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Institute of Biomedical Investigation Spanish Research Council, 28029 Madrid, Spain; and the Laboratory of Flow Cytometry, Faculty of Pharmacy (A.M.A.), and the Department of Biochemistry and Molecular Biology, Faculty of Medicine (A.S.), Complutense University of Madrid, Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Ana Perez-Castillo, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas, c/Arturo Duperier 4, 28029 Madrid, Spain. E-mail: APEREZ{at}Biomed.iib.uam.es or Dr. Angel Santos,
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Introduction |
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and ß, which
are differentially expressed in a tissue-specific manner (4, 5). One of the most important effects of thyroid hormone concerns neonatal brain development. The association of congenital hypothyroidism with abnormal development and mental retardation has been extensively documented (6). In humans, thyroid hormone deficiency has been associated with irreversible mental retardation and profound neurological deficits, including deafness and movement disorders (7). Also, experimental hypothyroidism in the rat results in impaired brain development with a diminished interneuronal connectivity, decreased myelination, defective cell migration, and alterations in levels of neurotransmitters (6). There is a critical period during which appropriate thyroid hormone levels are essential for normal brain development. In humans, this period was considered to begin late in gestation and to extend through 1–2 yr of age. In rats, this critical period was thought to occur during the first 15 days after birth. This is an important period for neuronal differentiation, maturation, and development of neuronal processes. We have observed that during this time, both the number of T3 receptors and the amount of receptor messenger RNA rise together with the level of T3 in brain (5, 8). More recently, however, the idea has arisen that thyroid hormone is required earlier in development (9, 10). Understanding the mechanisms behind thyroid hormone effects on brain development has been a challenge for many years, and despite all the descriptions of functional and morphological defects in the developing brain in hypothyroidism, only recently have several reports shown specific genes that are targets of T3 action in this tissue (10, 11, 12, 13, 14).
Thyroid hormone has been reported to play an important role in the regulation of mitochondrial function in several tissues, such as liver, kidney, and skeletal muscle. T3 stimulates mitochondrial metabolism, and T3 deficiency results in decreased respiratory activities (15, 16). In general, thyroid hormone causes an increase in the overall capacity of mitochondrial electron transport chain proteins. This regulation of mitochondrial function takes place in the adult animal as well as during development (16, 17, 18, 19, 20). In accordance with its role in growth and development, thyroid hormone also plays an important role in hepatic mitochondrial maturation (18, 19). At least in part, thyroid hormone can affect mitochondrial respiratory function by controlling the expression of nuclear-encoded respiratory genes as well as mitochondrially encoded transcripts (20, 21, 22). However, each tissue responds in a characteristic fashion, and not all thyroid hormone effects are observed in all responsive tissues. Brain mitochondria have been generally considered to be nonresponsive to thyroidal state, as no changes in O2 consumption in either adult or neonatal rats have been found (23). Nevertheless, some biochemical analyses have suggested that neonatal hypothyroidism causes an alteration of brain mitochondria (24, 25). Along this line, we have recently reported a significant decrease in mitochondrial gene expression during brain development as well as an induction after thyroid hormone treatment (10).
In view of these data, the present study was designed to determine
whether alterations in brain mitochondrial gene expression in
hypothyroid neonatal animals could result in alterations in
mitochondrial structure and function and, therefore contribute to the
well known dramatic consequences of thyroid hormone deficit during
brain development. To this end, we performed a flow cytometric analysis
together with electron microscopic studies of ultrathin sections of the
cerebral cortex, striatum, and hippocampus of 15-day-old control and
hypothyroid rats. Our results show a drop in mitochondrial
transmembrane potential (
m) in hypothyroid brain,
which is corrected after a long treatment with thyroid hormone. This is
associated with alterations in the number of cristae membranes and
large intracristal spaces in the neurons of all of the areas studied
besides the pyramidal neurons of the CA1 field of the hippocampus and
the nonneural population. In addition, to better define the role that
T3 plays in regulating mitochondrial transcription, we have
analyzed the DNA-binding capacities of brain mitochondrial extracts
from control, hypothyroid, and T3-injected 15-day-old
animals throughout an approximately 900-bp region containing well
characterized control elements within the displacement loop region of
the mitochondrial DNA (mtDNA). We have observed a
T3-dependent DNA-binding activity with a mtDNA sequence
corresponding to a conserved region called conserved sequence block II
(CSB-II).
