The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes. In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components. We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment. Experimental Physiology (2003) 88.1, 57-64.

We recently reported that a higher percentage of type I fibres in vastus lateralis and a greater peak oxygen uptake ([Vdot]O2) were associated with a greater initial rise in [Vdot]O2 (Δ[Vdot]O2 /ΔW, where W is work rate) following the onset of heavy constant power output exercise (above the lactate threshold, LT). It was unclear if these results were true only for heavy exercise, or if the association between fibre type and/or fitness and Δ[Vdot]O2 /ΔW would also be seen for moderate (< LT) exercise. The purpose of the present study was to compare the relationships between fibre type or peak [Vdot]O2 and Δ[Vdot]O2 /ΔW determined for moderate (< LT) and heavy (> LT) exercise intensities during incremental exercise. Nine healthy subjects performed an incremental ramp test on a cycle ergometer. The [Vdot]O2 /Wslope was calculated for the domain of power outputs up to the LT (S1), from the LT towards peak [Vdot]O2 (S2), and over the entire linear portion of the Δ[Vdot]O2 /ΔW response (ST), and compared to fibre type distribution determined from biopsy of the vastus lateralis, and to peak [Vdot]O2 (as ml kg-1 min-1). Significant correlations between Δ[Vdot]O2 /ΔW and the proportion of type I fibres were found for each exercise domain (r is 0.69, 0.71 and 0.84 for S1, S2 and ST, respectively, P < 0.05). S1 ranged between about 9 ml min-1 W-1 for a low proportion of type I fibres and 11 ml min-1 W-1 for a high proportion of type I fibres. Similar correlations were also found between S2 (r = 0.70) and ST (r = 0.76) and peak [Vdot]O2. These results are consistent with our previous findings during > LT constant power output exercise, and suggest that the proportion of type I fibres, and possibly fitness as indicated by peak [Vdot]O2, is associated with greater Δ[Vdot]O2 /ΔW during the initial adjustment to < LT as well as > LT exercise. These results do not appear to be explained by classical descriptions of the kinetics of adjustment of [Vdot]O2 following the onset of ramp or constant power output exercise. They might reflect enhanced motor unit recruitment in subjects with a greater percentage of type I fibres, and/or who are more aerobically fit. However, the underlying mechanism for these findings must await further study.

The sympathetic nervous system is believed to play a major role in regulating cardiovascular function during exercise. However, only a few direct measurements of sympathetic nervous activity during whole body dynamic exercise have been attempted. In the present study, we have established a method to allow routine measurement of renal sympathetic nerve activity (RSNA) and cardiovascular function during treadmill exercise in rats. We trained Wistar rats to run on the treadmill for a week before the surgery. At least 2 days before the experiment, electrodes for recording RSNA, electrocardiogram and electromyogram, and catheters for the measurements of systemic arterial and central venous pressures were implanted under aseptic conditions. Satisfactory signal to noise ratios were obtained in 80 %, 60 % and 40 % of the group at 1-3 days, 4-7 days and 8-10 days after the surgery, respectively. RSNA was successfully recorded without contamination by external noise during treadmill exercise. Treadmill exercise resulted in an abrupt increase in RSNA, by 82 % at 0.5 min, and then reached a stable level of ~40 % during the period of 5-30 min after the onset of treadmill exercise. This experimental model allows us to study the neural mechanisms involved in the regulation of cardiovascular function during dynamic exercise in rats. Experimental Physiology (2002) 87.1, 33-39.

Recent evidence indicates that endurance-trained athletes are able to increase their stroke volume (SV) throughout incremental upright exercise, probably due to a progressively greater effect of the Frank-Starling mechanism. This is contrary to the widely held belief that SV reaches a plateau at a submaximal heart rate (irrespective of fitness level), owing to a limitation in the time for diastolic filling. The purpose of this investigation was to evaluate whether endurance-trained athletes rely on a progressively greater effect of the Frank-Starling mechanism throughout incremental exercise. A secondary purpose was to evaluate the effects of postural position on the cardiovascular responses to incremental exercise. Ten male cyclists participated in this investigation. Left ventricular function was assessed throughout incremental exercise in the supine and upright positions (counterbalanced) using radionuclide ventriculography. Stroke volume increased in a linear fashion during incremental exercise in both the upright and supine positions. The increases in cardiac output (Q) throughout incremental to maximal exercise (in both the supine and upright positions) were significantly related to changes in heart rate, myocardial contractility and the Frank-Starling mechanism. Percentage changes in end-diastolic volume and SV were significantly greater in the upright position versus the supine position, reflecting an increased reliance on the Frank-Starling effect to increase Q. We conclude from this investigation that highly trained endurance athletes are able to make progressively increasing usage of the Frank-Starling effect throughout incremental exercise. Postural position has a significant effect on the relative contribution of heart rate, myocardial contractility and the Frank-Starling mechanism to the increase in Q during exercise conditions. Experimental Physiology (2002) 87.5, 613-622.

