrafts cannot achieve microgravity conditions of as high quality as those produced in drop-towers. This is because they are never completely in free fall and their drag forces are quite high. Nevertheless, they offer an important advantage over drop towers, experimenters can ride along with their experiments. They are used to study the effects of microgravity on animals as well as human cardiovascular system.
Sounding rockets follow suborbital trajectories and can produce several minutes of free fall. The period of free fall exists during its cost, after burn out and before entering the atmosphere. Microgravity of the order of 10-5 g and below can be created. The SPAR (Space Processing Application Rocket) rockets employed by NASA from 1975 to 1981 have been used for fluid physics, capillarity, liquid diffusion, containerless processing and electrolysis experiments. They could lift 300 kg payloads into free fall parabolic trajectories lasting 4-6 minutes.
Figure 1.4: Rocket parabolic flight profile (5)
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Figure 1.5: Sounding rocket for microgravity experiments (5)
Clinostats have become a very useful and important ground-based technique to study the effects of simulated microgravity (8, 9). In fact, important gravitational effects observed in space are reproducible, at least qualitatively, in the clinostat (10). The simplest kind of a clinostat is the 1-D clinostat in which the sample to be tested is fixed on the axis perpendicular to the gravity vector and rotated at speeds that are matched with the particular graviresponse time of the sample in question. Such rotations perform the function of nullification of the effects of gravity on the sample. The 2-D clinostat rotates the entire assembly about both axes normal to gravity. This is a better system for simulating microgravity than the 1-D clinostat as the gravity vector is nullified about two axes. Hoson has developed a 3-D clinostat in order to subject small biosystems (single cell cultures, unicellular organisms, plant seedlings) to vector-randomized gravity (12, 13). Probes are fixed as close as possible to the centre of a rotating
23
frame which rotates within another rotating frame. Both frames are driven by separate motors. Rotation of each frame is random and autonomous and regulated by computer software. Gravitometers are fixed to the frame in order to record the gravity vectors during rotation. These 3-D clinostats also know as Random Positioning Machines (RPM) address the question of the three dimensionality of biosystems. They are also perfect simulators of microgravity as in space (14). Schwarzenberg has shown that the RPM are better simulators of microgravity environment in space than the Free Fall Machine (FFM) (7). These simulate microgravity more effectively than 1-D and 2-D clinostats (13).
Orbiting spacecrafts are far advantageous over drop towers, parabolic flights and sounding rockets in the sense that they create microgravity conditions of the order of 10-5g for long durations (days, weeks, months and years). Having more time available for experiments allows researchers to investigate slower processes and more subtle effects.
Centrifuges are employed for studying hypogravity and hypergravity effects. On Earth, one can achieve only hypergravity as gravity is inherently present. If the centrifugal force C.F. has magnitude αg where α is a real number, then the resultant of the gravity vector and the centrifugal force vector is given by,
1.4 The magnitude is given by 1.5 and its direction is given by 1.6 as shown in Figure 1.6. With increasing α, R tends to equal CF in magnitude and direction.
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CF .
θ R
g
Figure 1.6: Forces in a centrifuge
Using a centrifuge in space hypogravity effects can also be studied as the only force would be the centrifugal force as there is “no” gravitational force. Combining a centrifuge with a clinostat or bioreactor, it is possible to study simulated hypogravity effects. Ground-based techniques used independently or in combination to minimize the adverse effects of altered gravity on the cardiovascular, musculoskeletal and vestibular systems are:
Long-term bed rest studies are used to test methods, which can counter the adverse physiological and psychological effects on the bed rest volunteers. These methods or countermeasures also hold similar beneficial effects for astronauts exposed to weightlessness. Bed rest studies are, therefore, used as an invaluable method of testing and developing countermeasures to the effects of weightlessness on astronauts through the use of, for example, exercise, physical therapy, medication or optimised nutrition.
Figure 1.7: Bed rest study
Courtesy ESA http://www.esa.int/esaCP/ESAF4UF18ZC_Life_1.html
Tilt-table test is used to evaluate how the blood pressure responds to gravitational stress. Cardiovascular parameters such as ECG, heart rate and blood pressure are
25
measured in the supine position which acts as a baseline. The minus 6° tilted bed rest model has been chosen as the best simulating the effect of changed gravity, similar to what astronauts experience when in space. This position reduces the effects of gravity on the body: in daily life, gravity is exerted in the longitudinal axis while in the HDT position; it is exerted in the transversal axis. Several factors of the space environment are reproduced during head-down tilted experiments: fluid shift, reduced physical activity and confinement.
Figure 1.8: -6° head down tilt bed rest
Figure 1.9: -6° head down tilt
Courtesy ESA http://www.esa.int/esaHS/ESAA1776K3D_index_1.html
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Figure 1.10: 70° head up tilt
Courtesy http://www.nymc.edu/fhp/centers/syncope/HUT%20and%20instrumentation.htm
Figure 1.11: Lower body negative pressure (LBNP) device.
Courtesy http://www.spacedoc.net/lower_body_negative_pressure.html
27
Figure 1.12: An astronaut lying in a Lower body negative pressure (LBNP) device.
Courtesy http://science.ksc.nasa.gov/mirrors/images/html/sl3.htm
Lower body negative pressure (LBNP) Device is a tool designed to apply a pressure lower than ambient pressure to a subject’s body from the hips down within the confines of the LBNP Bag. Decompression of the lower torso to negative pressures of -50 mmHg will simulate the 1g load normally experienced on Earth and will act as an orthostatic stressor. In this manner, the device can be used as a research tool to study deconditioning of the human cardiovascular system in space.
1.3 Impact of gravity on life All organisms
on Earth have evolved at unit gravity (1 g), and thus are
probably adapted to function optimally at 1 g. However, with the advent of space exploration, it has been shown that organisms are capable of surviving at much less than 1 g, as well as at greater than 1 g. Organisms subjected to increased g levels exhibit alterations in physiological processes that compensate for novel environmental
stresses,
such
as
increased
28
weight
and
density-driven
sedimentation. Weight drives many chemical, biological, and ecological processes on Earth. Altering weight changes these processes. Given these facts, it is not be surprising that changes in gravity could alter life, as we know it. If gravity causes changes to biology, then gravity must be a major physical environmental force shaping life on earth (5). Millions of years of evolution in an environment containing a constant factor, the unidirectional gravity vector, has led life forms on this planet to acquire characteristics that are not only shaped by this constant factor but are also adapted to utilize it for their benefit. For instance: large marine creatures like whales use their capacious lungs as buoyancy tanks and use their fins and tails to remain afloat or to be mobile. Without these adaptations, they would have quickly sunk to the seabed. Some species such as aquatic insects detect gravity using air bubbles trapped in certain passageways viz., tracheal tubes. However, most organisms use statocysts consisting of a fluid-filled chamber lined with a hairy, touch-sensitive epithelium containing statolith which is a solid granule of higher density (15). Interestingly, biological systems have been able to exploit their designs and molecular mechanisms to increase the sensitivity of their thresholds of detection. The vibrations detected by the Corti organ in a mammalian inner ear can be of the same order of magnitude or even less than thermal noise motion (16). Organisms have developed particular sizes and shapes which would make them more capable of efficient competition for survival. At least, in the short evolutionary time range, size and shape of organisms are strictly controlled internally. At most, the availability of nutrients is the main external parameter influencing them but there can be little doubt that had life been evolved in a different gravity environment, the sizes and shapes of organisms would have been different. Eggs and early cleavage cells are among the largest cells known. Till recently, physicists were of the opinion that changes in gravity would not affect cells as gravity is a weak force compared to other physical forces e.g. electrostatic, acting within cells (17). However, in spite of gravity being a weak force, its effects could be clearly detectable in them. Nevertheless, if one of the characteristics of complex organisms is more or less complete assimilation by endogenous (genetic)
29
regulatory mechanisms of processes that may have been originally directed by the external/internal non-genetic physicochemical processes, these effects may have become obscured by the more robust and recent autonomous genome-encoded mechanisms (18). The question then arises is about the evolutionary past. Fossil records are fragmentary and even the origin of Metazoan evolution is clouded with obscurity. The present consensus indicates that most or all current existing phyla appeared more or less simultaneously just before the Cambrian, around 600 million years ago, during the Vendian period of the latest Neoproterozoic Era, or even earlier if some estimates are correct (19, 20). Very less evidence is found on how development occurred and/or changed during evolution, particularly during Metazoan evolution. The few available data include recent findings of developing embryos in 600 million years old phosphorites in the Doushanto formation in Southern China (21). They show large eggs actively cleaving into smaller cells presumptive steroblastulae as the early embryonic structures of these primeval organisms. Hence it is possible to suggest that in the early Metazoan evolution, some eggs and early embryos became quite big, probably due to the ‘assimilation’ of oogenic storage processes that made the development of these early systems independent from external supplies. Big cells could be the targets of gravity-driven processes that at least initially could provide important signalling cues guiding the development of primitive Metazoans. Feeding efficiency may be affected by gravity if the animals feed by filtering suspended particles creating currents that carry the particulate food to their mouth opening. In normal gravity, the food particles tend to sink to the bottom and filter feeders must be able to suspend and collect the particles with some apparatus such as ciliary wreaths. In hypergravity, particle sedimentation rate increases thus reducing the animal filtering efficiency while in hypogravity, mainly microgravity, filtering rate will increase. Differently, some bacteriophagous animals do not possess structures to collect the food but commonly live and move into sediment and feed on the bacteria upon encounter. Hypergravity will apply higher pressure on their bodies and could force them to adhere to some surface and to reduce their
30
displacement while hypogravity, mainly microgravity could impede adhesion to the surface and make food item encounters improbable. Thus gravity perturbations may affect animal life – history traits such as survival or fecundity by influencing their feeding efficiency. Ricci and colleagues have studied these effects on the reproducing capacity of feeder organism (Macrotrachela quadricornifera, Rotifera Bdelloidea) and bacteriophagous (Panagrolaimus rigidus) (22). They found that microgravity did not impair rotifier and nematode survival or reproductive capacities while hypergravity affected the rotifiers and severely the nematodes. Organisms live in four dimensional space-time. The time dimension decides the life cycle – an expression of its development of each organism. The cycles of differentiation expressed in the timeframe of evolution has given rise to the biological diversity on Earth. In other words, phylogenesis can be understood as alterations in developmental processes. Thus, species of organisms are groupings of individuals that share developmental processes. The process of ontogeny occupies an appropriately central position in gravitational biology. It is thus natural and necessary to study the role of gravity in development. Hamsters exposed to hypergravity showed no apparent changes in locomotion but showed obvious disturbances in swimming ability (23). They swam more slowly than the controls and were observed to swim underwater and to circle while swimming. Some of them even succumbed to drowning. Direct effects of gravity on mammalian development are elusive as it involves bidirectional linkages between mother and offspring. A change in one may change the other. They are those that operate through a primary relation with the recipient organism or tissue. Gravitational forces are required for vestibular receptors to develop fully articulated afferent connections to the brainstem and for calcium metabolism to be continuously expressed during skeletal formation. Indirect effects of gravity are those that are expressed through avenues of the mammalian system e.g. rat pups grow slowly during spaceflight.
1.3.1 Cells Progress of gravitational cell biology research depends on the continuing evaluation of a wide variety of physical phenomena affected by gravity and their
31
roles in extracellular, intercellular and intracellular processes. Single cell functions are affected by perturbations in their internal and external environment by a variety of factors which includes gravity (24). Physical phenomena influencing cell function include sedimentation, buoyancy-driven convection, streaming potential, hydrostatic pressure and interactions among physical transport processes. Thermal motion and fluid viscosity play a significant role in all transport processes at the cellular level. The sedimentation of intracellular organelles is counteracted by the cytoskeleton. In microgravity, extracellular solutes must be transported by diffusion or active circulatory processes in the absence of a density gradient-driven convection and flocculation and coalescence are reduced due to lack of aggregates. All major physical components of gravity can have important effects on cell function. For instance, compression due to hydrostatic pressure, can affect the internal load-bearing structure of the cell (actin, microfilament, and microtubule cytoskeleton) making it more or less resistant to compression. In microgravity the cytoskeleton would be expected to become less prominent because of less need to support load due to hydrostatic pressure. Another prominent effect of hydrostatic pressure due to gravity is the adhesive compression of the cell against a rigid substrate or other cells. The cell's own weight is likely to have the same type of effects as hydrostatic pressure. The second physical component of gravity is differential acceleration of cellular organelles which is best documented by gravitropism studies of plant cells. In plants, starch containing amyloplasts (statoliths), are cellular organelles denser than the rest of the cytoplasm. As a result of this greater density at unit gravity, statoliths sediment to the bottom of the cell. The plant cell then detects the position of the statoliths and directs root cellular proliferation in that direction (25, 26). In microgravity this process is disrupted, but can be restored by artificial gravity (centrifugation) (27). The molecular mechanism for the signal transduction of the gravity information in plants is still not well understood though this is one of the clearer examples of a biological gravity sensor. In all eukaryotic cells, the nucleus is also a potential gravity sensor. The tightly packed DNA in the nucleus is about 20% denser than the rest of the cell. At 1 g, this
32
would tend to make the nucleus sink to the bottom of the cell; however the cytoskeleton actively maintains the nucleus in place. This process transfers a nuclear load to the cytoskeletal fibres adjacent to the nucleus and could be another way of transducing information about the direction and magnitude of the gravity field the cell is subjected to (24). Although this effect is postulated, no molecular mechanism for sensing nuclear positioning is known yet. The final physical component of gravity relevant to cells is thermal convection. At 1 g, heated fluids rise to the top along the gravity vector. Heated fluids are then replaced by cooler fluids, establishing a convection current that rapidly dissipates heat, renews nutrient supplies, and removes waste materials (24, 28). This factor is most important where no fluid flow (e.g. blood flow) exists to dissipate metabolic products and exchange nutrients around the cell. Without convection, slow diffusion processes are the only means for heat and nutrient exchange. This factor is likely to be most relevant in plants and single celled micro-organisms like some bacteria that have no motile structures like cilia or flagella. Although blood flows in animal tissues mostly overcomes this effect, this could still be a factor in cells localised where blood flow is minimal. Initial studies concentrated on the fine structure (morphology) of cells grown in microgravity and looked at issues like position of organelles, cytoskeletal density and organization, nuclear structure etc. (29, 30). Studies have also been performed at the cellular and tissue level focusing on how cells migrate and position themselves during development in frogs (31), how fertilization and development occurs (32-35), etc. The most prominent microgravity-induced cellular and tissue differences have been identified in bone tissue cells (29), muscle tissue cells (30) and in immune system cells (36). In all three cases, structural abnormalities correlate to bone, muscle and immune disorders observed as a result of spaceflight. Another class of cellular studies focused on metabolic and signalling pathways (37). These biochemical studies of cells have yielded results showing that many important pathways are affected by microgravity. Among these are basic energy metabolic pathways, and proliferating (mitogenic) pathways. As a
33
result in changes of these pathways, cellular functions like migration, growth/division, and survival are altered by microgravity. More recently, studies have been started to be conducted at the genomic level (38, 39). In these studies, gene arrays are used to determine which genes are expressed at the level of mRNA by cells in microgravity. This mRNA is ultimately translated into proteins that direct every function of the cell from signalling to structure. The graviresponses of Paramecium biaurelia consist of two components viz., gravitaxis and gravikinesis. Gravitaxis is defined as orientation with respect to gravity vector. Hypergravity increased gravitaxis as well as gravikinesis but there was no direct relation between the two (40). 0.16 g was found to be the lowest acceleration necessary to maintain a gravitactic response. Below this, random distribution of Paramecium biaurelia occurred. Long-term cultivation for up to 14 days in microgravity did not show dramatic changes in morphology of the protists although the proliferation rate increased. Behavioural changes due to the lack in gravity were restored after return to 1 g conditions. Hypergravity showed no saturation of gravireactions in ciliates up to 5 g conditions (1). Magnetically simulated gravity experiments showed that the gravikinetic response of Paramecium is linear from simulated -5 to 5 g and becomes nonlinear above 5 g (41). When the simulated gravity approaches 10 g, most Paramecia orient antiparallel to the force direction and on average propel without advancing, i.e., they stall. It may be speculated that perceiving of and reacting to gravitational forces is not the limiting factor for survival of ciliates on other planets whereas most remaining physical or chemical parameters (temperature, pressure, atmosphere, radiation) will probably be past endurance of protists. Experiments on E. coli in space showed a shortened lag phase, an increased duration of exponential growth and an approximate doubling of final cell population density compared to controls. This might be due to lack of convective fluid mixing and sedimentation (42). At least one set of signal transduction across the E. coli envelope, which is involved in osmoregulation, is functional in microgravity (43). The production of antibiotics gramicidin S by Bacillus brevis and microcin B17 by E. coli ZK650 was inhibited, although the test
34
strains grew faster in the simulated microgravity conditions compared to in normal gravity conditions (44). Simulated microgravity increased the virulence of E. coli and S. typhimirium (45). Microgravity is found to enhance the growth of planktonic bacteria. The speed of upward swimming euglena was found to be greater than horizontal and downward swimmers (46). Gravitactic simulation resulted in short period of hyperpolarisation followed by a massive depolarisation in Euglena gracilis (47). The membrane potential returned to initial values after a period of about 0.2 seconds. The possible sequence of events during gravitaxis begins with the force applied to the lower membrane by sedimentation of cell body resulting in the activation of environmentally sensitive calcium channels thus modifying the beating pattern of the trailing flagellum through changes in membrane potential (48). Understanding the mechanisms of graviperception in protists would possibly help to answer questions on the role of gravity in smaller cell systems such as mammalian cells (1). Experiments carried out in space with isolated cells have shown that eukaryotic cells can sense and respond to absence of gravity by modifying their reactions. The sensory mechanism seems to be related to the density of the cytoplasm and the involvement of stretch-sensitive receptors concentrated at specific points of the cellular membrane possibly through their linkage to the integral cytoskeletal framework (49). On the other hand, experiments related to the investigation of complex processes such as biological systems undergoing development under microgravity have been surprisingly unaffected by the space environment. This is a curious result as all organisms are evolutionarily adapted to the Earth’s gravitational field and hence should eventually change if this parameter will vary in a permanent manner. The small effects of the modifications in gravity on development in short-term experiments may be equivalent to the difficulties in detecting the involvement of other basic physical forces such as diffusion-controlled auto-organising reactions in currently developing biological systems. Establishing the role of gravity in plant requires information about how gravity regulates individual cell metabolism. Plant cells and tissues in vitro are