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Materials and Methods |
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-32P]deoxy-CTP, [
-32P]ATP, and DNA
labeling systems were purchased from Amersham Corp. (Aylesbury, UK) and
Pharmacia LKB Biotechnology (Piscataway, NJ), respectively. All other
chemicals were reagent or molecular biology grade.
Animal treatment
All animal experiments were conducted in accordance with the
Spanish Guidelines for the Care and Use of Laboratory animals. Female
Wistar rats were mated, and the day of appearance of the vaginal plug
was considered day 0 of fetal age. To induce fetal and neonatal
hypothyroidism, dams were given 0.02% methyl mercaptoimidazole in the
drinking water on day 9 of gestation. To assure very low levels of
T3 in the brain, 5 days after birth the pups were injected
with 131I (150 µCi/100 g BW) to destroy all thyroidal
tissue. Methyl mercaptoimidazole treatment was continued throughout the
period of lactation. With this treatment, T3 values were
below the level of detectability. For T3 treatment,
hypothyroid animals were daily injected with 200 µg
T3/100 g BW and killed 48 h (flow cytometry analysis)
or 72 h. (electron microscopy studies) later. After being weighed,
the animals from all groups were killed by decapitation on postnatal
day 15, and the brain was quickly removed and used for mitochondria
isolation or electron microscopy studies.
Isolation of mitochondria
Purified brain mitochondria were prepared as described
previously (26). The tissue was minced, washed, and homogenized
manually in 0.32 M sucrose, 1 mM EDTA, 10
mM Tris-HCl (pH 7.4), and 0.1% BSA. The homogenate was
centrifuged at 2,000 x g for 3 min. The precipitate
was discarded, and the supernatant was centrifuged again at 12,500
x g for another 10 min to yield the crude mitochondrial
pellet. This mitochondrial pellet was resuspended in 0.03 M
sucrose, 0.12 M manitol, 0.025 mM EDTA, 10
mM Tris-HCl (pH 7.4), and 3% (wt/vol) Ficoll. This
suspension (3 ml) was layered onto 7 ml of a discontinuous gradient of
Ficoll (7.5–13%) and centrifuged at 99,000 x g for
30 min. The pellet was washed twice, centrifuged at 12,500 x
g, and resuspended in homogenization medium.
Preparation of mitochondrial extracts
Mitochondrial extracts were prepared from brains of 15-day-old
control, hypothyroid, and T3-injected animals.
Mitochondrial pellets, isolated as described above, were resuspended in
lysis buffer [20 mM Tris HCl (pH 8.0), 0.2 mM
EDTA, 10% glycerol, 0.35 M NaCl, 1% Triton X-100, and 1
mM dithiothreitol] containing 1 mM
phenylmethylsulfonylfluoride. The mitochondrial suspension was
vigorously vortexed and allowed to stand on ice for 15 min. The
mitochondrial lysate was then spun at 45,000 rpm for 1 h. Protein
was determined in the supernatant, and fractions were frozen and stored
at -70 C.
Mobility shift assays
Three DNA probes were obtained by amplifying three different
sequences of the rat mitochondrial promoter spanning nucleotides
15,401–15,750, 15,751–16,012, and 16,065–16,298 and labeled using
Klenow DNA polymerase and [32P]deoxy-CTP. Two double
stranded oligonucleotides corresponding to two conserved sequence
blocks in the mitochondrial promoter (CSB-II and CSB-III) and one
oligonucleotide from nucleotides 16,167–16,192 were also obtained and
labeled using [-32P]ATP and T4
polynucleotide kinase. The gel shift mixture (20 µl) contained 10
mM Tris-HCl (pH 8.5), 10 mM MgCl2,
10 mM KCl, 1 mM dithiothreitol, 100 µg BSA, 2
µg poly(dI-dC), 10 µg mitochondrial protein, and 105
dpm of the indicated 32P-labeled DNA probes or
oligonucleotides. This mixture was incubated at room temperature for 30
min before electrophoresis on 4% nondenaturing polyacrylamide gels in
89 mM Tris, 89 mM borate, and 2 mM
EDTA. Gels were run at 200 V for 2–3 h, dried, and exposed for
autoradiography. The experiments were performed three times using
different animals.