The metabolic and functional diversity of animal mitochondria caused by different mitochondrial compositions due to tissue-specific mitochondrial pathways and tissue-specific differences in expression of isoforms of subunits of enzymes participating in oxidative phosphorylation will be reviewed here. Applying the concept of metabolic control analysis, the relevance of this diversity for the explanation of tissue-specific effects observed in mitochondrial diseases with homoplasmic mitochondrial DNA mutations and nuclear-encoded respiratory chain defects is discussed. Experimental Physiology (2003) 88.1, 149-154.

Breast volume and morphology of eight subjects were measured before conception and at intervals throughout pregnancy until 1 month of lactation. Breast volume before conception ranged from 293 to 964 ml. At the end of pregnancy the volume of breast tissue had increased by 145 ± 19 ml (mean ± S.E.M., n = 13 breasts, range 12-227 ml) with a further increase to 211 ± 16 ml (n = 12 breasts, range 129-320 ml) by 1 month of lactation. Urinary excretion of lactose increased at 22 weeks of pregnancy, signalling the capacity of the breast to synthesize lactose at this time. During pregnancy, both the change in breast volume and the change in cross-sectional area of the areola were related to the concentration of human placental lactogen in the plasma. The growth of the nipple and the rate of excretion of lactose were related to the concentration of prolactin in the plasma. During the first 3 days after birth, the rate of excretion of lactose was related to the rate of excretion of progesterone. There was no relationship between the growth of the breast during pregnancy and the amount of milk produced at 1 month of lactation.

Methods used to quantify the stress response in animals are vital tools in many areas of biology. Here we describe a new method of measuring the stress response, which provides rapid results and can be used in the field or laboratory. After a stressful event, we measure the capacity of circulating leukocytes to produce a respiratory burst in vitro in response to challenge by phorbol myristate acetate (PMA). During the respiratory burst leukocytes produce oxygen free radicals, and the level of production can be measured directly as chemiluminescence. When in vitro PMA-stimulated whole blood chemiluminescence is measured directly after a stressful event, we define the response as the leukocyte coping capacity (LCC). In an experiment badgers (Meles meles), which were caught as part of an on-going population study, were either transported to a central site prior to blood sampling or blood was collected at their site of capture. Transported animals had a significantly lower LCC and showed changes in leukocyte composition that were indicative of stress. We conclude that the stress of transport reduced LCC in badgers and that LCC serves as a quantitative measure of stress. Potential applications of this method are discussed. Experimental Physiology (2003) 88.4, 541-546.

Several lines of evidence suggest that central serotoninergic neurons may mediate fatigue signals during prolonged exercise. In this study we examined the effects of diet and ambient temperature on peripheral modulators and indices of serotoninergic function and their relationship to exercise performance. Six well-trained cyclists participated, in randomised order, in two diet and exercise regimens each lasting 8 days and comprising four cycle tests to exhaustion at 70 % of maximum oxygen uptake. On days 1 and 5, subjects exercised to exhaustion to deplete muscle glycogen. For 3 days after the first depletion trial a diet providing 10 % of energy in the form of carbohydrate (CHO) was consumed (low CHO), and for 3 days after the second depletion trial a diet providing 80 % (high CHO) of energy as CHO was consumed, and each diet was followed by a performance trial at the same ambient temperature, either 10 °C or 30 °C (days 4 and 8). This schedule was repeated after 1 week, but performance trials were carried out at the other ambient temperature. In the cold, cycling time increased (P < 0.01) from 89.2 (78.0-129.5) min (median (range)) in the low CHO trial to 158.2 (116.9-165.6) min in the high CHO trial. In the heat, cycling time increased from 44.0 (31.8-51.4) min in the low CHO trial to 53.2 (50.2-82.2) min on the high CHO trial (P = 0.02). The serum prolactin (Prl) concentration was higher at exhaustion during the two trials in the heat than in the two trials in the cold. Serum Prl levels were unrelated to the purported peripheral modulators of serotoninergic function (plasma concentrations of total tryptophan (Trp), free Trp, branched-chain amino acids (BCAAs), free Trp/BCAA ratio and total Trp/BCAA ratio) but were significantly related to the rectal temperatures measured during the two trials in the heat. This finding provides indirect evidence that the serotoninergic system may be involved in fatigue during exercise under conditions of heat stress. Experimental Physiology (2002) 87.2, 215-226.