35
valuable models for such purpose. Plant cells are enclosed with a cell wall which gives them characteristics different from animal cells. The cell wall provides the cells with structural rigidity thus directly determining their size and shape. Hence it has been assumed that the cell wall plays an important role in gravity resistance instead of the bones and muscles in the animal body (50-52). Evidence for this view has been obtained by micro- and hypergravity experiments (51, 52). Data obtained from non-numerous space and clinostat experiments with plant cells in vitro have demonstrated that their metabolism is sensitive to g-environment. Most experiments have shown a decrease in the biomass production and cell proliferation of spaceflight samples compared with ground controls (53). Noted ultrastructural arrangement in cells, mainly plastids and mitochondria, has been related to altered energy load and functions of organelles in microgravity. Also, changes in the lipid peroxidation under altered gravity supposed with modification of membrane structural-functional state (54). Sytnik has studied the activity of some enzymes of energetic and oxidative metabolism and the level of cell respiration in altered gravity (54). Altered gravity might decrease nucleolar functional activity in root meristematic cells (55-57). Spaceflight exposure does not impair isoflavonoid accumulation in developing soy bean tissues (58) but enhances it (59). Experiments performed in microgravity showed that the position of statoliths in root cap cells depends on two forces, the external gravitational force and the internal force exerted putatively by cytoskeletal proteins (60, 61). Gravisensitivity was found to be higher in the roots of lentil seedlings grown in microgravity than those grown in 1 g though the volume and number of amyloplasts were the same in both cases (62). In 1 g, statoliths grouped in the distal part of the cell while in microgravity, they were more disperse and located close to the centre. Gravity-oriented growth of plant organs such as roots is under the control of at least two signal transduction chains (60). The first of these is located at the site of stimulus transformation, i.e., in statocytes of the root cap while the second is at the site of differential growth i.e., in cells of the transition zone, where a major switch occurs concerning organization and distribution of cytoskeletal elements. In
36
the statocytes, located at the apical tip of the root in the centre of the root cap, polarity is expressed in the arrangement of the organelles (63). They show a vectorial polarity since the organelles have a specific location along the longitudinal axis of the cell. For most genera the nucleus is located near the proximal wall of the statocyte (closer to the meristem) whereas the ER is close to the distal wall (60). Polarity is also evident in the distribution of plasmodesmata, which are more numerous in the transverse walls than in the longitudinal walls. The statoliths or amyloplasts are the only organelles which do not contribute to the structural polarity of statocytes because they always sediment in the physically lowest part of the cell. When the root is vertical, the sedimentation of the amyloplasts exerts a force on the ER tubules, which are then pushed upwards along the longitudinal side walls. Vacuoles are small or absent. This is the main difference with stem statocytes, where a large vacuole occupies the cell centre. Microgravity experiments indicate that actomyosin-driven motion of statoliths counteracts the sedimentation process caused by the gravitational force. On the basis of this behaviour of statoliths, it can be hypothesized that gravity-perceiving cells transform the gravity stimulus by measuring the position of statoliths via tension within actomyosin networks. Grouping effects of statoliths as observed under microgravity might be important in this respect. Gravisensing occurs in plant cells which are not specialized for gravity suggesting the existence of another gravisensing mechanism or at least graviperception which is not likely to move amyloplasts in statocytes of the root cap (64). Short duration weightlessness has an influence only on epidermal cell wall structure in epidermis of mesophylls of Triticum leaves and epidermis and phloem of Impatiens hypocotyls and cotyledons (65). Clinorotation reduced the number of mesophyll cells in Arabidopsis and pea (66). However the mesophyll intercellular space increased for both the plants and there was not much difference in the number of chloroplasts. Clinorotated pea chloroplasts showed a decrease of thylakoids during grana development. Palisade cell chloroplasts showed an increase in the volume and accumulation of starch and plastoglobuli under clinorotation (66). Volume of mesophyll palisade cells increased in Brassica rapa
37
during spaceflight (67). Mesophyll cell volume and RuBisCo activity increased in pea plants clinorotated for 12 days (68). The area of central mesophyll cell section of wheat leaves decreased during spaceflight. The number and size of chloroplasts decreased while there was an increase in the number of mitochondria and peroxysomes. Also the number of granae decreased while the number of thylakoids in each grana and the volume of chromatin in the nucleus increased (69). The extent of stacking of thylakoid membranes decreased possibly due to a decrease of light-harvesting chlorophyll a/b-binding complex (LHCII) amount (67). Clinorotation reduces the efficiency of energy transformation in the photosynthetic process in pea (70) as well as Arabidopsis (71) chloroplasts. Calcium is an essential and major plant nutrient. It is required for structural, osmotic and signalling purposes. The pathway and molecular mechanisms by which Ca2+ enters the root and is delivered to the xylem and the roles of Ca2+ channels in mineral nutrition, intracellular signalling and polarized growth have been described by White (72). In tip-growing plant cells, the calcium gradient is the necessary condition for directed hair growth. The absence of the calcium gradient at the stage of a root hair protrusion means that certain direction of hair growth has not been established (73). Calcium plays an important role in gravitydependent processes of root hair apical growth of cress. Significant alterations in structure, orientation and Ca2+ balance of cress root hairs were found to take place under vector-free gravity (74).
It was found that cytoplasmic free calcium
concentration is a part of the gravity transduction mechanism in young Arabidopsis seedlings (75). Microgravity appears to impact the cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression (76) with involvement of regulatory Ca2+ messenger system (77). Low diffusion of Ca2+ in the cytoplasm is an important step of Ca2+ signalling because standing gradients of Ca2+ can be formed as in tip-growing plant cells. The maintenance of the Ca2+ gradient standing is essential for vesicle fusion and continued growth. In general, spatial segregation of (Ca2+) at defined sites in the cytoplasm can promote signalling specificity, one aspect of which can be the duration of the signalling itself. Increased cytosolic Ca2+ concentration may induce
38
auxin distribution, manifested as increased ethylene production, which is associated with stem bending and elongation (78). Alterations in calcium concentration are detected by a protein called calmodulin, which binds itself to the calcium molecule (79). Calmodulin is implicated in calcium-dependent processes like light, gravity, mechanical stress, phytohormones, pathogens, osmotic stress, salinity, heavy metals, xenobiotics, anoxia, oxidative stress, heat shock and chilling (80, 81). Calcium interacts with auxin though the mechanism of the interaction between calcium and auxin remains controversial (79). Auxin regulates a family of genes called SAURs. These genes generate proteins that are thought to play a role in plants’ ability to change their direction of growth in response to gravity. These SAUR proteins bind to the calcium/calmodulin molecule. Calcium/calmodulin is also involved in regulating cellular levels of hydrogen peroxide which is a highly reactive molecule that can cause cellular damage when present at higher than normal levels (82). It is also a signalling molecule involved in plants’ adaptation to environmental changes, including gravity. External load plays a critical role in determining muscle mass and its phenotype in cardiac myocytes. Cardiac myocytes have the ability to sense mechanical stretch and convert it into intracellular growth signals, which lead to hypertrophy. An increase in mechanical load of cardiomyocytes stimulates myocardial growth (83). This gives rise to the expectation that reduced mechanical load, for example under hypogravity, causes a reduction in myocardial mass. On neonatal cardiac cells, experimental hypergravity induces the expression of a marker of hypertrophied myocardium. Microgravity reduces creatine kinase activity in cardiomyocytes (84). Hypergravity causes cellular hypertrophy and changes in gene expression as found on Earth in hypertrophied myocardium in vivo and microgravity produces the opposite (85). Retinal spreading depression (rSD) waves are faster when travelling in the direction of gravity vector than when travelling against it. Even when the waves are perpendicular to gravity, it interacts with them in a non-linear fashion giving rise to increase in velocity with increasing g (86). In the Belousov-Zhabotinsky
39
reaction, relative propagation velocity decreased at high g-values when the gel surface was normal to the gravity vector. But when the gel surface was along the gravity vector, an increase in velocity with increasing g was observed significantly at an acceleration of 6 g (87). Hippocampal cells can form unique reliable representations of position on three orthogonal faces in microgravity but they may require a period of adaptation or more experience with the environment than is typically required in normal gravity. It remains to be determined whether the hippocampal code in microgravity can fully represent three dimensions, or whether the system adapts by developing independent twodimensional representations for each orthogonal surface. It is also unknown what cues drive the firing of place cells under these conditions (88). Cultured glial cells adapt to changes in gravity and restart normal cycling processes. In other words, microgravity induces only transient alterations in glial cells such that the functionality of the nervous tissue may not be permanently impaired during long space missions (89). Nerve-associated acetylcholine receptor patches (NARPs) from cultures in which nerve muscle contact was established before the onset of clinorotation were unaffected whereas cultures in which nerve contact took place during rotation showed a marked inhibition of NARPs (90). Also, the area of patch was significantly reduced compared with control in myocytes which exhibited NARPs. This suggests that the process of synapse formation is sensitive to the gravitational vector. Embryonic development of the nervous system, in space, may therefore be markedly different from that normally occurring on earth. The functional properties of acetylcholine (Ach) receptors were found to be unaffected by clinorotation for 1-2 days (91). Myocytes exposed to microgravity showed marked changes in distribution and organisation of actin filaments and a reduced incidence of acetylcholine receptor aggregates at the site of contact of polystyrene beads (92). Daily one hour per day standing is sufficient to prevent differential adaptational changes in function and structure of cerebral and hindquarter vessels during simulated microgravity in rats (93). Vandenburgh has flown fused myoblasts (i.e. muscle fibres) to investigate the effects of microgravity on cultured muscle fibres. He
40
found that flight muscle organoids were 10–20% thinner compared with ground controls due to decreased protein synthesis rather than increased protein degradation (94). Clinorotated osteoblastic ROS 17/2.8 cells underwent apoptotic cell death which might be due to mechanical unloading produced by clinorotation (95, 96). This suggests the possibility that osteoblasts require mechanical loading for survival and sustenance (95). This might be the cause of bone loss in astronauts during spaceflights. Simulated microgravity inhibited the differentiation of preosteoblasts to osteoblasts eventually leading to reduced bone formation (97). It is seen that accumulation of lactic acid occurs around animal cells in microgravity causing the environment of cells to acidify. This also might be another possible reason for bone loss in animals and humans in microgravity (28). Sensitivity to hypergravity appears highest at less mature stages of osteoblast differentiation, when cells are confluent but are not yet producing a matrix that is mineralized (98). It is seen that adhesion-dependent cells respond differently than T lymphocytes and Jurkat cells to altered gravity. Cell-cell and cell-adhesion adhesion are remarkably increased in clinostat while the reverse is true for neoplastic SGS/4A cells. Both types of adhesion are enhanced except cell-cell adhesion in syngenic fibroblasts FG (99). Short-term clinorotation of mouse spleen lymphocytes increases significantly cell esterase activity while short-term centrifugation of 10 g decreases it and also increases their activation (10, 100). In an experiment carried out in 1983 in Spacelab 1, it was discovered that mitogenic activation of T lymphocytes was nearly completely inhibited in microgravity (101, 102). This is due to a malfunction of monocytes acting as accessory cells. Lymphocytes were highly damaged under microgravity conditions while cells cultured at 1 g in flight on a 1 g reference centrifuge underwent mitosis and blastogenesis. Ultrastructural
changes
observed by electron
microscopy
suggested that apoptosis is increased in microgravity. This was later supported by the work of Lewis and colleagues (103). T-cells activated and cultured at 10 g were found to become motile earlier than those in normal gravity (104). White blood cells are capable of autonomous movements, of cell-cell contacts and of the
41
formation of aggregates in microgravity (10, 105). This was a surprising and unpredictable finding as it was thought that mammalian cells which lack special locomotion organelles can move only on a substratum and gravity is somehow driving the motion. It was also seen that the cytoskeleton which plays an important role in signal transduction, undergoes structural changes few seconds after its exposure to microgravity (106). Microtubules self organize in vitro in a reaction-diffusion process, which appears to be gravity-dependent. In a 13-minute flight on a sounding rocket, microtubules did not almost show any self-organization in the absence of gravity compared to samples in an in-flight 1 g-centrifuge (107). These findings are of primary importance because they point to a direct effect of gravity on basic structural elements of the cell. It can be assumed that these effects seen in lymphocytes may be detected also in other cells. It is important to notice that gravitational effects are seen mainly in cells undergoing differentiation rather than in those which are quiescent or non-differentiating. If differentiation is the response to the reception of a specific signal and consists of new expression of specific genes, we can expect that cancer cells which are dividing spontaneously are less or not at all dependent on gravitational changes. Clinorotation increased apoptosis promoters in thyroid carcinoma cells (108). On the other hand, cell cycling of pancreatic carcinoma cells increased in simulated microgravity (109). In hypergravity, but not at 1 g, concanavalin A bound to erythrocytes activates B lymphocytes in addition to T cells. Marco and colleagues have proposed that at a certain stage during the evolutionary emergence of multicellular organisms the cues laid by generic forces like gravity were involved in the evolutionary organisation of these primitive organisms (18). As evolution proceeded, it might be possible that these early mechanisms got obscured and/or were made redundant by the appearance of new internal environment-independent biological regulatory mechanisms. The mechanism of perception of gravity is still debated. Sedimentation leading to pathways triggered by activation of stretch-activated ion channels (1, 110- 113) and unloading of the cytoskeleton structure leading to change in cell
42
morphology (113-121) are the two possible mechanisms. Theoretical explorations on a qualitative level were done and are continuing on the formalism involved in the mechanics of the cell cytoskeleton (116-123). The major objective of several experiments performed in space was to establish whether single cells are sensitive to gravity. Certain cells showed reduced gravity leads to profound changes of a number of physiological functions like genetic expression, cell proliferation, signal transduction and cytoskeleton structure (101, 124). An experiment carried out in a sounding rocket showed that no self-organisation took place when microtubules were assembled in microgravity (125).
1.3.2 Plants Plant growth and development are affected by a lot of different environmental abiotic factors such as light, temperature and water supply. Immediately upon germination, another physical stimulus, gravity, strongly influences the growth of plant organs, root and shoot, in order to ensure their correct orientation in space and the survival of the young seedling. Since plants have evolved under the constant stimulus of gravity, its presence is one of the most important prerequisites for their growth and spatial orientation. The ability of plants to change their growth orientation in response to gradients in light and gravity maximizes their ability to obtain energy from light and moisture and nutrients from soil. Plants show two principal responses to gravity. One is gravimorphogenesis which enables plants to orient their leaves to sunlight for photosynthesis and their roots to soil for anchoring and absorbing water and minerals. The second is to resist the gravitational force by constructing a tough body. This graviresistance has been studied by centrifugation and space experiments (51, 52) and is very distinct from gravitropism (50). Plant organs such as shoots, roots, tendrils and runners often perform rhythmic movements often in a helical spiral fashion with a period that ranges typically from minutes to several hours. The path described by the moving tip can have a regular circular, elliptical or flat shape or have a more complicated shape, sometimes with superimposed irregularities. Short-period nutations with small amplitude are often conveniently called micronutations while the term
43
circumnutations often denotes periodic movements of larger amplitude and a typical period of 1 hour. However, there is no clear distinction and it might be that nutational phenomena cover a period range that is continuous. The two nutation types can exist simultaneously. Circumnutation and winding in plants are universal growth movements that allow plants to survive despite their sessile nature. These are gravity-dependent morphogenetic phenomena in plants where endodermal cells act as gravisensors (126). The amplitude and frequency of circumnutations in sunflower hypocotyls decreased and rotational change in direction occurred often in microgravity (127). In hypergravity, the amplitude and period of nutations increased (128). Clinorotation caused straightening of gravitropically curved roots in cress (129). Tomato plants grown under simulated microgravity exhibited a spreading growth and an increasing of the inter-node length. Total fruit yield, small fruit yield, leaf area, leaf dry weight, fruit dry weight, total dry weight and shoot – root ratio were lower in the clinorotated tomato plants than control. The number of flowers per plant increased in clinorotated plants while fruit setting was reduced under clinorotation (130). Microgravity stimulates elongation growth of rice coleoptiles (131), Arabidopsis hypocotyls (132) and etiolated pea seedlings (133) and also emergence of leaf in maize coleoptiles (133). Seed germination is faster in microgravity for flax (134) and rice (135). Also the root lengths were found to be higher (134, 135). Ivanova and colleagues have reported the non-production of seeds in wheat grown in space (136). Mechanical properties of the cell wall like elasticity moduli and viscosity coefficients were higher in space-grown roots than the controls suggesting that the capacity of the cell wall to expand decreases in space more or less (135). Cell wall extensibility is reduced by simulated microgravity in pea seedlings and maize coleoptiles (133). A decrease in gravitational force may influence the activity of mechanoreceptors, which could cause growth stimulation in rice roots in space (137). Elongation of Arabidopsis thaliana inflorescence stems was suppressed while dry weight of the inflorescence stems increased in hypergravity (138-140). Root length and fresh weight of roots and epicotyls of pea decreased as g increased
44
(141). Similar results have been obtained for azuki bean epicotyls (142) and wheat coleoptiles (143). Also primary and secondary cell wall contents (138-140) and shoot diameter (139, 140) increased in hypergravity. However, hypergravity reduced cell wall extensibility in Arabidopsis (139, 140), cress (144) and azuki bean epicotyls (142). Starch and total lipid concentrations are found to be dependent on gravity. Starch concentration in cotyledons of soybean seedlings decreased in microgravity and increased in hypergravity (145) while this was reverse for total lipid concentration (145). Starch accumulation is reduced in potato under microgravity conditions (146).