Flow cytometry analysis
Isolated mitochondria were analyzed on a FACScan flow cytometer
(Becton Dickinson, San Jose, CA), using CELLQUEST as the data acquiring
and analysis software. Except in kinetic experiments, in which time was
used as the limit of acquisition, 10,000 events were acquired by
sample. The mitochondrial fractions were first treated with propidium
iodide (5 µg/ml) to check the membrane integrity. All results
presented here represent the propidium iodide-negative mitochondrial
population. Rh-123 (5 nM) was added at the moment of
running the mitochondria in the flow cytometer, and events were counted
for 3 min. To study the effects of substrates on mitochondrial membrane
potential, mitochondria were incubated with Rh-123 for 10 min at room
temperature. After this, we began time acquisition for 5 min, adding
the substrates glutamate (2.5 mM) plus malate (2.5
mM), succinate (5 mM), and ADP (180
µM) every 90 sec. The mean fluorescence for each
substrate was obtained by gating by time. Values obtained for each
experimental group in the absence of substrates are called basal
values. All experiments were carried out three times using different
sets of animals.
Electron microscopy
Ultrastructural morphology of mitochondria was determined
by transmission electron microscopy. The brains were cut in the coronal
plane at 1-mm thickness, and sections of cerebral cortex, striatum, and
hippocampus were rapidly fixed with 4% paraformaldehyde-4%
glutaraldehyde in 0.1 M phosphate buffer. After fixation,
the sections were osmicated in 1% osmium tetroxide for 2 h at 4 C
and then dehydrated in an alcohol-acetone series. Tissues were embedded
in araldite resin, and semithin (2.5 µm) sections were removed for
optical microscopy. Ultrathin sections (50 nm) were mounted on copper
mesh grids and stained with uranyl acetate and lead citrate before
examination with a JEOL JEM 1010 electron microscope (JEOL, Peabody,
MA). All tissue samples were first inspected on 2.5-µm sections by
light microscopy so that the correct area could be determined. The most
internal area of the cerebral cortex and the dentate gyrus and CA1
field of the hippocampus were selected. The studies were performed on
three different groups of 15-day-old neonates [euthyroid, hypothyroid,
and T3 injected (200 µg/100 g BW)], each composed of
three different animals. Three grids were used for each animal. Four
different morphological groups of mitochondria were defined (see Fig. 3
). They were further divided into two groups according to the degree
of alteration observed: normal (N) and altered (A). After examining
them we assigned each mitochondrium to the closest morphological group.
This was performed without knowing the source of the tissue examined.
Values presented in the histograms of Figs. 4, 5, and 6 were obtained
after examining the morphology of about 200 mitochondria for each area
of the brain (cerebral cortex, striatum, and hippocampus) and for each
animal.
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, lower uptake of
Rh-123 was observed in brain mitochondria from hypothyroid neonates.
When average Rh-123-related fluorescence was calculated, a significant
decrease was detected in the hypothyroid group, 61% of the values
obtained in age-matched euthyroid animals (Fig. 1B). To evaluate
whether the loss in the fluorescence associated with the uptake of
Rh-123 would be reversible, hypothyroid neonates were injected 48
h before death with a dose of T3 that saturates the nuclear
receptor. As shown in Fig. 1, the drop in mitochondrial membrane
potential was totally reversed by T3 administration to
hypothyroid animals, and mitochondria in this experimental group
displayed a pattern of Rh-123 labeling similar to that observed for
control animals.
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). When control and hypothyroid
neonates were compared, we again found a significant decrease in Rh-123
fluorescence in the hypothyroid animals compared with that in the
control group. The values presented in Fig. 2B show 117% of the basal
signal in control animals, in contrast to 78% in hypothyroid neonates
(
17% of increment over basal values in both cases). When
hypothyroid animals were injected with a saturating dose of
T3 and killed 48 h later, not only was basal Rh-123
uptake normalized, but an increased response to the addition of
substrates was observed (42% over the basal values obtained for the
hyperthyroid group), suggesting a potentiation of mitochondrial
activity in the hyperthyroid animals, as a saturating dose of
T3 was used. In contrast with the observed effect after
48 h of T3 administration, no effect was noted at
shorter (2 h) times, and only a partial recovery was observed 18 h
later (data not shown). Taken together, these results suggest a
deficiency of mitochondrial respiratory capacity in hypothyroid
neonates that is corrected after thyroid hormone administration.