In this study we measured the central activation ratio (CAR) of the leg extensors and the elbow flexor muscles before and after exhaustive exercise in the heat to determine whether exercise-induced hyperthermia affects the CNS drive to exercised (leg extensors) and/or non-exercised (forearm flexors) muscle groups. Thirteen subjects exercised at fixed intensities representative of a percentage of peak power output (PPO) for 10 min periods (50 %, 40 %, 60 %, 50 %) and then at 75 % PPO until exhaustion in ambient conditions of 39.3 ± 0.8 °C and 60.0 ± 0.8 % relative humidity. Before and immediately following exercise subjects performed a series of maximal voluntary contractions (MVCs) with the leg extensors (exercised muscles) and forearm flexors (non-exercised muscles). The degree of voluntary activation during the sustained MVCs was assessed by superimposing electrical stimulation to the femoral nerve and the biceps brachii. Exercise to exhaustion increased the rectal temperature from 37.2 ± 0.2 to 38.8 ± 0.2 °C (P < 0.0001). The mean heart rate at the end of exercise to exhaustion was 192 ± 3 beats min-1. Leg extensor voluntary force was significantly reduced from 595 ± 143 to 509 ± 105 N following exercise-induced hyperthermia but forearm flexor force was similar before and after exercise. The CAR of the leg extensors decreased from 94.2 ± 1.3 % before exercise to 91.7 ± 1.5 % (P < 0.02) following exercise-induced hyperthermia. However, the CAR for the forearm flexors remained at similar levels before and after exercise. The data suggest that the central nervous system selectively reduces central activation to specific skeletal muscles as a consequence of exercise-induced hyperthermia. Experimental Physiology (2003) 88.6, 783-790.

Resting heart rate and heart rate variability of 33 postmenopausal women were compared with those of 50 premenopausal women of comparable activity level, none of whom had used hormone replacement therapy. Heart rate was measured as the mean of at least 600 consecutive R-R intervals obtained from electrocardiograph (ECG) records, and its variability as the standard deviation of these intervals. Activity levels were assessed by a scale modified from the Allied Dunbar National Fitness Survey (1992). There was a significant reduction in both mean R-R interval and the standard deviation in the postmenopausal women who had experienced their last menstrual period (LMP) 1 year or more prior to the observations being made, but no observable changes during the first year post menopause. Experimental Physiology 85.3, 327-330.

We compared estimates for the phase 2 time constant (τ) of oxygen uptake (V˙O2) during moderate- and heavy-intensity exercise, and the slow component of V˙O2 during heavy-intensity exercise using previously published exponential models. Estimates for τ and the slow component were different (P < 0.05) among models. For moderate-intensity exercise, a two-component exponential model, or a mono-exponential model fitted from 20 s to 3 min were best. For heavy-intensity exercise, a three-component model fitted throughout the entire 6 min bout of exercise, or a two-component model fitted from 20 s were best. When the time delays for the two- and three-component models were equal the best statistical fit was obtained; however, this model produced an inappropriately low ΔV˙O2/ΔWR (WR, work rate) for the projected phase 2 steady state, and the estimate of phase 2 τ was shortened compared with other models. The slow component was quantified as the difference between V˙O2 at end-exercise (6 min) and at 3 min (ΔV˙O2 (6-3 min); 259 ml min-1), and also using the phase 3 amplitude terms (truncated to end-exercise) from exponential fits (409-833 ml min-1). Onset of the slow component was identified by the phase 3 time delay parameter as being of delayed onset ~2 min (vs. arbitrary 3 min). Using this delay ΔV˙O2 (6-2 min) was ~400 ml min-1. Use of valid consistent methods to estimate τ and the slow component in exercise are needed to advance physiological understanding. Experimental Physiology (2001) 86.5, 667-676.