This might be possibly due to reduced starch
synthesis by ADP-glucose pyrophosphorylase in microgravity and vice versa (145). However, a recent report has mentioned otherwise (147). Polysaccharide synthesis decreased in microgravity. Lysis of cuticle cellulose microfibrilles and matrix polysaccharides led to wall loosening and increased cuticular transpiration during weightlessness (65). Simulated microgravity enhanced respiration in oat seedlings (148). Metabolic activity was also enhanced which was observed in increased root lengths. Cell wall polysaccharides as well as hemicellulosic polysaccharides decreased in space which might be the cause of the decrease in apparent cell wall extensibility of rice coleoptiles (131) and Arabidopsis hypocotyls (132) in space. Development of metaxylem is promoted by hypergravity (139, 140). Lignin formation and cell wall polysaccharide content in cress hypocotyls (144) and hemicellulosic
polysaccharides
in
wheat
coleoptiles
(143)
increased
in
hypergravity. Similar results have been obtained for maize coleoptiles and mesocotyls (149) and azuki bean epicotyls (150) exposed to hypergravity. Hypergravity increased sterol levels in azuki bean epicotyls while no change was observed in fatty acid compositions in phospholipid and glycolipid (142). Hypergravity causes growth inhibition possibly by thickening cell walls (138, 144, 150) as well as by modifying xyloglucan metabolism making the cell wall mechanically rigid (150) while microgravity does the reverse (131, 132). Foliar amount of carotenoids, and chlorophyll a and b, were substantially reduced in tomato plants under simulated microgravity conditions (130). Size, composition and function of the photosynthetic apparatus were found to be
45
affected on the clinostat in Arabidopsis thaliana plants. Chlorophyll content decreased while carotenoid content increased under clinorotation. Photosynthetic activity also decreased in simulated microgravity (71). Chlorophyll a/b ratio increased in Brassica rapa during spaceflight. Photo-inhibition of PSI is induced by microgravity without any photo-damage of PSII (67). CO2 saturation, CO2 compensation point, photosynthetic photon flux compensation point and quantum yield of canopy net photosynthesis rate in wheat were not negatively affected by microgravity during first 24 days of canopy development suggesting that microgravity, as such, is not a significant environmental stress affecting canopy photosynthesis (151). Short-term microgravity increases leaf temperature and decreases net photosynthetic rate in barley leaves (153). Chlorophyll a content increased significantly in Arabidopsis during clinorotation while no such change was observed for pea. Biosynthesis of chlorophyll b was inhibited in clinorotated pea plants while no change was observed for Arabidopsis (66). Leaf senescence in oats is promoted by both clinorotation (152, 154) and centrifugation (154). Centrifugation is found to increase production of nitric oxide and hence DNA fragmentation and cell death in Kalanchoe daigremontiana and Taxus brevifolia (155, 156). Nitric oxide is involved in DNA damage leading to cell death by apoptosis in leaves and therefore might be an important signalling molecule in plants. Centrifugation is found to increase production of nitric oxide and hence DNA fragmentation and cell death in Kalanchoe daigremontiana and Taxus brevifolia (155, 156). The evidence for a central role for auxin in control of the differential growth patterns of plants has been summarized (157-159). A balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone between hypocotyls and root resulting in lateral placement of a peg in cucumber seedlings (160). Clinorotated and vertically grown cucumber seedlings did not form pegs suggesting that gravity is needed for peg formation possibly by auxin redistribution (161, 162). Mitochondrial cristae volume and matrix density in potato minitubers increased in microgravity (146). However, under controlled conditions including controlled ethylene levels, no differences
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were observed in the ultrastructure of potato tubers grown in space (163). Ethylene plays a positive effector role in the gravitropic response of maize roots by modulating brassinolide-increased gravitropic response (164). Seeds of Brassica rapus showed an increase in biomass after 5-25 days in clinostat. This increase was attributed to early initiation of secondary roots followed by elongation of primary and secondary roots. Also concentrations of Indole-3-acetic acid (IAA), abscisic acid (ABA) and zeatin were increased on the clinostat (165). Starch statoliths could act as gravisensors in plants (166). Plants, grown in space, are more susceptible to pathogenic attacks leading to diseases. This is a cause of concern as it is more difficult to control diseases in space. Understanding why plants become vulnerable to diseases in space could yield strategies to control plant diseases not only in space but also on earth (167).
1.3.3 Invertebrates Sea urchin sperm are sensitive to small changes in gravitational forces. More importantly this sensitivity has an effect on the ability of the sperms to fertilize eggs (34). Hypergravity decreased the hatching rate of eggs of C. elegans. Oocyte meiotic division for exclusion of polar bodies shortly after fertilization is the most susceptible aspect to hypergravity. In contrast, the oocyte maturation just before fertilization and the embryogenesis that proceeds after the meiotic division were unaffected by hypergravity (35). Genes involved in the responses of C. elegans to increased gravity have been identified (168). Patch-clamp experiments in microgravity on leech neurons by Klauss and Hanke show that the whole membrane is a gravity sensor and not just the ion-channel protein (169). Microgravity increases the rate of production of statoconia in Biomphalaria (170, 171). Hypergravity decreased the size of the statoliths and also the number and size of statoconia (172). Experiments on Drosophila melanogaster carried out in space and on ground indicate that behavioural responses that may be important in setting life-spans of organisms, for example, may still be readily susceptible to manipulation by external cues (18). Drosophila melanogaster adults exposed to microgravity markedly increase their motility. This change in behaviour might be a cause of faster aging
47
on the ground after their recovery from space (173). Short-lived strains show high levels of motility from the very beginning of their lives while long-lived ones show reduced motility during initial part of their life spans reaching higher levels similar to short-lived ones in their later stages of their lives (174). Drosophila melanogaster is able to sense the gravity vector showing a clear negative geotactic response (173). The altered behavioural response to microgravity may have something to do with this gravitactic response as very young flies i.e., the ones immediately hatched out from the pupal case, show higher responses both behavioural and of accelerated aging. Studies on Drosophila activity using an infrared system (177) have shown that during parabolic flight, male flies were found to be more active than females during hypergeotaxis (178). Exposing Drosophila to 2.5-7 g for short periods increased the longevity of the male flies but not the female flies (175). Young flies are affected longer by changing gravitational field than mature flies. Activity level of the flies was stimulated more by microgravity than by hypergravity while there was almost no difference in continuous velocity in microgravity as well as hypergravity (179). Studies on beetles in microgravity suggest that the circadian rhythm is sensitive to gravity (180, 181).
1.3.4 Vertebrates Studying the vestibular system is important as it would shed light on the developmental and evolutionary relationships between gravity and life. It would also help us better understand and perhaps counteract the effects of living, working and even growing up in altered gravity. In vertebrates (including humans) altered gravitational environments such as weightlessness can induce malfunction of the inner ears due to a mismatch between canal and statolith afferents. This leads to an illusionary tilt because the inputs from the inner ear are not confirmed by the other sensory organs, which then results in intersensory conflict. Vertebrates in orbit therefore face severe orientation problems. Fish have proven the most suited vertebrates for research into gravitational effects (182, 183). Fish use visual and vestibular cues for postural equilibrium maintenance and orientation as other vertebrates and invertebrates. They often
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show abnormal swimming behaviour like downward or upward pitching, inward looping, spinning movements, etc. especially during the transition from normal to microgravity. This is similar to the space adaption syndrome seen in humans. Zebrafish exposed to microgravity during only 24-72 hours after fertilisation show significant long-lasting functional deficits in their vestibular apparatus. These defects are possibly permanent though zebrafish also display various behavioural effects making it difficult to determine this. Swimming ability is particularly affected such that the fish are not effective in feeding themselves thus reducing their life spans to 10-14 days (184, 185). During parabolic flights, blinded goldfish showed downward pitching during pull-up hypergravity phase, looping behaviour during microgravity phase and upward pitching during push-down hypergravity phase showing that fish experience severe orientation problems in microgravity. Larval cichlid fish that were allowed to complete their development in hypergravity were affected regarding neither their morphogenetic development nor the onset and performance of their swimming behaviour. However, as soon as the centrifuge was stopped, many of the young fish revealed looping responses and spinning movements as observed after the transfer from 1 g to microgravity conditions in fish grown at 1 g which normally disappears (183, 186). Early amphibian embryogenesis is not dependent on gravity (187). On Earth, the fertilized frog egg rotates upon sperm penetration, and this rotation is thought to be essential for normal development. Upon fertilization, the egg begins to divide and form the embryo that, after an appropriate time, emerges from the jelly-like egg as a tadpole. Fertilised eggs sent to space showed that embryos can develop normally in weightlessness (188, 189) and that locomotion in weightlessness is strongly disturbed (188). Female frogs were sent into space and induced to shed eggs that were artificially inseminated. The eggs did not rotate and yet, surprisingly, the tadpoles emerged and appeared normal. After return to Earth within 2–3 days of hatching, the tadpoles metamorphosed and matured into normal frogs. However tadpoles raised in microgravity tended to remain underwater and also had smaller lungs than normal. The optomotor responses were stronger for tadpoles raised in microgravity (187, 190) while the tadpoles
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raised in hypergravity showed weaker optomotor responses (190). No appreciable changes were observed in bone and cartilage of chickens developed in microgravity (191). There are critical development periods during which biomechanical loading of limbs is essential to give cues to nerves. Without the cues, brain development and limb innervations may not occur normally and animals may develop an abnormal walking behaviour (192). Head-up tilt increased arterial blood pressure while stroke volume and cardiac output decreased significantly in dogs (193). 21 days of head-down tilt increased femoral and mesenteric venous compliance and decreased jugular venous compliance in rabbits. However, a daily 4 hour head-up tilt helped in preventing the changes of venous compliances in different anatomical regions of the rabbit due to simulated weightlessness (194). Orientation of the head with respect to gravity plays an important role in orienting and tuning the vertical angular vestibo-ocular reflex gain in monkeys (195). Renal blood flow and terminal aorta blood flow reduced significantly even more than the cardiac output. Marked effect of microgravity on cardiac rhythm responses to otolith stimulation were observed in rhesus monkeys (196). Head-down tilt induced hypovolemic and hypoadrenergic states in rhesus monkeys with reduced LBNP which is associated with a decrease in CO and SV (197). Microgravity does not alter cardiac contractility and can cause increase in ventricular compliance in monkeys (198). Clinorotation induces apoptosis and suppression of the production of progesterone in rat luteal cells possibly due to dysfunction of mitochondria (199, 200). In rats, the heart was found to undergo mitochondria changes in response to short-term microgravity which are more drastic than in skeletal muscles (201). Head-down tail suspension elevates cerebral vascular resistance in rats (202). Exposure to microgravity elicited by parabolic flights induces decrease of abdominal aortic pressure (AAP) in anesthetized rats (203-205). Daily gravitational loading by standing for 1 hour and also by head-up tilt for 4 hours is found to prevent differential adaptation changes in structure and function of vessels of rats exposed to simulated microgravity by tail suspension (206). Exposing rats to 2 g for
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two weeks impaired their spatial learning ability suggesting that a constant gravity is needed for spatial learning (207). Electrophysiological studies suggest that the hypothalamus might be the most sensitive region of the rat limbic system (208). Highly significant correlation between mammary metabolisms in micro and hypergravity was observed (209). It was found that beta blocker prevents bone loss induced by vestibular lesion suggesting that the vestibular system controls bone mineralization partly via the sympathetic nervous system. Similar bone loss is induced by spaceflight indicating that vestibular system mediates bone loss in weightlessness. Thus this may help in solving the problem of bone loss during space missions by using beta blockers (210). Mammary metabolic activity in pregnant rats increased in microgravity (211) but decreased in hypergravity (212) showing an exponential increase with g load and a continuum from micro- to hypergravity environments (212). This is possibly due to gravity-related changes in the hormone prolactin (209). Sustained hypergravity acceleration, especially along the long axis of the body of animals and humans (Gz), produces significant mal-effects on subjects. The most common syndromes of Gz application are cardiovascular deconditioning, black-out and red-out and loss of consciousness, which can finally lead to death. Sustained +2 and +3 -Gz produces cardiovascular deconditioning and decrease in CBF alongwith reduction of VEP (213). Soleus muscle mass is significantly larger while capillary density was significantly reduced for rats exposed to 3 g (214). Heart mass and mitochondrial volume were also larger in these rats. Fully matured vestibular epithelium of rats is not affected by 2.5 g exposure for 9 months starting at the age of 1 month with only a decrease in cell size (215, 216). On the other hand, exposing rats to hypergravity from embryonic stage till the age of 14 weeks increased the cell size without affecting the mechanosensory transduction in the vestibular system (217, 218). This suggests that possibly a gravity-dependent mechanism is present during a particular development stage (218). However, development of vestibular reflexes was delayed under hypergravity (219). Vestibular induced compensatory eye reflexes are affected by hypergravity during development (220, 221). Significant changes occur in myosin heavy chain (MHC)
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isoform expression under hypergravity in rats (222). Hypergravity during the foetal period and varying periods of time thereafter induces a delay in particular aspects of motor development, particularly in those reflexes dependent on vestibular inputs such as contact-righting, air-righting and negative geotaxis (223).
1.3.5 Humans An important physiological phenomenon occurring in microgravity is the shift in body fluids towards the cardiopulmonary compartment. More than half of the astronauts experience space sickness (in fact, a form of motion sickness), the signs and symptoms of which are stomach discomfort, nausea, pallor, cold sweating and vomiting. Lack of gravitational loading affects multiple physiological systems, especially fluid flow, balance, and support structures that are particularly vulnerable to change or injury during re-entry and renewed exposure to gravitational
forces.