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). Some of them, which we
call normal, have the same basic architecture of typical neuronal
mitochondria, with a highly folded internal membrane circumscribing a
mitochondrial compartment filled with a dense matrix. On the other
hand, we also observed some mitochondria with a morphology that we call
altered, with a marked decrease in the area of the inner membrane, a
reduction in the number of cristae, a destructurization of the matrix
compartment, and clear vacuolization.
These two classes of mitochondria were visible in the three brain
areas studied (Figs. 4-6A). Quantitation
data of the different types of mitochondria are shown in
Figs. 4–6B.
The majority of neuronal mitochondria from the cerebral cortex of
control animals presented a normal morphology, with a highly packed
inner membrane and a regular disposition of the cristae; only a minor
fraction (12%) belonged to the altered type. However, a great
proportion of hypothyroid mitochondria harbor dramatic alterations in
the mitochondrial structures, and in hypothyroid neonates, a
significant increase in the number of altered mitochondria was observed
compared with those found in age-matched euthyroid animals (57% and
12%, respectively; Fig. 4B). The changes observed consist mainly of a
marked reduction on the surface of the inner membrane and vacuolization
of the matrix compartment. Different stages of membrane disintegration,
from minor disarrangements of the inner membrane to empty vesicular
structures, can be seen. Similar results were obtained with the neurons
of the striatum (Fig. 5) and the granular
neurons of the dentate gyrus (Fig. 6). In
contrast, we did not observe any difference in the ratio of normal to
altered mitochondria between control and hypothyroid rats in the
pyramidal neurons of the hippocampal field CA1 (data not shown). In
these neurons, about a 50% of the mitochondria were normal, and the
other 50% belonged to the altered type in both control and hypothyroid
neonates. On the other hand, nonneuronal cells, such as astrocytes and
oligodendrocites, did not present a high proportion of altered
mitochondria in the hypothyroid animals, and the ratio of normal to
altered was the same in both control and hypothyroid groups, with a
great percentage (80–90%) of mitochondria with no morphological
alterations (data not shown). The fact that hypothyroidism did not
affect the CA1 neurons and the nonneuronal cells suggests that the
effect of thyroid hormone is specific and differential. Also, the
aberrant mitochondrial structures were not preparation or fixation
artifacts, as the integrity of other subcellular organelles (not shown
in the pictures) affirms good subcellular preservation. Thyroid hormone
administration to hypothyroid neonates resulted in a normalization of
mitochondrial morphology. Quantitation of the number of altered
mitochondria, compared with normal (
Figs. 4–6B), resulted in a similar
percentage of both populations between control and hyperthyroid
animals. No effect of T3 injection was observed in the
neurons of the CA1 hippocampal field or in nonneuronal cells (data not
shown).
These morphological alterations are in accordance with the flow cytometry data, showing a decrease in membrane potential, which is a reflection of a reduction in the respiratory activity of the mitochondria.
Gel retardation analysis of the mtDNA promoter region
Mammalian mitochondrial DNA is bidirectionally transcribed from a
heavy strand and a light strand promoter. These promoters are both
localized to the mayor regulatory region for mtDNA transcription and
replication in the only extensive noncoding area, called the
displacement loop (D-loop) (29). To gain further insight into how
thyroid hormone could regulate mitochondrial gene expression, we next
performed electrophoretic mobility shift assays to determine whether
thyroid status could influence the binding of some mitochondrial
protein to this regulatory region of the mtDNA. As indicated in
Materials and Methods, three different DNA fragments,
covering the region of the D-loop from nucleotides 15,401–16,298, were
obtained. Brain mitochondrial extracts were prepared from euthyroid,
hypothyroid, and hypothyroid T3-injected animals and tested
for DNA-binding activity. As shown in Fig. 7A, binding activity was highest in the
extracts from control rat brain and was lowest in the hypothyroid
extract when DNA fragment 16,065/16,298 was used. These differences
were consistently observed. The binding activity was recovered 24
h after thyroid hormone administration to hypothyroid neonates. On the
contrary, no differences were detected when the other mtDNA regulatory
sequences, DNA fragments 15,401/15,750 and 15,751/16,012, were tested
(data not shown). We next investigated more precisely the DNA sequence
responsible for this DNA-binding activity. To this end, we designed
three different oligonucleotides corresponding to two conserved
sequence blocks in the D-loop region (CSB-II and CSB-III) as well as an
oligonucleotide from nucleotides 16,167–16,192, all of them within the
region that caused a retardation dependent on thyroid state. As shown
in Fig. 7B, we observed that the T3-dependent protein-DNA
binding is localized to the CSB-II region. The other two
oligonucleotides tested did not present any T3-dependent
DNA-binding activity. A 200-fold molar excess of unlabeled
oligonucleotide abolished the formation of the shifted band, indicating
specificity of binding. Unrelated oligonucleotides (CSB-III and
oligonucleotide from nucleotides 16,167–16,192) did not prevent
formation of the retarded band (data not shown).