The mechanism of human parturition is not understood and further research into this important physiological process is needed. Preterm labour remains a major cause of perinatal mortality and morbidity and there is controversy about the effectiveness of current tocolytic agents. In some species, notably the sheep, parturition is preceded by an activation of the fetal hypothalamic-pituitary-adrenal axis. However, in primates this axis has a supportive, rather than essential, role. A fall in maternal progesterone levels is a prerequisite for parturition in most mammals and this takes place either through increased conversion of progesterone to oestrogens in the placenta, or through the demise of the corpus luteum of pregnancy, depending on the species. In primates and guinea-pigs parturition occurs without an apparent fall in maternal progesterone levels. Gene targeting experiments in mice have demonstrated the critical role of prostaglandin FP receptors, necessary to mediate the luteolytic effect of PGF2α before parturition. Prostaglandin synthesis is required for the onset and progress of labour as demonstrated by experiments with cPLA2- and PGHS-1-deficient mice. The importance of local tissue conversion of progesterone to reduced androgens in the regulation of cervical ripening has been demonstrated in 5α-reductase-deficient mice. The chronic and ubiquitous gene inactivation obtained with conventional methods has disadvantages, in that it may allow the activation of compensating pathways, making the interpretation of results difficult. This problem may be overcome by using pulsed and tissue-selective gene knockout strategies. The study of human parturition is complicated by the lack of access to direct experimentation, whereas the endocrine differences between species make it difficult to extrapolate animal data to humans. However, the development of genomic/proteomic technologies that allow the simultaneous screening of thousands of genes and gene products in small samples of tissue, and new methods to study the biochemistry of receptors and proteins involved in smooth muscle physiology promise new insights into the control of human labour. Nevertheless, the integration of rapidly expanding knowledge into a complete understanding of the roles of the mother and the fetus in the initiation of parturition, and the development of selective medication for the effective management of preterm labour remain an arduous challenge for the next decade. Experimental Physiology (2001) 86.2, 213-222.

In this study we tested the hypothesis that, compared to young adult rats, senescent rats have a reduced ability to respond to muscle unloading. Unloading of the muscles was induced by hindlimb suspension (HS) of young adult and senescent rats for 21 days. Plantaris muscles from young adult rats had significantly higher levels of myogenin mRNA and protein (890 % and 314 %, respectively, P < 0.05) than plantaris muscles from senescent rats and also a higher MyoD mRNA level (280 %, P < 0.05), but ageing did not increase MyoD protein levels. Although HS did not increase plantaris mRNA or protein levels of myogenin or MyoD in senescent rats (P = 0.22), myogenin mRNA and protein levels increased by 850 % and 580 % respectively, and MyoD mRNA and protein levels by 235 % and 1600 %, respectively in young adult rats (P < 0.05). Soleus muscles from senescent rats had 150 % and 85 % greater myogenin and MyoD mRNA levels, respectively (P < 0.05), than soleus muscles from young adult rats, whereas protein levels of myogenin were similar (P > 0.05) and MyoD protein levels were 60 % lower in the muscle of senescent rats (P < 0.05). In young rats, soleus muscle mRNA levels of myogenin and MyoD were not altered by HS but myogenin protein levels decreased by 57 % (P < 0.05) whereas MyoD protein levels increased by 187 % (P < 0.05). In senescent rats, HS decreased soleus muscle myogenin mRNA and protein levels by 42 % and 26 % respectively (P < 0.05), but MyoD protein and mRNA levels were not changed. MRF4 levels were not affected by ageing in either muscle. These data suggest that ageing reduces the ability of fast muscles to increase myogenin protein levels, and prevents both fast and slow muscles from increasing MyoD protein levels during muscle unloading. Experimental Physiology (2001) 86.4, 509-517.

If we are to develop new strategies for the treatment and management of preterm and dysfunctional term labour, it is imperative that we improve current understanding of the control of human uterine activity. Despite many studies of animal pregnancy, there is a paucity of knowledge relating to the complex control of human myometrium during pregnancy. It is hypothesized that human myometrium is relatively quiescent during the majority of pregnancy and that as term approaches there is cascade of molecular events that prepare the uterus for labour. This review will consider the cellular mechanisms involved in the regulation of human myometrial activity and the modulation of these by hormonal and mechanical signals. In particular, the contribution of calcium homeostatic pathways to the control of human myometrial contractility during gestation will be discussed. Experimental Physiology (2001) 86.2, 247-254.