Exposure
to
microgravity,
besides
affecting
the
neurovestibular and respiratory systems, greatly alters the dynamics of the circulation and leads to bone demineralization and muscle atrophy. When taken together, circulatory deconditioning and muscle atrophy lead to a reduced exercise capacity and intolerance. Orthostatic intolerance after spaceflight can be attributed to decreases of cardiac filling pressure and stroke volume during orthostatic stress due to decreased blood volume. It is generally believed that the above modifications are completely reversible upon re-entry to normal 1 g conditions even if it is still a matter of debate whether this is really the case after very long space flights. Spaceflight causes a fluid shift from the legs toward the head, producing a puffy face and bird-like legs. The fluid shift increases the amount of blood in the chest region, causing the heart and fluid-volume sensors in the neck to detect an increase in fluid volume. The increased chest fluid initially increases heart size (i.e. amount of blood), but regulatory mechanisms quickly kick in and return the fluid to an appropriate, lower level. The loss of fluid results in a reduced plasma or blood volume. To keep blood thin, the decrease in plasma volume triggers a destruction of newly synthesized, immature, red blood cells, probably by a mechanism of programmed cell death or apoptosis (224). The shift of fluids to the
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upper body and the distended facial veins noted in astronauts suggest that central venous pressure should increase. Surprisingly, it decreases, suggesting that our concepts of pressure and volume regulation need revision (225). These changes are appropriate for the spaceflight environment. However, upon return to Earth, many crew members have difficulty standing, usually due to the rush of blood to the feet that can cause fainting. This re-adaptation to Earth’s gravitational force following spaceflight could pose a problem if crews are expected to stand and function normally immediately after landing on any planetary body. Head-down tilt decreased thermoregulatory responses to heat stress (226). The gain of the aortic-cardiac baroreflex was increased during head-down tilt (227) while plasma volume was reduced (228, 229). Colour processing and opponent mechanisms for blue vs yellow are affected by head-down rest for 24 hours (230). Blood flow in the brachial and femoral arteries decreased during both lower body negative pressure and head-up tilt. The decrease for the former was found to be lesser while it was the reverse for the latter (231). Both create blood volume shifts to the lower body though their physiological aspects are not entirely equivalent (232). Cardiac atrophy was found to occur under microgravity conditions (233, 234). Cardiac volume increases in microgravity while central venous pressure (CVP) decreases. This is the result of an increase in cardiac transmural pressure in microgravity equalling CVP-intrathoracic pressure (235, 236). Mean arterial pressure (MAP) decreases during short-term weightlessness to below that of 1-G supine simultaneously with an increase in left artrial diameter (LAD). Distension of the heart and associated central vessels during microgravity might induce hypotensive effects through peripheral vasodilatation (237). Studies using parabolic flights have shown a decrease in the heart rate and arterial pressure (238-240). Central venous and oesophageal pressures decreased during parabolic flights (241). HR and HRV decreased during spaceflight (242, 243). This might probably due to lack of postural baroreflex stimulation (243). Long duration spaceflight reduces vagal-cardiac nerve traffic and decreases vagal baroreflex gain (244). However, sympathetic baroreflex gain is not reduced during parabolic flights (245). A simulated lunar trip consisting of a 4 day trip to
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the moon using -6° bed rest followed by a 6 day stay on the moon using +11° bed rest and a 4 day trip back to earth using -6° bed rest showed that lower limb haemodynamics adapted differently in various phases of simulation. Exposure to microgravity induced an increase in leg vein haemodynamics and slight modification in venous emptying parameters which tended to stabilise in partial gravity equivalent to that of the moon (246). On earth, using gravity measured by the otoliths as an external reference is an optimal strategy to determine self-orientation with respect to the environment (247). However, in microgravity, this strategy fails since the external reference is no longer available, but is apparently still assumed to exist by the central nervous system. Hence the information about changes of self-orientation detected by the vestibular system can cause a sensory conflict. Such conflicting information about the change of direction of gravity either given by otolith input or due to the internal processing, and from the canals about the change of self-orientation, can lead to spatial disorientation and illusions about actual body position and perceived motion. The brain uses an internal model of gravity to supplement sensory information (248). In space, the eyes send signals that confuse the brain because the visual references that we rely on for stability are missing. These mismatched sensory inputs may be one cause of ‘Space Adaptation Syndrome’ (SAS), an adaptive process that often involves nausea and can lead to vomiting (42). Another possible cause of SAS is sensor adaptation to a novel gravitational environment to increase the gain of sensory cells, possibly by increasing the number of synapses (249). Astronauts also often experience different types of spatial orientation illusions though performance on spatial tasks improves over time indicating that adaptation to microgravity does occur (250). The visual system possibly relies on the otolith and somatosensory information for 3D perception (251). Reflexes, associated with posture and balance, are slowed even on short-duration missions (42). With long-duration flights, changes in reflexes, visual perception, and eye/hand coordination may become major issues for reentry and re-adaptation to Earth. Intracranial pressure increases during transient weightlessness (252). Moderate horizontal sinusoidal linear acceleration applied
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in sitting human subjects can induce different muscle sympathetic nerve activity responses than those induced by caloric vestibular stimulation supporting the idea that otolith organs contribute to sympathetic regulation in humans (253). Sympathetic neural outflow increases moderately in space (254). A decrease in the performance of higher cognitive functions was observed during spaceflight (255). Under 2.5 Gz acceleration, coherences between both cerebral hemispheres tend to increase implying an improvement in functional coordination of both hemispheres and an enhancement in activation level (256). Gravity-related sensory input from muscle plays an important role in the regulation of the movement of the muscle itself (257). The musculoskeletal system is highly responsive to load. Without gravitational load, muscles and bones associated with posture and weight-bearing become weaker. With the fluid shifts and decreased bone loading, calcium is lost from bone and calcium excretion increases. The higher calcium load presented to the kidneys is of concern for potential kidney stone formation. During spaceflight, the amount of mineral in some bones, including the head, may increase to offset losses from other sites. Bone and muscle are lost only in the legs, back, and neck indicating that the musculoskeletal changes are site-specific i.e. loss does not occur throughout the entire body. Bone loss primarily occurs at sites in weight-bearing bones where muscles (that are also losing mass) attach to that bone. The muscles that help maintain posture are most severely affected and change phenotype. Muscle sympathetic nerve activity (MSNA) was found to be suppressed by microgravity while it was enhanced by hypergravity during parabolic flight (258, 259). The sympathoadrenal system is not impaired by exposure to microgravity (260). Microgravity is found to greatly reduce sleep-related apnoea and hypopnoea and snoring in healthy individuals suggesting that gravity plays a dominant role in the increase in upper airway resistance and obstruction which occurs after the transition to the supine posture and during all stages of sleep (261). Astronauts show low thyroid hormone levels after spaceflight possible due to increase of apoptosis in thyrocytes because of microgravity (108).
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By altering gravity, we are able to investigate those biological systems that were developed to detect or oppose this unique force. Decreasing gravity on earth for more than several seconds is impossible with existing technology. Until Sputnik was launched in October 1957, we had little opportunity to study how lowering this physical force influenced life. By decreasing gravity through spaceflight, we are beginning to understand that not only gravity, but also the physical changes that occur in the absence of gravity, may have profound effects on evolution of species and their ecologies. By going into space, we can gain a better understanding of how gravity shaped life on the Earth.
In this thesis, effort has been done to study the effects of different gravity conditions on insect, plant and the human cardiovascular system. A Java-based program developed for ECG analysis has also been discussed.
1.4 Thesis Outline Gravity
is the only physical parameter that has remained constant
throughout the history of evolution and hence could have played a major role in the evolutionary process. All biological systems have been developed in accordance with this constant force. Gravitational force is a pervasive environmental parameter that affects directly or indirectly, virtually all life on this planet. Experiments have shown that living systems respond and adapt to gravity on cellular and molecular levels. This has resulted into the development of gravireceptors and graviresponding systems in both lower and higher organisms. Aquatic organisms are affected by gravity largely indirectly by means of depthdependent hydrostatic pressure. Terrestrial organisms are subjected to increasing gravitational disturbance of body fluids necessitating dramatic modifications of fluid regulation and musculoskeletal systems related to support and locomotion which became increasingly important with evolution of bigger and taller organisms. The roots of plants are always directed towards gravity and hence exhibit positive geotropism. The shoots, on the other hand, show negative
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geotropism as they grow against the gravitational force towards sunlight. In humans, the brain is situated in the head region which is about 180 cm while the heart is situated about 150 cm from the ground. The heart, therefore, has to pump blood about 30 cm against gravity in order to supply blood to the brain. All veins, below the heart, are provided with valves in order to prevent backflow of the blood which is being carried back to the heart against gravity. Missions to space and the moon have put focus on the need of studying the effects of altered gravity on the various developmental and physiological processes. The science of gravitational biology took a giant step forward with the advent of the space program. It provided the first opportunity to examine living organisms in gravity environments lower than could be sustained on Earth. Organisms ranging in complexity from single cells through humans are responsive to Earth’s gravity and hence these organisms are most likely to be affected by changes in gravity. Our knowledge of the biological consequences of decreased gravity (i.e. spaceflight) has increased significantly since 1957, yet we only have snapshots of biological changes in multiple species. Although many physiological systems appear to be affected by spaceflight, only the human cardiovascular system is covered in this thesis. This thesis focuses primarily on altered gravity responses of insects, plants and the human cardiovascular system. The thesis has a total of seven chapters. The contents are briefly presented below: Chapter 1 – This chapter gives the introduction and also this deals with the relevant theoretical background and literature review. Chapter 2 – This chapter is dedicated to the design and development of the 1-D clinostat and clinostat-centrifuge for studying effects of altered gravity on plants and insects. Studies carried out on insects in order to test the clinostat have also been described. Chapter 3 - This chapter deals with the effects of microgravity on plant systems. Initial studies on the nature of graviresponse by bending of shoots and roots have also been described. Chapter 4 – This chapter describes the studies carried out on the effects of gravity and posture on the human cardiovascular system. These include both theoretical
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and experimental studies. A simple electric circuit model was developed for the change in blood pressure to change in g. The experiments done to study these changes on Sheersasana volunteers have been discussed. It also includes the design and development of a tilt-table which can be rotated through 360° in steps of 5°, 10°, 15°, etc. A Java-based program developed for ECG analysis has also been discussed. Chapter 5 - This chapter summarises the results of the work done and its future scope. Bibliography – This contains the list of references cited in this thesis.
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CHAPTER 2
Chapter 2: Clinostat and ClinostatCentrifuge 2.1 Introduction
The advent of space laboratories and of sophisticated instrumentation for biological research in altered gravity conditions has given great impetus to the research in gravitational physiology at the cellular level. Most of the work done to study the effects of microgravity on plants and cell cultures has been done in space labs like Mir (Russian space station) and the International Space Station (ISS). The limited access to real microgravity in space in terms of flight opportunities and the cost of such endeavours strongly recommends performing experiments in earthbound facilities producing conditions of simulated or of real short-term microgravity. Ground-based studies like parabolic flights are too expensive and the effects can be studied only for a period of few seconds. However, the low-cost approaches are required to be tested for biological systems which are sensitive to the gravity stimulus, to prepare spaceflight investigations and to conduct systematic studies in gravitational biology. Since the past three decades or so, the clinostat has become a very useful and important ground-based technique to study the effects of simulated microgravity (8, 9). In fact, considerable knowledge on the gravitational effects on single cells has been gained in clinostats and centrifuges (10, 11). The simplest kind of a clinostat is the 1-D clinostat in which the sample to be tested is fixed on the axis perpendicular to the gravity vector and rotated at speeds that are matched with the particular graviresponse time of the sample in question. Such rotations perform the function of nullification of the effects of gravity on the sample. The 2-D clinostat rotates the entire assembly about both axes normal to gravity. This is a better system for simulating microgravity than the 1-D clinostat as the gravity vector is nullified about two axes.
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Hoson T. has developed a 3-D clinostat in order to subject small biosystems (single cell cultures, unicellular organisms, plant seedlings) to vector-randomized gravity (12, 13). Probes are fixed as close as possible to the centre of a rotating frame which rotates within another rotating frame. Both frames are driven by separate motors. Rotation of each frame is random and autonomous and regulated by computer software. Gravitometers are fixed to the frame in order to record the gravity vectors during rotation. These 3-D clinostats also known as Random Positioning Machines (RPM) address the question of the three-dimensionality of biosystems. They are also perfect simulators of microgravity as in space (14). Schwarzenberg M. has shown that the RPM are better simulators of microgravity environment in space than the Free Fall Machine (FFM) (6). These simulate microgravity more effectively than 1-D and 2-D clinostats (13). However, they require very precise designing and hence are more costly compared to the 1-D and 2-D clinostats.
2.1.1 The Simulation of Microgravity The simulation of microgravity depends on a variety of factors, the response time, external factors such as buoyancy, temperature, light etc. If the gravistimulus changes constantly and faster than the response time, the system is unable to respond to the stimulus. However, the compensation of gravity by rotation must be limited to avoid centrifugal forces that may result in unrelated effects on the system of interest. Of all the possible ways of simulating microgravity or zero-gravity conditions on Earth - parabolic flights that allow utmost a few seconds of free-fall, sounding rockets, drop-towers that would have to be of sky-scraping heights to provide enough time to collect any data, buoyancy tanks, the clinostat and its variants are the most practical and economical means within the reach of any experimentalist. The simplest kind of a clinostat is the 1-D clinostat in which the sample to be tested is fixed on the axis perpendicular to the gravity vector and rotated at speeds that are matched with the particular graviresponse time of the sample in
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question. Such rotations perform the function of nullification of the effects of gravity on the sample.
As shown in Figure 2.2, this rotation seen from the
reference frame of the cell is akin to rotating the gravity vector centred at the centre of the cell and rotated with the angular velocity of the horizontal assembly. Thus the time-averaged gravity vector is zero.
Figure 2.1: A ‘stationary’ cell
Figure 2.2: Cell rotated about an axis passing through its centre
2.2 Instrumental Design of Clinostat A 1-D clinostat had been designed and developed in this laboratory to study the effects of simulated microgravity on plant and insect systems. Figure 2.3 shows the skeletal design of this clinostat. The clinostat consists of the following parts.
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4
3
6
5
1 2
Figure 2.3: Schematic sketch of the 1-D clinostat
1.
0-10 rpm (0-12 V variable DC, Imax = 3 A) motor
2.
Motor mount
3.
Connectors
4.
Bearing block made of Aluminium
5.
Bearings
6.
Sample holder All parts except the sample holder were made of aluminium so that the
clinostat would be light. The sample holder was made of mild-steel in order to make it robust. The diameter of the sample holder was 5 cm, the diameter of a 100 ml beaker. Beakers of dimensions (outer diameter = 7.5 cm, inner diameter = 6.5 cm, height = 10 cm) made of Perspex/acrylic with a base diameter of 5 cm were used for studies on plants. For studies on insects, Perspex beakers of dimensions (outer diameter = 6.5 cm, inner diameter = 5 cm, height = 7 cm) were used. The developed clinostat is shown in the figure below.
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Figure 2.4: The 1-D clinostat designed and developed in the laboratory
Calibration of clinostat motor
12
y = 0.895x - 0.321
10
ω rpm
8 6 4 2 0 0
2
4
6
8 V Volts
10
12
14
Figure 2.5: Calibration curve (RPM vs. Voltage) of clinostat
2.3 Testing of clinostat 2.3.1 Introduction Experiments on Drosophila melanogaster carried out by Marco and colleagues in space and on ground indicate that behavioural responses that may be important in setting life-spans of organisms, for example, may still be readily susceptible to manipulation by external cues (18). Drosophila melanogaster adults exposed to microgravity markedly increase their motility. This change in behaviour might be a cause of faster aging on the ground after their recovery from space (173). Their
64
group also detected that short-lived strains show high levels of motility from the very beginning of their lives while long-lived ones show reduced motility during initial part of their life spans reaching higher levels similar to short-lived ones in their later stages of their lives (174). Drosophila melanogaster is able to sense the gravity vector showing a clear negative geotactic response (173). The altered behavioural response to microgravity may have something to do with this gravitaxic response as very young flies i.e., the ones immediately hatched out from the pupal case, show higher responses both behavioural and of accelerated aging. In this section, the effects of clinorotation on the development of Chironomus ramosus have been discussed. Chironomus is the scientific name of non-biting midge (262, 263). It has been selected for experimental purpose for the following three important reasons. 1.
Eggs of Chironomid midge are very transparent and hence, it is very easy to
see the effect of stimulus as changes in egg structure inside egg sac. 2.
Hatching period is only 48 hours at 25 ± 1 ºC. Hence it is easier to see the effect
of applied stimulus within a short time. 3.
Eggs of harlequin fly are easily available in stagnant and running water bodies. Hence, by applying altered gravity conditions on Chironomus, the changes
within the eggs were directly observed taking advantage of their transparency. Chironomus has been established as an excellent organism to carry out behavioural studies for exploring innate behavioural traits. The embryonic developmental stages of Chironomus have been studied (264, 265). Also its chromosomes have been characterised (266). Also effects of stresses on Chironomus like heat shock (267) have been studied. The life-cycle of Chironomus consists of 4 stages viz., egg, larva, pupa and adult. Of these, only the adult stage is non-aquatic. An adult female lays about 300500 eggs which are enclosed in a jelly-like mass. The jelly can be dissolved by sodium hypochlorite when necessary. Normal eggs at early stages have a layer of
65
transparent cytoplasm on the surface and the interior of the eggs is filled with retractile oil droplets and brown ‘yolk’ granules. In sections, besides the superficial cytoplasmic layer, many cytoplasmic islands are connected with each other and also with the superficial layer by means cytoplasmic strands.
Egg mass
Larva
Pupa
Adult Figure 2.6: Life-cycle of Chironomus
66
Table 2.1: Stages of embryonic development in Chironomus
Stage
Time in hours at 22 0C water Developmental events temperature
1
0-6
Homogeneous nature of egg cytoplasm
2
6-7½
Pole cells form
3
7½ - 8½
Syncytial blastoderm
4
8½ - 11
Cellularization of the blastoderm
5
11-12
Gastrulation
6
12-14
Germ band elongation (early phase)
7
14-16
Germ band elongation (late phase)
8
16-19
Contraction of germ band
9
19-24
Organogenesis (initial phase)
10
24-30
Organogenesis (mid phase)
11
30-36
Organogenesis (final phase)
12
36-42
Beginning Periodic contraction & undulation
Hatching
42-48
Hatch to first instar Larva
Embryonic Development Stages 1)
Stage 1 - Homogenous nature of egg cytoplasm.
2)
Stage 2 - Pole cell formation.
3)
Stage 3 – Syncytial blastoderm.
4)
Stage 4 - Cellularization of blastodem.
5)
Stage 5 - Gastrula.
6)
Stage 6 - Germ band elongation.
7)
Stage 7 - Late phase of germ band elongation.
8)
Stages 8-10 – Organogenesis phases.
9)
Stages 11-12 – Pre-hatching stages.
67
1)
2)
3)
4)
5)
6)
7)
8)
9)
Figure 2.7: Embryonic Development of Chironomus
2.3.2 Materials and Methods Egg masses collected from the fields were observed under a microscope. Stage 2 (pole cell formation) of the eggs were identified using a map shown in Figure 3.2 (265). Then each egg mass was divided into two equal halves. One half was placed in the clinostat while the other half was kept in normal 1g conditions, which served as control. The clinostat rotation was kept at about 2.7 rpm as it was experimentally observed that the eggs would just begin to float when the speed of clinorotation is 2.5-3 rpm. The eggs were clinorotated for durations of 6, 12, 18 and 24 hours. After clinorotation, the eggs were observed under a microscope. Both control and clinorotated eggs were observed for any change in morphology and then allowed to develop under normal conditions.