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The general organization of the mitochondrial inner membrane cristae into the typical transverse alignment in control animals was largely absent in the mitochondria isolated from hypothyroid brain. These mitochondria were characterized by highly fractured and degenerated cristae and a clear vacuolation. The drop in mitochondrial membrane potential, observed by flow cytometry in total brain mitochondria of hypothyroid animals, is probably a reflection of the decreased surface area of the inner membrane observed in neuronal cells in the electron microscopic study because in this membrane all enzymes of the respiratory chain are present.
As we noted above, until recently it has been generally considered that brain mitochondria were insensible to the action of T3. However, there were some reports indicating that mitochondrial alterations could be occurring in the brains of hypothyroid animals. An alteration in phospholipid composition and membrane fluidity of rat brain mitochondria has been reported (30) as well as some alterations of oxidative phosphorylation (31). We recently found that differences in thyroidal state during development result in profound alterations of mitochondrial gene expression (10). All of these data together with the results of the present study clearly indicate that mitochondrial function can be severely impaired in the hypothyroid brain, and that T3 can counteract this event. These findings provide a biochemical basis for the known alterations of brain function as a consequence of the lack of thyroid hormone during brain development. This is particularly important in view of the growing evidence that primary defects in mitochondrial function are implicated in over 100 diseases, and that the clinical manifestations most often involve the central nervous system (Kearns-Sayre syndrome, MERFF, MELAS, etc.). In addition, mitochondrial dysfunction have now been found to occur in aging and in age-related degenerative diseases affecting brain function, such as Parkinson’s and Alzheimer’s diseases (32).
Alterations in mitochondrial morphology caused by hypothyroidism have
been described in other tissues, such as liver and skeletal muscle (33, 34), and the modifications are essentially the same as those described
here for the developing brain. In the liver, Jakovcic et al.
(35) also reported hypothyroid mitochondria to be shorter and wider
than those in controls, and some swelling after T3
administration has also been observed (33). In skeletal muscle,
the administration of T3 to hypothyroid animals results in
an increase in the number of cristae, accompanied by some hypertrophy
(33). The results of the flow cytometric analysis and electron
microscopic studies shown here also revealed small differences in size
between the brain mitochondria of control and hypothyroid neonates.
Although the alterations found in mitochondrial morphology in the
hypothyroid animals are essentially the same between liver and brain,
our data, showing a drop in brain mitochondrial transmembrane potential
in these animals, are different from the situation in the liver. There
are several reports showing a lowering of the mitochondrial membrane
potential after T3 administration to euthyroid rats (36, 37). It has been proposed that the decrease in m in
hyperthyroid liver mitochondria is the result of a nonspecific thyroid
hormone-induced increase in proton conductance of the mitochondrial
inner membrane (proton leak). Thus, there is a clear difference in the
response of brain and liver mitochondria to thyroid hormone status,
suggesting a tissue-specific effect of thyroid hormone on brain
mitochondria.
An interesting point is the observation of a clear differential responsiveness to T3 in the mitochondria of the different neurons. Whereas granular neurons mitochondria of the dentate gyrus respond to T3 in the same fashion as those of the cerebral cortex or striatum, mitochondria of the pyramidal neurons from the CA1 field of the hippocampus are not sensitive to thyroidal state. These results together with recent reports showing a differential effect of thyroid hormone on brain gene expression depending on the area studied and the age of the animal (14, 38) point to a very complex effect of hypothyroidism on brain development, where different kinds of neurons respond to this hormone in very different fashions. The variability in the response to thyroid hormone suggests a modulation of T3 action on gene expression for other cellular factors differentially expressed in different brain cell types.