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50 % IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 ± 1 and 18 ± 4 mmol (kg dry mass)-1, respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP]. Experimental Physiology (2001) 86.3, 411-415.

Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O2 for 6 weeks) adult male Wistar rats (final body mass ~355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia. Experimental Physiology (2002) 87.3, 287-291.

It has been shown that cardiac fibroblasts of the human heart are electrically non-excitable and mechanosensitive. The resting membrane potential of these cells is -15.9 ± 2.1 mV and the membrane resistance is 4.1 ± 0.1 GΩ. Rhythmic contractions of the myocardium associated with stretch of the surrounding tissue produce reversible changes in the membrane potential of cardiac fibroblasts. These mechanically induced potentials (MIPs) follow the rhythm of myocardial contractions. Simultaneous recording of the action potential of cardiomyocytes and MIPs of cardiac fibroblasts demonstrates a delay of 40.0 ± 0.4 ms after the action potential before the appearance of the MIP. Contraction produces a MIP which is more positive or more negative than the reversal potential - the membrane potential due to current injection at which the MIP reverses its direction. Regardless of the initial orientation of the MIP, intracellular polarization increases the amplitude towards the reversal potential if the background MIP had depolarized the membrane or away from the reversal potential if the initial background MIP had hyperpolarized the membrane. Artificial intracellular polarization changed the amplitude but not the frequency of the MIP. The pool of electrically non-excitable mechanosensitive cells, which change their electrical activity during contraction and relaxation of the heart, may play a role in the mechano-electrical feedback mechanism which has to be taken into account in the normal function of the heart as well as in pathological processes.

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0·01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 ± 8 ms in subendocardial (n = 30 myocytes), 251 ± 7 ms in midmyocardial (n = 30) and 204 ± 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1·59 ± 0·16 pA pF-1, n = 16) than in either subendocardial (1·16 ± 0·12 pA pF-1, n = 17) or midmyocardial (1·13 ± 0·11 pA pF-1, n = 21) myocytes (P < 0·05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0·50 ± 0·04 pA pF-1, n = 11) than in subendocardial (0·25 ± 0·05 pA pF-1, n = 9) or in midmyocardial myocytes (0·38 ± 0·05 pA pF-1, n = 11, P < 0·05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0·1 µM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35·7 ± 7·1 %, n = 11) than in midmyocardial (15·7 ± 3·8 %, n = 10) and subepicardial (20·2 ± 4·3 %, n = 11) myocytes (P < 0·05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.

The role of the paraventricular nucleus (PVN) of the hypothalamus in the cardiovascular response induced by blood volume expansion was examined in anaesthetised rabbits, in which simultaneous recordings were made from a renal sympathetic nerve and one of other sympathetic nerves, the inferior cardiac nerve, a splanchnic nerve and an adrenal nerve. Activation of PVN neurones, by discrete injections (25-100 nl) of d,l-homocysteic acid (DLH, 0.2 M) produced one main pattern of sympathetic nerve activity accompanying a pressor response (57-86 % of PVN sites). This was a decrease in renal sympathetic activity (27 +/- 12 %) and an increase in splanchnic (60 +/- 12 %), adrenal (31 +/- 9 %) and cardiac (42 +/- 8 %) sympathetic activity. Sites in the PVN from which these combinations of nerve activity were obtained were not confined to a specific subnucleus. An increase in renal sympathetic activity which was reversed to a decrease by reducing the volume of DLH injected was obtained at 10 sites in the PVN. These sites were mainly located in the dorsal parvocellular subnucleus. Varying combinations of sympathetic activation were obtained at a minority of sites. It is concluded that the PVN can evoke a differential pattern of sympathetic discharge which may be functionally significant in the control of blood volume regulation, as it mimics that seen on acute volume expansion.

Presently, many researchers have adopted a reductionistic approach to physiology. Specifically, this involves studies on acutely isolated or cultured cells. While this is essential for our understanding of important cellular and subcellular mechanisms, it should not be forgotten that physiology is about the function of an integrating body. One view point is that definitive interpretations can only be made by crossing boundaries of physiology from the molecular/ cellular to the systems/behavioural level. An aim of this lecture will stress the importance of a multidisciplinary approach to physiology, that is, 'putting the cell back into the system'. The lecture attempts to demonstrate why this is important for enhancing the functional significance and relevance of physiological data as well as for validating predictions.