2.3.3 Results Eggs exposed to 6 hours of clinorotation hatched about 6 hours earlier than the normal 48 hours. For clinorotation of 12 hours, hatching of eggs occurred in about 30 hours while eggs hatched within 28 hours in case of 18 hours of clinorotation. When the eggs were clinorotated for 24 hours, around 75-80% of the
68
eggs had hatched after 24 hours while the remaining where in last stages prior to hatching (Figure 2.11). Figure 2.12 shows the comparison of hatching periods for different durations of clinorotation. From Figure 2.8, it can be seen that the eggs clinorotated for 6 hours are in stage 6 in 12 hours compared to the normal 14 hours and are still slightly ahead of the control till hatching. Eggs clinorotated for 12 hours have reached stage 10 in 18 hours as compared to the normal 30 hours (Figure 2.9) while the eggs clinorotated for 18 hours are in stage 11 within 24 hours instead of the normal 36 hours (Figure 2.10). Eggs clinorotated for 12 and 18 hours are much ahead of the control till hatching. 24 hours of clinorotation resulted in hatching of about 80% of the eggs while the remaining eggs where in stages 11 and 12 (Figure 2.11). No apparent deformities were observed in the larva hatched from the clinorotated eggs. Also the undulation frequency was 1-2 undulations per second which is normal. Control Clino
6 hours clinorotation
48
Time (hours)
36
24
12
0 0
2
4
6
8
10
12
14
Stages
Figure 2.8: Stages of development after 6 hours of clinorotation upto hatching
69
Control Clino
12 hours clinorotation
48
Time (hours)
36
24
12
0 0
2
4
6
8
10
12
14
Stages
Figure 2.9: Stages of development after 12 hours of clinorotation upto hatching
Control Clino
18 hours clinorotation 50
Time (hours)
40
30
20
10
0 0
2
4
6
8
10
12
14
Stages
Figure 2.10: Stages of development after 24 hours of clinorotation upto hatching
70
Figure 2.11: Development after 24 hours of clinorotation
Hatching period (hours)
48
36
24
12
0 0
6
12
18
24
Duration of clinorotation (hours)
Figure 2.12: Histogram showing variation in hatching period with duration of clinorotation. Zero hours of clinorotated sample represents control.
2.3.4 Discussion It was observed that simulated microgravity speeded up the development of the eggs. As the duration of simulated microgravity increased, the development of eggs became faster which can be seen from the decrease in hatching period. As no apparent deformities were observed, it can be said that microgravity only enhances faster development of Chironomus eggs.
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2.4 Instrumental Design of Clinostat-Centrifuge A clinostat-centrifuge system can be used to study the effects of varied gravity environments including hypogravity and hypergravity on plants, insects and cell cultures. For this purpose, a clinostat-centrifuge has been designed and developed. The system consists of two clinostats mounted on a centrifuge. The design of the clinostat part has been described in section 2.2. For the centrifuge part, a 20-1500 rpm (0-180 V variable DC, Imax = 0.8 A) motor was used. All the components were made of aluminium in order to reduce load on the motor. Figure 2.13 depicts the skeletal design of this clinostat-centrifuge. The speed of rotation of the centrifuge motor was set using Eqn. 1.5 with the help of a tachometer for measuring the angular speed (ω) in rpm.
Figure 2.13: Schematic sketch of the clinostat-centrifuge
The clinostat-centrifuge consists of the following parts.
72
1) Motor Drive for centrifuge 2) Clinostat Motors 3) Sample Holders 4) Gears 5) Pulleys 6) Chamber 7) Supports 8) Rotating contacts 9) For Tachometer
a) For tachom eter 0-1500 rpm m otor Sample holder Perspex beaker
b)
0 -10 rp m m o to r
S am p le h o ld er
Figure 2.14: The clinostat-centrifuge designed and developed in the laboratory
a) Top view b) Side view
73
Calibration of clinostat-centrifuge
1200 1000
y = 6.5046x - 100.52
ω rpm
800 600 400 200 0 -10
10
30
50
70
90
110
130
150
170
-200 V Volts
a) Calibration of clinostat-centrifuge
5
y = 0.0002x2 - 0.0105x + 0.1152 4
ω rpm
3
2
1
0 0
20
40
60
80 100 V Volts
120
140
160
b) Figure 2.15: Calibration curve of clinostat-centrifuge a) ω vs. Voltage b) g vs. Voltage
In this thesis, however, only the clinostat has been used. Studies on hypoand hypergravity have not been included.
2.5 Conclusions This chapter describes the design and development of a 1-D clinostat to study the effects of simulated microgravity on insects, plants and cell cultures. It was seen that clinorotation enhances the development of Chironomus (non-biting midge) eggs. Also the design and development of a clinostat-centrifuge to study the effects of simulated hypogravity and hypergravity has been described.
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CHAPTER 3
Chapter 3: Effects of Altered Gravity Environments on Plants 3.1 Introduction
Several ground-based
studies have been carried out using clinostats to
study the effects of simulated microgravity on plants. For example, clinorotation caused straightening of gravitropically curved roots in cress (129). Microgravity stimulates elongation growth of rice coleoptiles (13, 131), Arabidopsis hypocotyls (13, 132) and etiolated pea seedlings (133) and also emergence of leaf in maize coleoptiles (133). Seed germination is faster in microgravity for flax (134) and rice (135). Also the root lengths were found to be higher (134, 135). Ivanova and colleagues have reported the non-production of seeds in wheat grown in space (136). Polysaccharide synthesis decreased in microgravity. Lysis of cuticle cellulose microfibrilles and matrix polysaccharides led to wall loosening and increased cuticular transpiration during weightlessness (65). Simulated microgravity enhanced respiration in oat seedlings (148). Metabolic activity was also enhanced which was observed in increased root lengths. Foliar amount of carotenoids, and chlorophyll a and b, were substantially reduced in tomato plants under simulated microgravity conditions (130). Chlorophyll content decreased while carotenoid content increased for clinorotated Arabidopsis thaliana plants. Photosynthetic activity also decreased in simulated microgravity (71). Chlorophyll a/b ratio increased in Brassica rapa during spaceflight. Photo-inhibition of PSI is induced by microgravity without any photodamage of PSII (67). Short-term microgravity increases leaf temperature and decreases net photosynthetic rate in barley leaves (153). Chlorophyll a content increased significantly in Arabidopsis during clinorotation while no such change was observed in pea. Biosynthesis of chlorophyll b was inhibited in clinorotated
76
pea plants while no change was observed for Arabidopsis (66). Leaf senescence in oats is promoted by both clinorotation (152, 154) and centrifugation (154).
3.2 Initial studies on the nature of graviresponse Initial experiments were carried out using vertical and horizontal tilt positions. These experiments helped to establish that plants can be used to observe the changes under altered gravity conditions.
3.2.1 Materials and Methods Seeds of Brassica (mustard) were used due to their short germination period (1-2 days) and relatively fast growth rate as compared to other species commonly used for such experiments (Arabidopsis, Soy bean). Response to gravity was studied under different conditions of incident light for roots and stems. Stems were allowed to develop in flasks and roots in Petri dishes as the roots do not grow as straight as desired in the agar gel and were easier to study by marking the growth on the lid of the Petri dish. The stems: Taking a plumb line as a reference, sectors were drawn on a plain white sheet of paper which was then placed behind the flask containing the stems. The initial positions and orientations of the stems were marked on the flask. Distortions due to refraction taking place through the glass flask were ignored. The roots: The initial positions and orientations of the seeds and roots were marked on the glass lid of the Petri dish and subsequent positions were marked as points that were joined later using a fine-tipped permanent marker. Guided by previous observations, different parts of the roots (the middle, the tip) were marked differently.
3.2.2 Results It was found that the responses for both roots and stems differed in the nature and time of response. Stems appeared to respond faster to gravity and the speed of response reduced only in the absence of sunlight. The speed of response of different regions of the roots was different. The speed of response decreased with increasing distance from the root tip. The growth
77
rate of both stems and roots did not appear to be affected much by the reorientation of the plants. Stems in Ambient Sunlight 100
90
80
Curvature(degrees)
70
60
50
40
30
20
10
0 0
2
4
6
8
10
12
14
16
Time (hrs)
Figure 3.1: Graviresponse of stems grown in ambient sunlight with time
Rate of Growth 5.2
Control
4.7
Length cms
4.2
3.7
3.2
Test 2.7
2.2 0
2
4
6
8
10
12
14
16
18
20
Time(hrs)
Figure 3.2: Rate of growth of shoot grown in ambient sunlight with time
78
Stems grown in darkness 90
80
curavture(degrees)
70
60
50
40
30
20
10
0 0
2
4
6
8
10
12
14
16
18
20
time (hrs)
Figure 3.3: Graviresponse of stems grown in darkness with time
Rate of Growth 3.1
2.9
Control 2.7
Test
Length cms
2.5
2.3
2.1
1.9
1.7
1.5 0
2
4
6
8
10
12
14
Time(hrs)
Figure 3.4: Rate of growth of stems grown in darkness with time
79
16
Root graviresponse 100
90
80
curvature(degrees)
tip 70
60
middle 50
40
30
20
10 2
4
6
8
10
12
14
16
time(hrs)
Figure 3.5: Graviresponse of roots grown in ambient sunlight with time
root growth 0.7
0.6
Control
Growth ( cms )
0.5
test 0.4
0.3
0.2
0.1
0 0
2
4
6
8
10
12
14
16
time ( hrs )
Figure 3.6: Graviresponse of roots grown in ambient sunlight with time
80
Soyabean stems in ambient sunlight 100 90
80
70
curvature
60
50 40
30
20
10 0 0
5
10
15
20
25
30
time (hrs)
Figure 3.7: Graviresponse of Soy bean stems grown in ambient sunlight with time
This data is representative of 8 samples for stems and 10 samples for roots of Brassica and 3 samples for Soy bean. Graviresponse at the roots and the stems differed widely. The orientation of the stem towards the vertical was more pronounced than the orientation of the root which tended to ‘sag’ rather than bend in the downward direction. The rate of growth in both the cases slowed after reorientation of the plant. Differences in the growth rate were more pronounced in the case of the stems.
3.2.3 Discussions The bending of the organ is caused by the different growth rates (cell expansion) at the top and the bottom of the organ. In the case of roots, the growth is inhibited at the bottom and exaggerated at the top, hence the root bends down. Conversely, in the case of stems, growth is inhibited at the top and accelerated at the bottom leading to an upward bend. It was also noted that reorientation of the plants produced the same effects i.e. the same rate of curvature in all cases. Repeated reorientation too did not appear to affect the graviresponse in any way. The responses in the two species that were tested differed. Bending in the case of the Soy bean stems took more time than bending in Brassica.
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3.3 Preliminary experiments on the clinostat Based on the results obtained from the studies described in the previous section, the 1-D clinostat designed and developed in the laboratory was used to carry out experiments on plants under simulated microgravity conditions.
3.3.1 Materials and Methods Rice seeds had been used. The seeds were imbibed in distilled water for a period of 24 hours in a sealed container and were then sterilized using 10 percent ethanol solution to avoid fungal infection. The containers used were also sterilized using 10 percent ethanol. The seeds were then mounted on 0.8 percent agar gel as substrate in two beakers made up of Perspex. One beaker was placed in the horizontal axis clinostat and was rotated at 2.7 rpm for 12 hours while the other beaker acted as control. After 12 hours, the beaker was removed from the clinostat and both of these beakers were placed in normal gravity for 2 weeks and the seedlings were allowed to grow. The experiment was conducted in a room where the air temperature was about 25-30 °C. The number of germinated seeds was counted every 12 hours till the last day. Also length of shoot and root of each seedling were measured in the interval of every 12 hours. From the fourth day, UV-Vis absorption spectra of chlorophyll isolated from the shoots of the rice plants in both clinorotated and control were taken for each alternate day. Chlorophyll was isolated by crushing shoots in 80 percent acetone using a mortar-pestle so that the concentration of the solution would be the same.
3.3.2 Results 3.3.2.1 Direction of roots and shoots It was seen that the direction of root in normal gravity was downward direction and vertical while the direction of roots in microgravity condition was not exactly vertically downward but was slightly inclined to vertical direction. Also the number of secondary roots in the microgravity samples was less
82
compared with those in control. But shoot growth was in the vertically upward direction in both the samples. 3.3.2.2 Growth of roots and shoots It was seen that the initial growth of shoots in microgravity samples was higher than that of in control samples for the day set. But it was seen that after 9th day, there was almost no difference in the growth of shoots in the microgravity and control samples. However, for the night set, there was almost no difference in the initial growth of shoots in the microgravity and control samples. After the 9th day, shoot growth was higher in the microgravity samples compared to control. In case of night sown seeds, root growth was lower in the microgravity samples as compared with that in the control samples. But in case of day sown seeds, root growth was higher in the microgravity samples as compared with that in the control samples. The graphs of these results are shown in Figure 3.8 below.
83
micro control 20
Shoot length vs Days
16
16
12
12
Shoot length cm
Shoot length cm
20
micro control
8
8
4
4
0
0
0
4
8
12
Shoot length vs Days
0
16
4
8
12
16
Days
Days
micro control 9
micro control 9
Root length vs Days
6
Root length cm
Root length cm
6
Root length vs Days
3
3
0
0 0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.5
4.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Days
Days
Night set
Day Set
Figure 3.8: Shoot and root lengths measured every 12 hours after 12 hours of clinorotation
84
4.5
3.3.2.3 UV-Vis Spectra Chlorophyll was extracted from the shoots of rice plants of both control and microgravity samples and their absorption spectra were recorded. From the recorded spectrum, it was seen that, there was no change in the position of the main absorption peaks in both microgravity and control samples. But, there was significant change in the absorbance. The peak positions occurred at wavelengths 435 nm and 660 nm. This indicates the presence of chlorophyll b and chlorophyll a respectively in the samples. From the spectra recorded on 6th, 8th, and 10th day in microgravity and control samples for day and night set, absorbance for chlorophyll b and chlorophyll a was measured. These spectra are shown in Figure 3.9 below. From the table, it is clear that the absorbance is less in microgravity samples as compared with those in control in both day and night experiments.
Table 3.1: Measurement of absorbance
Day
Wavelength nm
Day Set Absorbance Control Micro
Night Set Absorbance Control Micro
6
435 665
0.21 0.13
0.55 0.43
0.04 0.19
0.08 0.1
8
435 665
0.05 0.03
0.017 0.001
0.21 0.24
0.22 0.25
10
435 665
0.17 0.14
0.11 0.10
0.09 0.1
0.13 0.15
85
0.6
control micro
Day 6
0.6
0.4
Absorbance (a.u.)
0.4
Absorpance
control micro
Day 6
0.2
0.2
0.0
0.0
400
500
600
400
700
500
0.6
600
700
λ (nm)
λ nm
control micro
Day 8
0.4
Day 8
0.3
Absorbance (a.u.)
Absorpance
0.4
0.2
Control micro 0.2
0.1
0.0 0.0
450
550
650
400
750
500
Day 10
0.6
0.6
control micro
0.5
Day 10
control micro
0.4
Absorbance (a.u.)
Absorpance (a.u.)
700
0.5
0.4
0.3
0.2
0.3
0.2
0.1
0.1
0.0
0.0
350
600
λ (nm)
λ nm
450
550
650
400
750
500
600
700
λ (nm)
λ (nm)
Night set
Day Set
Figure 3.9: Absorption spectra of chlorophyll recorded every alternate day from the 6th day since 12 hours of clinorotation
86
3.3.3 Discussion The seeds sown in day showed more shoot and root growth in the microgravity samples as compared with control. The seeds sown at night also showed similar results for shoot growth. But root growth was higher in the microgravity samples for seeds sown in day while it was lower for those sown at night as compared with control. This means that the time (day or night) at which the seeds are sown might affect the growth of shoots as well as roots. Day set gave more consistent results and also showed fewer deviations as compared with night set for root and shoot lengths. This may be reasoned on the basis of the biological rhythms of plant systems. As the absorbance obtained from absorption spectra is proportional to chlorophyll content, it can be said that chlorophyll content in microgravity samples was higher than in those of control for both the sets on the 6th day. Chlorophyll content remained higher in microgravity samples for the night set while it was lower in that of control for the day set on the 8th and 10th day. This can be possibly attributed to faster senescence in microgravity samples compared to those in control.
3.4 Experiments with the clinostat for longer periods 3.4.1 Materials and Methods Rice (Oryza sativa v. PRH 10 obtained from National Seeds Corporation, Govt. of India) seeds were kept in 0.5 percent fungicide (Uthane) for 5-10 minutes and washed thoroughly with distilled water to remove traces of fungicide. They were then imbibed in distilled water for a period of 24 hours in a sealed beaker. The seeds were then mounted on 0.8 percent agar gel as substrate in two beakers made up of Perspex. The beakers used were also sterilized using 0.5 percent fungicide (Uthane) for 5-10 minutes and washed thoroughly with distilled water to remove traces of fungicide. One of the beakers was placed in the 1-D clinostat and was rotated for durations of 3, 5 and 7 days at speeds calculated using the equation derived by
87
Dedolph (268) while the other beaker acted as control which was also kept horizontally. The experiment was conducted under ambient conditions of temperature and humidity and a light intensity of 1250 Lux for 16 hours photoperiod per day. On the last day, root and shoot lengths were measured for both control and clinorotated samples.
Also weights of shoots were taken.
Chlorophyll was isolated by crushing shoots in 80 percent acetone using a mortarpestle so that the concentration of the solution would be 5 mg/ml. UV-Vis absorption spectra and fluorescence spectra were then recorded for both the samples. Chlorophyll content was calculated using Arnon’s formula (269).