The next step toward a better understanding of the role of T3 in mitochondrial function must include studies of the molecular mechanisms underlying T3 action. There are some reports showing very rapid effects of thyroid hormone on hepatic mitochondrial activity in vivo (39), which have been suggested to be mediated by a direct effect of the T3 metabolites 3,3'-diiodothyronine and 3,5'-diiodothyronine on the mitochondria (40). This is in contrast to the effect of thyroid hormone that we have observed on brain mitochondrial activity, as this one requires a much longer period to be observed, suggesting that it is related to changes in mitochondrial gene expression (10). Although the presence of thyroid hormone receptors has been recently described in rat liver mitochondria (41, 42), a better explanation of the T3 effects on these organelles could be the existence of a T3-regulated mediator between T3 nuclear receptors and the mitochondria, as the effects of thyroid hormone on the expression of nuclear and mitochondrially encoded genes are coordinate (10, 43). Good candidates for mediators are the nuclear respiratory factor-I protein (44), which is known to regulate the mitochondrial transcription factor A (mtTFA) (45), the mtTFA itself, or another protein necessary for mitochondrial transcription and not yet characterized.
In an attempt to know the possible existence of one of these mediators, in this work we have analyzed the protein-DNA binding activity on the regulatory region of the mtDNA and its response to thyroidal status. The mammalian mitochondrial DNA possesses a region called the D-loop, which is the main noncoding region and the most variable part of vertebrate mitochondrial genomes. This region contains the origin of heavy strand DNA replication and the promoters for heavy and light strand transcription (29). Within this region there are three conserved sequence blocks, called CSB-I, CSB-II, and CSB-III, that are thought to be involved in mtDNA replication and transcription. Our findings show that rat brain mitochondrial extracts contain a protein(s) that binds to a specific sequence (CSB-II) in the mitochondrial regulatory region. The binding activity of the protein(s) was influenced by the thyroid status of the animal, suggesting that T3 regulates mitochondrial gene expression through the control of proteins that bind to regulatory sequences responsible for controlling mitochondrial transcription. However, additional studies will be necessary to identify these proteins and establish their function and whether they could act as a link between the nuclear receptor for T3 and its action in the mitochondria.
In summary, this work provides evidence that the changes in mitochondrial gene expression during development in the hypothyroid neonatal brain, previously reported by our laboratory (10), result in an impaired mitochondrial function and in the appearance of a population of neuronal mitochondria with severely altered structure. This work thus shows for the first time that the lack of thyroid hormone in the developing brain can severely alter mitochondrial structure and function and, in consequence, provides a cellular basis for the observed dependency on T3 of the developing brain. Finally, our findings supply initial information about the mechanism by which thyroid hormone can influence brain mitochondrial gene expression.
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Acknowledgments |
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Footnotes |
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2 Predoctoral fellow from the Communidad de Madrid.
3 Predoctoral fellow from the Fondo de Investigaciones Sanitarias de
la Seguridad Social.
Received February 26, 1997.
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and ß thyroid hormone receptor genes. EMBO J 9:1519–1528[Medline]
and ß-related T3 receptors in rat
liver mitochondria. Eur J Cell Biol 62:105–113[Medline]
1 is located in the
mitochondrial matrix of rat liver. J Biol Chem 270:16347–16354This article has been cited by other articles:
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  B. Martinez, T. B. Rodrigues, E. Gine, J. P. Kaninda, A. Perez-Castillo, and A. Santos Hypothyroidism Decreases the Biogenesis in Free Mitochondria and Neuronal Oxygen Consumption in the Cerebral Cortex of Developing Rats Endocrinology, August 1, 2009; 150(8): 3953 - 3959. [Abstract] [Full Text] [PDF]
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M. Garcia-Fernandez, G. Delgado, J. E. Puche, S. Gonzalez-Baron, and I. Castilla Cortazar Low Doses of Insulin-Like Growth Factor I Improve Insulin Resistance, Lipid Metabolism, and Oxidative Damage in Aging Rats Endocrinology, May 1, 2008; 149(5): 2433 - 2442. [Abstract] [Full Text] [PDF]
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