3.4.2 Results 3.4.2.1 Direction of roots and shoots It was seen that direction of roots and shoots in the clinorotated samples was horizontal while that of control was vertical. 3.4.2.2 Growth of roots and shoots Primary root length was higher for samples clinorotated for 3 and 7 days than control while it was lower for samples clinorotated for 5 days. Similar results were obtained for shoot length as well as shoot weight. Primary root length (cm)
Control 10
Clino
8 6 4 2 0 3 days
5 days
7 days
Days
Figure 3.10: Primary root length vs. duration of clinorotation
Control shoot length (cm)
5
Clino
4 3 2 1 0 3 days
5 days
7 days
Days
Figure 3.11: Shoot length vs. duration of clinorotation
88
Control
Shoot Wt. (gm)
0.4
Clino
0.3
0.2
0.1
0
3 days
5 days
7 days
Days
Figure 3.12: Shoot weight vs. duration of clinorotation
3.4.2.3 Chlorophyll content Absorption and fluorescence spectra showed that chlorophyll a content was lower for all clinorotated samples than control. Chlorophyll b content and total chlorophyll content were also lower for all clinorotated samples than control.
0.5
0.5
0.4
Absorbance (a.u.)
0.4
0.3
0.2
0.1
0.3
0.2
0.1
0.0
0.0 400
450
500
550
600
650
700
750
400
λ nm
450
500
550
600
650
700
7 50
λ nm
a)
b) 0.5 C ontrol C lino 0.4
Absorban ce (a .u.)
Absorbance (a.u.)
Control Clino
C ontrol C lino
0.3
0.2
0.1
0.0 400
500
600
700
λ nm
c)
Figure 3.13: Absorption spectra of chlorophyll for different durations of clinorotation a) 3 days b) 5 days c) 7 days
89
1000
1000 Control Clino
Control Clino 800
Fluorescence (a.u.)
Fluorescence (a.u.)
800
600
400
200
600
400
200
0
0 600
650
700
750
600
650
700
λ nm
λ nm
a)
b)
750
1000 C ontrol C lino
Fluorescence (a.u.)
800
600
400
200
0 600
650
700
750
λ nm
c)
Figure 3.14: Fluorescence spectra of chlorophyll for different durations of clinorotation a) 3 days b) 5 days c) 7 days
Table 3.2: Chlorophyll content calculated using Arnon’s Formula (269)
Days
Chl a content
Chl b content
Total Chl content
mg/litre
mg/litre
mg/litre
Control
Clino
Control Clino
Control
Clino
3
0. 453
0. 299
0.654
0.359
1.25
0.739
5
1.2
0.55
0.68
0.38
2.075
1.034
7
2.74
2.33
0.93
0.83
4.013
3.452
3.4.3 Discussion Simulated microgravity enhances the growth and development of rice seeds. Spaceflights also show similar results for flax (134) and rice (135). The decrease in chlorophyll content for clinorotated samples suggest that simulated
90
microgravity promotes faster senescence in rice. This is in agreement with the results reported for pea (66), Arabidopsis (66, 71) and tomato (130).
3.5 Conclusions Preliminary studies of plant gravitropism were conducted wherein protocols for setting up conditions conducive for the growth of the samples were identified and the basic methods of studying gravitropism were explored. Studies on the clinostat showed that simulated microgravity enhances the growth and development of rice seeds. Interestingly, simulated microgravity also enhances faster senescence in rice.
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CHAPTER 4
Chapter 4: Effects of Gravity on the Human Cardiovascular System 4.1 Introduction
An important physiological phenomenon occurring in microgravity is the shift in body fluids towards the cardiopulmonary compartment. The fluid shift increases the amount of blood in the chest region, causing the heart and fluidvolume sensors in the neck to detect an increase in fluid volume. The increased chest fluid initially increases heart size (i.e. amount of blood), but regulatory mechanisms quickly kick in and return the fluid to an appropriate, lower level. The loss of fluid results in a reduced plasma or blood volume. To keep blood thin, the decrease in plasma volume triggers a destruction of newly synthesized, immature, red blood cells, probably by a mechanism of programmed cell death or apoptosis (224). These changes are appropriate for the spaceflight environment. However, upon return to Earth, many crew members have difficulty in standing, usually due to the rush of blood to the feet that can cause fainting. This re-adaptation to Earth’s gravitational force following spaceflight could pose a problem if crews are expected to stand and function normally immediately after landing on any planetary body. Orthostatic intolerance after spaceflight can be attributed to decrease of cardiac filling pressure and stroke volume during orthostatic stress due to decreased blood volume. It is generally believed that the above modifications are completely reversible upon re-entry to normal 1g conditions even if it is still a matter of debate whether this is really the case after very long space flights. Studies show that the gain of the aortic-cardiac baroreflex increased during head-down tilt (227) while plasma volume was reduced (228, 229). Blood flow in the brachial and femoral arteries decreased during both lower body negative pressure and head-up tilt. The decrease for the former was found to be lesser while it was the reverse for the latter (231). Both create blood volume shifts
93
to the lower body though their physiological aspects are not entirely equivalent (232). Cardiac atrophy was found to occur under microgravity conditions (233, 234). Cardiac volume increases in microgravity while central venous pressure (CVP) decreases. This is the result of an increase in cardiac transmural pressure in microgravity equalling CVP-intrathoracic pressure (235, 236). Mean arterial pressure (MAP) decreases during short-term weightlessness to below that of 1-G supine, simultaneously with an increase in left artrial diameter (LAD). Distension of the heart and associated central vessels during microgravity might induce hypotensive effects through peripheral vasodilatation (237).
Studies using
parabolic flights have shown a decrease in the heart rate and arterial pressure (238-240). Central venous and oesophageal pressures decreased during parabolic flights (241). Central venous pressure during short (20 seconds) and longer (3 hours) periods of microgravity is close to or below that of the supine position on the ground (242). HR and HRV decreased during spaceflight (235, 242, 243) probably due to lack of postural baroreflex stimulation (243). Long duration spaceflight reduces vagal-cardiac nerve traffic and decreases vagal baroreflex gain (244). However, sympathetic baroreflex gain is not reduced during parabolic flights (245).
4.2 Modelling the Cardiovascular System In order to study the effects of gravity on the cardiovascular system, it is useful to have a model of the cardiovascular system. Hence, in order to gain a deeper understanding of the cardiovascular phenomenon, an effort was made to develop a model analogous to an electric circuit for the blood flow in a blood vessel. In this model, the blood pressure is considered equivalent to the voltage, the flow velocity to the current, the peripheral resistance to the electrical resistance and the compliance (elasticity) of the blood vessel to the capacitance. This is shown in Figure 4.1.
94
ri∆z
ri∆z
A
Ii (z, t)
Km∆
Cm∆z
Vc
ri∆z
B
Ii (z+∆z, t)
Km∆
Cm∆
Figure 4.1: Electric circuit model of cardiovascular system
ri: Resistance per unit length cm: Capacitance/compliance per unit length of the vessel wall km: Current/flow per unit length in the membrane Vc: Pressure/voltage across the membrane
k m ∆z = c m ∆z
∂Vc ( z , t ) ∂V ( z , t ) ∂V ( z , t ) ⇒ k m = cm c = cm i ∂t ∂t ∂t
(4.1)
as Vc = Vi assuming the resistance of cm: is zero. Applying Kirchoff’s law at junction A, we get,
Ii ( z , t ) = Ii ( z + ∆z, t ) + k m ∆z
or
∂I i ( z , t ) ∂V ( z , t ) = − k m = −c m i ∂z ∂t
(4.2)
Vi ( z , t ) = Vi ( z + ∆z, t ) + ri ∆zIi ( z + ∆z, t )
or ∂Vi ( z , t ) = −ri I i ( z , t ) ∂z
Differentiating Eqns. 4.2 and 4.3 partially again with z, we get,
95
(4.3)
∂ 2Vi ( z , t ) ∂I ( z , t ) = − ri i 2 ∂z ∂t
(4.4)
Substituting Eqn. 4.2 in Eqn. 4.4, we get, ∂ 2Vi ( z , t ) ∂V ( z, t ) = ri cm i 2 ∂z ∂t
(4.5)
The left-hand side of Eqn. 4.5 is independent of t while the right-hand side is independent of z. Hence they can be separated if we assume ri and cm to be constants independent of z and t. In other words, Vi ( z , t ) = Viz ( z )Vit (t )
(4.6)
Substituting this in Eqn. 4.5, we get, Vit (t )
∂ 2Viz ∂V (t ) = ri cmViz ( z ) it 2 ∂z ∂t
or
1 ∂ 2Viz ri c m ∂Vit (t ) = Viz ∂z 2 Vit ∂t
(4.7)
We can therefore solve for Viz and Vit independently.
1 d 2Viz = −a 2 2 Viz dz
(4.8a )
and ri c m dVit (t ) = −a 2 Vit dt
Rearranging Eqn. 4.8a, we get,
d 2Viz + a 2Viz = 0 dz 2 This has a solution of the form
Viz = V0 iz e iaz If a = k +
i
α
, we get,
96
(4.8b) (4.8)
Viz = V0iz eikz e − z / α As eikz = cos kz + i sin kz , we have
Viz = V0iz (cos kz + i sin kz )e − z / α At z = 0 , Viz = V0iz while at z =
π 2k
, Viz = iV0iz e −π / 2α k . Hence only the real part of Viz is
acceptable.
Viz = V0 iz cos kze − z / α
(4.9)
Rearranging Eqn. 4.8b, we get, dVit (t ) a2 =− Vit dt ri c m
or dVit (t ) a2 =− dt ri c m Vit
Integrating both sides we get, ln Vit = −
a2 t +b ri c m
At t = 0 , Vit = V0it ⇒ b = ln V0it . ln
Vit a2 =− t V0 it ri c m
or −
Vit = V0it e a=k+
i
α
∴ Vit = V0 it e
a2 t ri c m
⇒ a2 = k 2 + −( k 2 +
1
α2
α2
) t ri c m
Substituting for Viz and Vit in Eqn. 4.6, we get,
97
1
(4.10)
Vi ( z , t ) = V0 izV0 it cos kze
− z /α
−( k 2 +
e
1
α2
) t ri c m
or
Vi ( z , t ) = VS cos kze
− z /α
−( k 2 +
e
1
α2
) t ri c m
where VS represents the systolic blood pressure. After 1 systole of the heart, the blood pressure drops to the diastolic blood pressure VD, i.e. at t = t s , Vi = VD . Heart level is taken as the zero point and hence z = 0 .
∴ VD = VS e ∴ (k 2 +
1
α
2
)
−( k 2 +
1
α2
) tS ri c m
tS V = ln S ri c m VD
or k2 +
1
α
2
=
ri c m VS ln tS VD
Vi ( z , t ) = VS cos kze
− z /α
− ln(
e
(4.11) VS )t tS VD
(4.12)
Incorporating the fluid/hydrostatic pressure of blood in Eqn. 4.12, we get the net pressure of blood as below.
V( z , t ) = VS cos kze
− z /α
− ln(
e
VS ) t tS VD
− ρ gz
(4.13)
Eqn. 4.13 gives the variation of blood pressure with distance z and time t suggesting a linear relationship of V with g. Figure 4.2 shows the variation of V as a function of z and t for different values of g (gsinθ for different values of θ).
98
Figure 4.2: V as a function of z and t with z = 0 to 1000 mm and t = 0 to 1000 ms
4.3 Initial Experiments On the basis of the understanding gained through the theoretical exercise which provides the nature of variation in blood pressure as a function of g, the following experiment was planned. This experiment was carried out to explore changes caused due to variation of g in a tilting position. To begin with experiments were carried out using a tilt-table facility in a gym whose tilt could be adjusted between +90º (upright position) and -90º (upside position).
4.3.1 Materials and Methods Subjects were attached to a platform which could be rotated from +90º (standing position) to -90º (upside down position). Only lead I of the electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor). Readings were first taken when the subjects were in the sitting position. The subjects were then attached to the platform and the readings for standing position (+90º) were taken.
99
The platform was rotated by 15º and readings were taken for each position till the platform was -90º i.e. the subjects were upside down. Readings were taken for the reverse direction as well. All the subjects were healthy in the age group of 18-25 and who regularly practiced Sheersasana (which is a Yogic posture wherein the person stands upside down balancing on the hands). Only five subjects were involved in the experiment. Only Sheersasana volunteers were studied as there was no idea as to how the experiment would affect the volunteers.
Figure 4.3: Experimental Setup of tilt-table rotatable from +90º to -90º available at Hemangi’s Aero Gym, Pune
4.3.2 Results and Discussion Figure 4.4 and Figure 4.5 show that the heart rate (HR), systolic blood pressure, diastolic blood pressure and pulse pressure (PP) is almost linear with gsinθ. Systolic blood pressure, diastolic blood pressure and pulse pressure (PP) also show a similar trend.
100
Systole BP(+90 to -90)
Effect of g on SBP and DBP
130
Diastole BP(+90 to -90) Systole BP(-90 to +90)
120
Diastole BP(-90 to +90)
SBP(m mHg) DBP(m m Hg)
110
100
90
80
70
60
50 -1100
-880
-660
-440
-220
0 gsinθ
220
440
660
880
1100
Figure 4.4: Effect of g on systolic and diastolic blood pressure
HR(+90 to -90)
HR,PP vs g
HR(-90 to +90)
90
PP(+90 to -90) PP(-90 to +90)
80
HR (beats/m in) PP (m m Hg)
70
60
50
40
30 -1100
-880
-660
-440
-220
0 gsinθ
220
440
660
880
1100
Figure 4.5: Effect of g on heart rate and pulse pressure
The only change seen in the ECG records was an increase in T-P interval from upright to supine position and then a slow decrease in T-P interval from supine to inverted position (not shown). This implies a decreased heart rate in upright to supine position and a slow increase in heart rate from supine to upside down position.
101
4.4 Later Experiments The information obtained was used to plan experiments using more controlled parameters and under the supervision of trained doctors. However, here also the tilt experienced by the volunteer was in the range 0º (supine position) to +90º (upright position) and back and then 0º (supine position) to -90º (upside position) and back. This experiment also included controls along with the persons doing Sheersasana.
4.4.1 Materials and Methods Subjects were attached to a platform which could be rotated from 0o to 90o. Data viz., ECG, heart rate and blood pressure were first taken for the supine position. The subjects were then rotated in steps of 15 upto 90 and back to 0 in o
o
o
two directions from 0° (supine position) to 90° (upright position) and 0° (supine position) to -90° (upside down position). Corresponding data were acquired for each position. Data were taken for the reverse direction as well. The duration of each position was 150 seconds of which 45-60 seconds were needed for the cardiovascular system to get stabilised as could be seen from the ECG records. All the subjects were healthy males in the age group of 18-25. The subjects were divided in two groups. One group consisted of those who regularly practiced Sheersasana till the past two years while the other group had never practiced Sheersasana and was taken as control group. There were 10 subjects in the control group and 5 subjects in the other. Only Lead I of the electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor) and also by the standard cuff method.
102
a)
b)
c) Figure 4.6: Experimental setup of tilt-table which can be rotated from 0º to 90º available at Sancheti Hospital, Pune
4.4.2 Results and Discussion The results were plotted in the form of histograms for the differences in heart rate, systolic blood pressure, diastolic blood pressure and pulse pressure between two consecutive positions. The normal group shows a comparatively large spread than those in the Sheersasana group which can be seen in Figures 4.7 – 4.10. This suggests that regular practice of Sheersasana might possibly help in tuning the cardiovascular system to the pooling of the blood in the head as in early days of spaceflight. Table 4.1: Class intervals for histograms for variations in heart rate and blood pressure
a
b
c
d
e
f
g
h
i
j
-24 to -20
-19 to -15
-14 to -10
-9 to -5
-4 to 0
1 to 5
6 to 10
11 to 15
16 to 20
21 to 25
103
HR(0 to 90) freq(C)
HR
50
FRE QUE NCY
F RE QUE NCY
SBP(0 to 90) freq(S)
40
40 30 20 10
30
20
10
0
0
a
b
c
d
e
f
g
h
i
j
a
b
c
d e f g h CLASS INTERVALS
CLASS INTERVALS
DBP(0 to 90) freq(C)
DBP
50
50
i
j
PP(0 to 90) freq(C)
PP
DBP(0 to 90) freq(S)
PP(0 to 90) freq(S)
40 FRE QUE NCY
40 FRE QUE NCY
SBP(0 to 90) freq(C)
SBP
50
HR(0 to 90) freq(S)
30 20 10
30 20 10
0
0 a
b
c
d
e
f
g
h
i
j
a
CLASS INTERVALS
b
c
d
e
f
g
h
CLASS INTERVALS
Figure 4.7: Histograms for variations in heart rate and blood pressure for 0° to 90°
104
i
j
50
HR(90 to 0) freq(C)
HR
50
30 20 10
30 20 10
0
0 a
b
c
d
e
f
g
h
i
j
a
b
c
CLASS INTERVALS
50
d
e
f
g
h
i
j
CLASS INTERVALS
DBP(90 to 0) freq(C)
DBP
50
PP(90 to 0) freq(C)
PP
DBP(90 to 0) freq(S)
PP(90 to 0) freq(S)
40 FRE QUE NCY
40 F RE QUE NCY
SBP(90 to 0) freq(S)
40 FRE QUE NCY
FRE QUE NCY
40
SBP(90 to 0) freq(C)
SBP
HR(90 to 0) freq(S)
30 20 10
30 20 10
0
0 a
b
c
d
e
f
g
h
i
j
a
CLASS INTERVALS
b
c
d
e
f
g
h
CLASS INTERVALS
Figure 4.8: Histograms for variations in heart rate and blood pressure for 90° to 0°
105
i
j
50
HR(0 to -90) freq(C)
HR
50
30 20 10
30 20 10
0
0 a
b
c
d
e
f
g
h
i
j
a
b
c
CLASS INTERVALS
50
DBP
d
e
f
g
h
i
j
CLASS INTERVALS
DBP(0 to -90) freq(C)
50 40 F RE Q UE NCY
30 20
PP(0 to -90) freq(C)
PP
DBP(0 to -90) freq(S)
40 FRE QUE NCY
SBP(0 to -90) freq(S)
40 F RE QUE NCY
F RE Q UE NCY
40
SBP(0 to -90) freq(C)
SBP
HR(0 to -90) freq(S)
PP(0 to -90) freq(S)
30 20 10
10
0
0 a
b
c
d e f g h CLASS INTERVALS
i
a
j
b
c
d
e
f
g
h
CLASS INTERVALS
Figure 4.9: Histograms for variations in heart rate and blood pressure for 0° to -90°
106
i
j
50
HR(-90 to 0) freq(C)
HR
50
FRE QUE NCY
F RE QUE NCY
SBP(-90 to 0) freq(S)
40
40 30 20
30 20 10
10
0
0 a
b
c
d e f g h CLASS INTERVALS
i
a
j
b
c
d
e
f
g
h
i
j
CLASS INTERVALS
DBP(-90 to 0) freq(C)
DBP
50
50
PP(-90 to 0) freq(C)
PP
DBP(-90 to 0) freq(S)
PP(-90 to 0) freq(S)
40 FRE QUE NCY
40 F RE QUE NCY
SBP(-90 to 0) freq(C)
SBP
HR(-90 to 0) freq(S)
30 20
30 20 10
10 0
0 a
b
c
d
e
f
g
h
i
j
a
CLASS INTERVALS
b
c
d
e
f
g
h
i
j
CLASS INTERVALS
Figure 4.10: Histograms for variations in heart rate and blood pressure for -90° to 0°
4.5 Design and Development of Tilt-Table In the initial experiments, though the table could be rotated from +90° to -90°, it had some drawbacks. The subjects had to support themselves by holding to the handles at the sides of as they were not strapped. The table had to be held and supported at the desired angle by a third person. This may cause errors in recording the parameters due to the possibility of a sense of insecurity in the volunteers. In the later experiments, the subjects were strapped and the table rotated by means of a rotating shaft and stopped at the desired angle. However, it could be rotated only from 0° to 90°. The volunteers had to get up and then change their
107
position for Sheersasana posture after supine to upright position. This was a major drawback as the cardiovascular parameters would have changed in this shifting. In order to overcome these drawbacks, a tilt-table which can be rotated through 360° in steps of 5°, 10°, 15°, etc has been designed in this laboratory and developed by Hi-Q Electronics, Pune. The schematic sketch of the tilt table is shown in Figure 4.11 below.
Figure 4.11: Schematic sketch of the Tilt-table
The developed tilt-table is shown in Figure 4.12.
(a)
108
(b) Figure 4.12: Tilt-table designed in the laboratory and developed by Hi-Q Electronics, Pune (a) Side View (b) Front View
The tilt-table can be operated both electronically as well as manually.
4.6 Experiments on Tilt-Table 4.6.1 Materials and Methods
Figure 4.13: Experimental setup
109
Subjects were attached to the table. The subjects were divided in three groups. One group consisted of those who regularly practiced Sheersasana, the second group consisted of those who regularly had exercise (not Yogic Asanas) while the last group included those who never did any of these and was taken as control group.
There were 4 subjects in each group. Only lead I of the
electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor) as well as the standard cuff method. Readings were first taken when the subjects were in the supine position. The table was then rotated by 15º and readings were taken for each position till the platform was 360º i.e. back to supine position. All the subjects were healthy males in the age group of 18-25.
4.6.2 Results and Discussion Systolic blood pressure appears to be steady for the control and Sheersasana groups compared to the exercise group (Figure 4.14). Diastolic blood pressure and heart rate tend to be steadier for the control and Sheersasana groups compared to the exercise group which shows a lot of variations (Figure 4.14 and 4.15). Similar results are obtained for pulse pressure as well (Figure 4.15). PR and QT intervals are more stable for the Sheersasana group in comparison with the control and exercise groups (Figure 4.16). This suggests that both atrial and ventricular contractions and relaxations are more stabilised for the Sheersasana group than the other groups.
110
SBP
DBP
C
125
E
85
S 120
S
80 75
DBP (mm Hg)
SBP (mm Hg)
C
90
E
115
70 65
110
60 55
105
50
-1100
-880
-660
-440
-220
0 220 gsinθ (cm/s2)
440
660
880
-1100
1100
-880
-660
-440
-220
0 220 gsinθ (cm/s2)
440
660
880
1100
Figure 4.14: Variations in Systolic and Diastolic Blood Pressure vs. gsinθ
HR
PP
C
90
C E
55
S
E
85
S
50
45
75 PP (mm Hg)
HR (bpm)
80
70 65
40
60 35 55 50
30
-1100
-880
-660
-440
-220
0 220 gsinθ (cm/s2)
440
660
880
1100
-1100
-880
-660
-440
-220
0 220 2) g sinθ( θ(cm/s θ(
440
660
880
1100
Figure 4.15: Variations in Heart rate and Pulse pressure vs. gsinθ
C
PR
C
QT
E
200
E
450
S
S
Stabilising Time (s)
Stabilising Time (s)
175
150
400
350
125
100 -1100
300 -880
-660
-440
-220
0 220 gsinθ (cm/s2)
440
660
880
1100
-1100
-880
-660
-440
-220
0 220 gsinθ (cm/s2 )
440
660
880
Figure 4.16: Variations in PR and QT intervals obtained from Lead I of ECG vs. gsinθ
111
1100
The difference histograms for systolic blood pressure shows that the Sheersasana group exhibits a better normal distribution than both the exercise and control groups in the direction of 0º to 180 º, i.e., head-up positions (Figure 4.17 a) as well as for the head-down positions, i.e., from 180º to 360º (Figure 4.19 a). This suggests that the systolic blood pressure is relatively stable for Sheersasana group as compared to the other groups. Difference histograms for diastolic blood pressure (Figure 4.17 b and Figure 4.19 b) and pulse pressure (Figure 4.17 d and Figure 4.19 d) also show a similar trend. However, the difference histograms for heart rate show a better normal distribution in the control and exercise groups for the head-up positions, i.e., from 0º to 180º (Figure 4.17 c) while a better normal distribution is observed in the Sheersasana and exercise groups for the head-down positions, i.e., from 180º to 360º (Figure 4.19 c). 25
o
o
o
20
20
15
15
10
10
5
5
0
0 a
b
c
d
e
f
g
h
i
j
k
a
b
c
d
Class Intervals
e
f
g
h
i
j
k
Class Intervals
a)
b)
25
o
o
25
C E S
HR Histogram (from 0 to 180 ) 20
20
15
15
10
5
o
C E S
o
PP Histogram (from 0 to 180 )
Counts
Counts
C E S
o
DBP Histogram (from 0 to 180 )
Counts
Counts
25
C E S
SBP Histogram (from 0 to 180 )
10
5
0
0 a
b
c
d
e
f
g
h
i
j
k
a
Class Intervals
b
c
d
e
f
g
h
i
j
Class Intervals
c) d) Figure 4.17: Histograms for variations in blood pressure and heart rate (0º to 180º)
112
k
25
o
o
20
20
15
15
10
5
o
o
C E S
QT Histogram (from 0 to 180 )
Counts
Counts
25
C E S
PR Histogram (from 0 to 180 )
10
5
0
0 a
b
c
d
e
f
g
h
i
a
Class Intervals
b
c
d
e
f
g
h
i
j
Class Intervals
a)
b)
Figure 4.18: Histograms for variations in PR and QT intervals obtained from Lead I of ECG (0º to 180º)
Difference histograms for PR interval show normal distribution only in the Sheersasana group for the head-up positions, i.e., from 0º to 180º (Figure 4.18 a) as well as for the head-down positions, i.e., from 180º to 360º (Figure 4.20 a). This suggests that the atrial contractions and relaxations of those who regularly practise Sheersasana are stable in the tilting environment in both directions. Histograms for QT intervals show normal distribution for the control and exercise groups for the head-up positions, i.e., from 0º to 180º (Figure 4.18 b). On the other hand, normal distribution is seen in the Sheersasana group for the head-down positions, i.e., from 180º to 360º (Figure 4.20 b). This suggests that the ventricular contractions and relaxations are the most stable in the case of control and exercise groups for the head-up positions while the same are most stable in the case of Sheersasana group for the head-down positions. These results suggest that regular practice of Sheersasana might possibly help in tuning the cardiovascular system to the pooling of the blood in the head as in early days of spaceflight.
113
Table 4.2: Class intervals for 0º to 180º
Class Intervals For SBP, DBP, HR and PP For PR intervals For QT intervals a
-27 to -23
-51 to -42
-45 to -35
b
-22 to -18
-41 to -32
-34 to -24
c
-17 to -13
-31 to -22
-23 to -13
d
-12 to -8
-21 to -12
-12 to -2
e
-7 to -3
11 to -2
-1 to 9
f
-2 to 2
-1 to 8
10 to 20
g
2 to 7
9 to 18
21 to 31
h
7 to 12
19 to 28
32 to 42
i
12 to 17
29 to 38
43 to 53
j
17 to 22
k
22 to 27
54 to 64
114
Table 4.3: Class intervals for 180º to 360º
Class Intervals For SBP, DBP, HR and PP For PR intervals For QT intervals a
-27 to -23
-160 to -135
-60 to -50
b
-22 to -18
-134 to -109
-49 to -39
c
-17 to -13
-108 to -83
-38 to -28
d
-12 to -8
-82 to -57
-27 to -17
e
-7 to -3
-56 to -31
-16 to -6
f
-2 to 2
-30 to -5
-5 to 5
g
2 to 7
-4 to 21
6 to 16
h
7 to 12
22 to 47
17 to 27
i
12 to 17
48 to 73
28 to 38
j
17 to 22
74 to 99
39 to 49
k
22 to 27
100 to 125
50 to 60
l
126 to 151
61 to 71
m
152 to 177
72 to 82
n
178 to 203
115
20
o
20
C E S
o
SBP Histogram (from 180 to 360 )
C E S
o
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4.7 Program for ECG Analysis 4.7.1 Introduction With the advent of the computational infrastructure the medical informatics has assumed an important role in the medical diagnosis and the treatment. Different types of analytical tools have been developed based on biochemical, electrical and structural aspects of different organs and the parts of human body. Image analysis carried out using the techniques such as x-rays, CT scan, MRI and the PET has become a routine practice. Many commercial packages are available for this purpose. Measurement and analysis of biopotentials such as ECG, EEG, EMG, ERG and visual evoked potentials have also been developed and are being used. But many of these techniques are platform-dependent and also do not consider multi parametric analysis. Current programs are limited to specific ailments like cardiac ischemia (270), cardiac arrhythmias (271), R-R interval analysis (272), T-wave analysis (273) and QRS detection (274). Neural networks have also been developed for ECG analysis (275). On this background, an effort was made to develop a Java-based multiparametric analysis program, which is user-friendly and can help the physician to detect the irregularities in the ECG signal.
4.7.2 Materials and Methods A Java-based program using Java Swings as the programming tool had been developed which can analyse previously acquired ECG signals with data available in digital form. Since it is Java-based, it is platform-independent. The ECG records, corresponding to each of the leads, which are stored in files in ASCII format with the end of the name of each file having name corresponding to each lead. This program reads data from any of the files (leads) selected and reconstructs the ECG signal for all the leads. The program detects the P, Q, R, S and T peaks and obtains PR, QT, RR and TP time intervals and the corresponding peak amplitudes. The mean and standard deviation values for each of the
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parameters are calculated. The program also plots the histograms for each of the above parameters. It pops the analysis performed onto the window and also writes it to a printable file thus helping for quick assessment of ECG records and indicating possible irregularities related to heart rate variability and the variation in the amplitudes.
4.7.3 Results and Discussion The flow chart for the program is shown below (Figure 4.21). The program reads data from the files (leads) that are obtained using data acquisition, reconstructs the ECG signal and analyzes the signal for all of the leads. The result of the analysis is popped up onto the window and also written to a printable file creating the report. The analysis gives possible indications of ailments that can be diagnosed using ECG. This is depicted in the Figures 4.22 a - j. Figure 4.22 a shows the basic menu system that is designed. Selection of the file (lead) is shown in Figure 4.22 b which results in the reconstruction of the ECG signal and popping up of the analysis performed onto the window as shown in Figure 4.22 c. Figure 4.22 d-h show the plotting of histogram for a particular peak of a particular lead. Figure 4.22 i and Figure 4.22 j show the result of selection of a wrong file. Current programs are limited to specific ailments like cardiac ischemia (270), cardiac arrhythmias (271), R-R interval analysis (272) and QRS detection (274). Also they are not platform-independent.
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Start Open and read required file. Find the extremities (maxima and minima) of the data in the file. Find the period of each extremity.
for i=0, i
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for i=1, i
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Find minimum and maximum for each extremity. define n,count,counts[].
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Figure 4.21: Flow Chart of the Java based program for ECG analysis
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Figure 4.22: Output of the Java based program developed for ECG analysis
4.8 Conclusions In this chapter, the effects of gravity on the human cardiovascular system have been studied. An electric circuit model of the cardiovascular system has been discussed. Preliminary experiments using a tilt-table which could be rotated from 0º to 90º and back showed that regular practice of Sheersasana might possibly help in tuning the cardiovascular system to the pooling of the blood in the head as in early days of spaceflight. This has been confirmed by experiments on the tilt-table which could be rotated from 0º to 360º.
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A Java-based program has been developed for multi parametric analysis of digitally stored ECG signal records. As the program is Java-based, it is platformindependent. The program detects the P, Q, R, S and T peaks and obtains PR, QT, RR and TP time intervals and the corresponding peak amplitudes. The mean and standard deviation values for each of the parameters are calculated. The program also plots the histograms for each of the above parameters. It pops the analysis performed onto the window and also writes it to a printable file thus helping for quick assessment of ECG records and indicating possible irregularities related to heart rate variability and the variation in the amplitudes. Currently the program analyses ECG signals that are already acquired in digital form. It has to be developed further so that it can analyse signals that are acquired online. Also it can be used only on a standalone PC. Further development has to be done so that it can be used in a LAN network. This program can also be further developed for applications in other fields of medicine.
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CHAPTER 5
Chapter 5: Conclusion and Future Scope 5.1
Summary
The science of gravitational biology took a giant step forward with the advent of the space program. It provided the first opportunity to examine living organisms in gravity environments lower than could be sustained on Earth. Organisms ranging in complexity from single cells through humans are responsive to Earth’s gravity; thus, these organisms most likely would be affected by a lack of gravity. To fully appreciate the effects of altered gravity on biological species, multiple generations must be studied at that gravity level. Subtle biological changes due to altered gravity are difficult to define over a single generation. Acute changes can be studied in less than one generation; the duration of most altered gravity experiments. Also it will be interesting to study how the evolutionary response of biological systems would be (e.g. linear, logarithmic, or degraded) at gravity levels other than 1 g. Clinostats have been established as a ground-based technique to study the effects of altered gravity environments on plants and cell cultures by simulating microgravity. For the same, a 1-D clinostat had been designed and developed in the laboratory. Combining a clinostat and centrifuge would help in attaining simulated hypo- and hypergravity environments for ground-based studies. For the same, a clinostat-centrifuge had been designed and developed.
5.1.1
Studies on insects Chironomus is the scientific name of non-biting midge. It has been
established as an excellent organism to carry out behavioural studies for exploring innate behavioural traits. The embryonic developmental stages of Chironomus have been well studied and its chromosomes have been characterised. Eggs of harlequin fly are very transparent and hence, it is very easy to see the effect of stimulus as changes in egg structure inside egg sac. Also, hatching period is only 48 hours and hence not too long unlike Drosophila (7 days). Hence it is easier to see the effect of
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applied stimulus as early as possible. Eggs of harlequin fly are easily available in marshy lands. Hence, by applying altered gravity conditions on Chironomus, the changes within the eggs were directly observed taking advantage of their transparency. Chironomus eggs were exposed to different durations of simulated microgravity and then allowed to develop under normal gravity conditions. Their development was observed in terms of the hatching period and changes in morphology. As the duration of clinorotation increased, the time required for all the eggs to hatch reduced. However, there was no change in morphology of the eggs for all the durations of clinorotation or any apparent deformities in the hatched larva.
5.1.2
Studies on plants Plant growth and development are affected by a lot of different
environmental abiotic factors such as light, temperature and water supply. Immediately upon germination, another physical stimulus, gravity, strongly influences the growth of plant organs, root and shoot, in order to ensure their correct orientation in space and the survival of the young seedling. Since plants have evolved under the constant stimulus of gravity, its presence is one of the most important prerequisites for their growth and spatial orientation. The ability of plants to change their growth orientation in response to gradients in light and gravity maximizes their ability to obtain energy from light and moisture and nutrients from soil. The nature of graviresponse in plant seedlings had been studied by orienting the developing seedlings vertically and horizontally. In the case of roots, the growth is inhibited on the bottom and exaggerated at the top, hence the root bends down. Conversely in the case of stems growth is inhibited at the top and accelerated at the bottom leading to an upward bend. It
was
also
noted
that
reorientation of the plants produced the same effects i.e. the same rate of curvature in all cases. Repeated reorientation too did not appear to affect the graviresponse
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in any way. The responses in the two species that were tested differed. Bending in the case of the Soy bean stems took more time than bending in Brassica. Rice seeds were clinorotated for 12 hours and then allowed to grow normally for 2 weeks under ambient conditions of light, temperature and humidity. There were two sets of experiments viz., day set (seeds were sown during the day) and night set (seeds were sown at night). The seeds sown during the day showed more shoot and root growth in the microgravity samples as compared with control. The seeds sown at night also showed similar results for shoot growth. But root growth was higher in the microgravity samples for seeds sown in day while it was lower for those sown at night as compared with control. Chlorophyll content in microgravity samples was higher than in those of control for both the sets on the 6th day. Chlorophyll content remained higher in microgravity samples for the night set while it was lower in that of control for the day set on the 8th and 10th day. Rice seeds were clinorotated for durations of 3, 5 and 7 days. The experiment was conducted under ambient conditions of temperature and humidity and a light intensity of 1250 Lux for 16 hours photoperiod per day. On the last day, root and shoot lengths were measured for both control and clinorotated samples. Also weights of shoots were taken. Chlorophyll was isolated and UV-Vis absorption spectra and fluorescence spectra were recorded for both the samples. Chlorophyll content was calculated using Arnon’s formula. Primary root length was higher for samples clinorotated for 3 and 7 days than control while it was lower for samples clinorotated for 5 days. Similar results were obtained for shoot length as well as shoot weight. Absorption and fluorescence spectra showed that chlorophyll a content was lower for all clinorotated samples than control. Chlorophyll b content and total chlorophyll content were also lower for all clinorotated samples than control.
5.1.3
Studies on humans A simple electric circuit model of the cardiovascular system was developed.
In this model, the blood pressure is considered equivalent to the voltage, the flow
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velocity to the current, the peripheral resistance to the electrical resistance and the compliance (elasticity) of the vessel to the capacitance. Subjects were attached to a platform which could be rotated from +90o (standing position) to -90o (upside down position). Only lead I of the electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor). Readings were first taken when the subjects were in the sitting position. The subjects were then attached to the platform and the readings for standing position (+90o) were taken. The platform was rotated by 15o and readings were taken for each position till the platform was -90o i.e. the subjects were upside down. Readings were taken for the reverse direction as well. All the subjects were healthy in the age group of 18-25 and who regularly practiced Sheersasana (which is a Yogic posture wherein the person stands upside down balancing on the hands). Only five subjects were involved in the experiment. Only Sheersasana volunteers were studied as there was no idea as to how the experiment would affect the volunteers. Systolic and diastolic blood pressure showed an almost linear decrease with gsinθ towards the supine position from the upright position while there was no particular trend towards the upside position where θ is the tilting angle. Heart rate and pulse pressure also showed a similar trend. Subjects were attached to a platform which could be rotated from 0º to 90º. ECG, heart rate and blood pressure were first taken for the supine position. The subjects were then rotated in steps of 15º upto 90º and back to 0º in two directions from 0° (supine position) to 90° (upright position) and 0° (supine position) to -90° (upside down position). Corresponding data were acquired for each position. Readings were taken for the reverse direction as well. All the subjects were healthy males in the age group of 18-25. The subjects were divided in two groups. One group consisted of those who regularly practiced Sheersasana till the past two years while the other group had never practiced Sheersasana and was taken as control group. There were 10 subjects in the control group and 5 subjects in the
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other. Only Lead I of the electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor) and also by the standard cuff method. The results were plotted in the form of histograms for the differences in heart rate, systolic blood pressure, diastolic blood pressure and pulse pressure between two consecutive positions. The normal group shows a comparatively large spread than those in the Sheersasana group. In the initial experiments, though the table could be rotated from +90° to 90°, it had some drawbacks. The subjects had to support themselves by holding to the handles at the sides of as they were not strapped. The table had to be held and supported at the desired angle by a person. This may cause errors in recording the parameters due to the possibility of a sense of insecurity in the volunteers. In the later experiments, the subjects were strapped and the table rotated by means of a rotating shaft and stopped at the desired angle. However, it could be rotated only from 0° to 90°. The volunteers had to get up and then change their position for Sheersasana posture after supine to upright position. This was a major drawback as the cardiovascular parameters would have changed in this shifting. In order to overcome these drawbacks, a tilt-table which can be rotated through 360° in steps of 5°, 10°, 15°, etc had been designed in this laboratory and developed by Hi-Q Electronics, Pune. Subjects were attached to the table. The subjects were divided in three groups. One group consisted of those who regularly practiced Sheersasana, the second group consisted of those who regularly had exercise (not Yogic Asanas) while the last group included those who never did any of these and was taken as control group. There were 4 subjects in each group. Only lead I of the electrocardiograph was taken using a portable ECG device. Blood pressure and heart rate were simultaneously measured using a handheld digital portable device (Omron MX2 Automatic Digital Blood Pressure Monitor) as well as the standard cuff method. Readings were first taken when the subjects were in the supine
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position. The table was then rotated by 15° and readings were taken for each position till the platform was 360° i.e. back to supine position. All the subjects were healthy males in the age group of 18-25. Systolic blood pressure appeared to be steady for the control and Sheersasana groups compared to the exercise group. Diastolic blood pressure and heart rate tend to be steadier for the control and Sheersasana groups compared to the exercise group who showed a lot of variations. Similar results were obtained for pulse pressure as well. PR and QT intervals were more stable for the Sheersasana group in comparison with the control and exercise groups. This suggests that both atrial and ventricular contractions and relaxations are more stabilised for the Sheersasana group than the other groups. The difference histograms for systolic blood pressure showed that the Sheersasana group exhibits a better normal distribution than both the exercise and control groups in the direction of 0º to 180º, i.e., head-up positions as well as for the head-down positions, i.e., from 180º to 360º. This suggests that the systolic blood pressure is relatively stable for Sheersasana group as compared to the other groups. Difference histograms for diastolic blood pressure and pulse pressure also show a similar trend. However, the difference histograms for heart rate shows a better normal distribution in the control and exercise groups for the head-up positions, i.e., from 0º to 180º while a better normal distribution is observed in the Sheersasana and exercise groups for the head-down positions, i.e., from 180º to 360º. Difference histograms for PR interval show normal distribution only in the Sheersasana group for the head-up positions, i.e., from 0º to 180º as well as for the head-down positions, i.e., from 180º to 360º. This suggests that the atrial contractions and relaxations of those who regularly practise Sheersasana are stable in the tilting environment in both directions. Histograms for QT intervals show normal distribution for the control and exercise groups for the head-up positions, i.e., from 0º to 180º. On the other hand, normal distribution is seen in the Sheersasana group for the head-down positions, i.e., from 180º to 360º. This suggests that the ventricular contractions and relaxations are the stable in the
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control and exercise groups for the head-up positions while the same are stable in the Sheersasana groups for the head-down positions. However, it was very difficult to get volunteers as the number of males who regularly practise Sheersasana in the age group of 18-25 are very few. It was also difficult to get volunteers from the control and exercise groups as many were unwilling to volunteer due to fear psychosis. Also the volunteers in this age group were mostly students and so many were reluctant to miss their lectures and volunteer for this experiment. A Java-based program had been developed for multi-parametric analysis of digitally stored ECG signal records. As the program is Java-based, it is platform-independent. The program detects the P, Q, R, S and T peaks and obtains PR, QT, RR and TP time intervals and the corresponding peak amplitudes. The mean and standard deviation values for each of the parameters are calculated. The program also plots the histograms for each of the above parameters. It pops the analysis performed onto the window and also writes it to a printable file thus helping for quick assessment of ECG records and indicating possible irregularities related to heart rate variability and the variation in the amplitudes. Currently the program analyses ECG signals that are already acquired in digital form.
In the present thesis, the problems which were dealt with were related to establishing facilities to perform experiments which may bring out similar effects under altered gravity conditions in space. It was planned that the experiments should be carried out in insects, plants and human beings. Accordingly, experimental facilities were designed and developed in the laboratory. In some cases, existing facilities were used. The nature of graviresponse in plant seedlings was studied by orienting the developing seedlings vertically and horizontally. These results showed that plants can be used to study effects of altered gravity conditions on biological organisms. In order to study the effects of microgravity on insects and plants, a
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one-dimensional clinostat was designed and developed for this purpose. Rice seeds were subjected to simulated microgravity by clinorotating the seeds for different durations and effects on growth of roots and shoots and chlorophyll content were observed. The results obtained for the chlorophyll in rice have not been reported earlier. These observations showed that simulated microgravity promotes the growth and development of rice seeds. This observation is consistent with the observations made during space flights. Simulated microgravity was also found to enhance faster senescence in rice. To see the effects of development in case of insects, the system which has been used is that of Chironomus. Earlier studies have been carried out on Drosophila and beetles in terms of behavioural aspects. However Chironomus has not been used so far for studies related to altered gravity. This system has already been standardised in many respects in the Department of Zoology, University of Pune. The various stages in the life cycle of these insects starting from egg mass to larva to pupa to adult have been well established. These eggs are easy to handle and hatching is completed in 48 hours at 25 ºC. The experiments with these insects under microgravity conditions show the faster development. The results obtained with this system certainly can be used to carry out further studies. This has also given us direction to develop a clinostat-centrifuge. In order to establish a relation between gravitational acceleration and the blood pressure, a theoretical model was developed and a relation was established between blood pressure and the gravitational acceleration. The experiments carried out with human beings are also very interesting. In this study, a new concept of rotating a human body through 360º was used. The experiments were planned in a careful manner. To begin with, facilities from a gymnasium were used to establish the path by rotating through +90º to -90º does cause variations in blood pressure and vascular parameters. This study provided the basis to carry out study in a more controlled manner with the help of Sancheti Hospital by rotating subjects through 0º to 90º. In this particular study, volunteers were subjected to different orientations. The control group and Sheersasana group (those practising
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Sheersasana regularly) were studied. This study indicated that the persons doing Sheersasana regularly were most responsive as compared to the control group. This observation would certainly help to plan the type of training to be given to astronauts before they sent to space. This study also indicated that a platform could be required which can be rotated through 360º in a controlled manner. Such a platform was designed in the laboratory and developed by Hi-Q Electronics, Pune. The experiments performed by using this platform provided the data where a controlled person can be compared with persons doing exercise and Sheersasana. Thus the present thesis has established that one can undertake studies which can simulate microgravity conditions in a laboratory. In space, the blood in a human body is accumulated in the upper part of the body due to lack of gravitational acceleration whereas in the case of our experiments, blood is accumulated in the upper part of the body because of the inclination of the person. The results obtained show that accumulation of blood under gravitational conditions show similar effects as observed in microgravity. Under such circumstances, the present study has given sufficient indications to carry out further experiments which could be further explored to study the effects of microgravity and altered gravity conditions.
5.2 Conclusions
From the work presented in this thesis, the following conclusions can be made. 1)
Development of insects and plants is promoted by simulated microgravity.
2)
Senescence in plants is promoted by simulated microgravity.
3)
Regular practice of Sheersasana might help astronauts to adapt easily to the
pooling of blood in the upper part of the body during initial days of spaceflight.
5.3 Future Scope
Many
challenges lie ahead. An important aspect is helping our
understanding of how living systems emerged on our planet and how they may
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change if transferred permanently into environments very different from those in which they have evolved. Also we can extrapolate these results to other biological organisms and find out the tolerance range. The clinostat-centrifuge system can be used to study the effects of simulated hypo- and hypergravity environments on the development of plants and insects. Also work can be carried out at the cellular, sub-cellular and genetic levels. This may throw light on how life can adapt to varied gravity conditions as on other planetary bodies. The tilt-table can be used to study the effects of gravity and posture on other physiological systems such as neurovestibular, musculoskeletal and thermoregulatory systems. These studies can give an idea as to how astronauts can be trained for long-term space missions and missions to other planets. The Java based program can be developed further so that it can analyse signals that are acquired online. Further development can also be done so that it can be used in a LAN network. This program can also be further developed for applications in other fields of medicine.
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List of Publications 1) Santosh Bhaskaran and Pandit B. Vidyasagar, "Java based program for multi parametric ECG analysis", Ind. J. Med. Info., 2004, 1(1): http://www.iami.org.in/journal1/java.asp
2)
P.B. Vidyasagar, Santosh Bhaskaran and Salil Bidaye, "Gravitational effects on
blood circulation", Phys. Edu., 2004, 21(1): P43-49 3)
Santosh B., Sabnis S.M., Joshi V., Razia R.N., Vidyasagar P.B., "Effect of gravity
on the cardiovascular system", J. Grav. Physiol., 13(1): 35-36
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List of Conferences Attended/Presented 1) Student Participant in the a)
National Symposium on Biophysics, 21-23 Feb 2003
b) International Workshop on Education and Capacity Building in Biophysics: Needs of the Asian African Region, 24-25 Feb 2003 at Indian Institute of Technology, Roorkee, India 2) Student participant in the Workshop on Genes, Genomics and the Development of Behaviour: The Development of Function in the Nervous System. at the abdus Salam International Centre for Theoretical Physics, Trieste, Italy, 14 July-1 Aug 2003 3) Santosh Bhaskaran, P.B. Vidyasagar, "Java based program for multi parametric ECG analysis", IAMI-2003, Biennial Conference of the Indian Association for Medical Informatics, Post-Graduate Institute for Medical Education & Research, Chandigarh, India, 18-19 Oct 2003 4)
Student Participant in the International Workshop on Clinical Data
Management at Institute of Bioinformatics and Biotechnology, University of Pune, Pune, India, 11-13 Dec 2003 5) Santosh Bhaskaran, P.B. Vidyasagar, "Multi parametric ECG analysis to study the effect in microgravity", RMC-2004, Department of Physics, University of Pune, Pune, India, 26-27 Feb 2004 6) Student Participant in the 45th Annual Conference of the Indian Society of Aerospace Medicine at Institute of Aerospace Medicine, Bangalore, India, 17-19 Nov 2004 7) Santosh B. and P.B. Vidyasagar, Poster titled “Effect of gravity on the cardiovascular system”, IBS-2005, University of Pune, Pune, India, 22-25 Jan 2005 8) Santosh B., Sabnis S.M., Joshi V., Razia R.N., Vidyasagar P.B., "Gravitational effects on the cardiovascular system", RMC-2006, Department of Physics, University of Pune, Pune, India, 24-25 Feb 2006
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9) Santosh B., Sabnis S.M., Joshi V., Razia R.N., Vidyasagar P.B., "Effect of gravity on the cardiovascular system", 27th Annual Gravitational Physiology Meeting, Osaka University, Osaka, Japan, 23-28 Apr 2006 10) P.B. Vidyasagar, Santosh Bhaskaran and Sarah Thomas, “Possible Role of Thermoluminescence under microgravity conditions”, at Photosynthesis in the Post Genomic ERA II: Structure and Function of Photosynthesis, Puschino, Moscow, Russia, 20-26 Aug 2006 11) A.A. Babrekar, B.B. Nath, S. Bhaskaran and P.B. Vidyasagar, Poster titled “Developmental Effects of Altered Gravity Environments in Chironomus”, ISDB2006, Agarkar Research Institute, Pune, India, 23-25 Nov 2006 12) Santosh B., Jagtap S., Kamble S. and P.B. Vidyasagar, Poster titled “Effect of altered gravity on plant growth and development”, at IBS 2007, New Delhi, India, 13-15 Feb 2007 13) Santosh B., A.A. Babrekar, B.B. Nath and P.B. Vidyasagar, Poster titled “Effect of altered gravity on insect development”, at IBS 2007, New Delhi, India, 13-15 Feb 2007 14) Jagtap S.S., Santosh B., Kamble S. and Vidyasagar P.B., Poster titled “Microgravity effects on plant growth and development”, at RMC-2007, Department of Physics, University of Pune, Pune, India, 23-24 Feb 2007 15) Pandit Vidyasagar, Sagar Jagtap, Amit Nirhali, Santosh Bhaskaran and Vishakha Hase, Poster titled “Effects of hypergravity on the chlorophyll content and growth of root and shoot during development in rice plants”, at Photosynthesis 2007, Glasgow, UK, 22-27 July 2007 16) Santosh B., Jagtap S.S., Nirhali A.A., Hase V.R. and Vidyasagar P.B., “Microgravity effects on chlorophyll content”, at International Astronautical Conference 2007, Hyderabad, India, 24-28 September 2007 17) Jagtap S.S., Hase V., Amit A.N., Santosh B. and Vidyasagar P.B., Poster titled “Effects of simulated microgravity on growth and chlorophyll content during development in rice”, at IBS 2007, Chandigarh, India, 15-17 Nov 2007
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18) Jagtap S.S., Vidyasagar P.B. Nirhali A.A., Hase V. and Santosh B., “Preexposure effects of hypergravity on growth and development in rice”, at RMC2008, Department of Physics, University of Pune, Pune, India, 23-24 Feb 2008
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