J. Anim Sci. 2008. 86:2888-2896. doi:10.2527/jas.2008-1062
© 2008 American Society of Animal Science
OPEN ACCESS ARTICLE
ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Adenosine monophosphate-activated protein kinase involved in variations of muscle glycogen and breast meat quality between lean and fat chickens1
V. Sibut*,
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E. Le Bihan-Duval,
S. Tesseraud,
E. Godet,
T. Bordeau,
E. Cailleau-Audouin,
P. Chartrin,
M. J. Duclos and
C. Berri,2
* Institut Technique Avicole, F-37380 Nouzilly, France; and and
INRA, UR83 Recherches Avicoles,F-37380 Nouzilly, France
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Abstract
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The present study was aimed at evaluating the molecular mechanisms associated with the differences in muscle glycogen content and breast meat quality between 2 experimental lines of chicken divergently selected on abdominal fatness. The glycogen at death (estimated through the glycolytic potential) of the pectoralis major muscle and the quality of the resulting meat were estimated in the 2 lines. The fat chickens exhibited greater glycolytic potential, and in turn lower ultimate pH than the lean chickens. Consequently, the breast meat of fat birds was paler and less colored (i.e., less red and yellow), and exhibited greater drip loss compared with that of lean birds. In relation to these variations, transcription and activation levels of adenosine monophosphate-activated protein kinase (AMPK) were investigated. The main difference observed between lines was a 3-fold greater level of AMPK activation, evaluated through phosphorylation of AMPK
-(Thr172), in the muscle of lean birds. At the transcriptional level, data indicated concomitant down- and upregulation for the 
1 and 2 AMPK subunit isoforms, respectively, in the muscle of lean chickens. Transcriptional levels of enzymes directly involved in glycogen turnover were also investigated. Data showed greater gene expression for glycogen synthase, glycogen phosphorylase, and the subunit of phosphorylase kinase in lean birds. Together, these data indicate that selection on body fatness in chicken alters the muscle glycogen turnover and content and consequently the quality traits of the resulting meat. Alterations of AMPK activity could play a key role in these changes.
Key Words: adenosine monophosphate-activated protein kinase • chicken • fatness • glycogen • meat quality • pH
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INTRODUCTION
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With changes similar to those that occurred in the swine industry, the poultry market is now characterized by an increasing diversity of further processed products (Mead, 2004
), and the improvement of meat processing ability has become a prevalent concern for meat companies. In both species, postmortem pH appears to be a key factor controlling poultry meat quality. In chickens as in pigs, stress and behavioral activity before slaughter are largely involved in the variations of pH in the early stages of rigor (Debut et al., 2003). The final pH of meat depends on the muscle glycogen content at the time of slaughter (Berri et al., 2005, 2007). Several studies highlighted the major role of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) in regulating the level of muscle glycogen (Carling and Hardie, 1989; Jørgensen et al., 2004; Figure 1) and postmortem metabolism (Scheffler and Gerrard, 2007).
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Figure 1. Function of studied enzymes in the regulation of muscle glycogen metabolism. AMPK = adenosine monophosphate-activated protein kinase; GSK3 = glycogen synthase kinase; PHK = phosphorylase kinase; GYS = glycogen synthase; PYG = glycogen phosphorylase.
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In the present study, we compared the muscle glycogen content and breast meat quality traits from 2 experimental chicken lines divergently selected for abdominal fatness (i.e., fat and lean lines; Leclercq et al., 1980). To evaluate the possible role of AMPK to explain variations in pectoralis major (P. major) muscle glycogen content observed between the fat and lean lines, we characterized the mRNA expression of the PRKA genes (coding for the catalytic , regulatory β and AMPK subunits) by quantitative reverse transcription PCR. In addition, we evaluated the level of AMPK activation by performing immunoblotting with a phospho-specific antibody. We also determined the gene expression of several enzymes directly involved in glycogen synthesis and degradation (Figure 1
); that is, glycogen synthase (GYS), glycogen phosphorylase (PYG), and their upstream kinases glycogen synthase kinase 3 (GSK3) and phosphorylase kinase (PHK; composed of one catalytic subunit and 3 regulatory subunits , β, and 
that inhibit the phosphotransferase activity of the subunit).
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MATERIALS AND METHODS
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All animal procedures and care were performed with due regard to legislation governing ethical treatments of animals. Principal investigators were certified by the French government to experiment on live animals.
Birds
The chickens originated from 2 experimental lines divergently selected for high abdominal fatness (fat line) or low abdominal fatness (lean line) at 9 wk of age (Leclercq et al., 1980). They were reared under similar conditions in a conventional poultry house at the INRA experimental poultry unit (UE1295, Nouzilly, France). They were given ad libitum access to standard diets throughout the growth period. At 9 wk of age, the birds were individually weighed 1 d before slaughter. After 7 h of feed withdrawal, 60 male birds in each line were slaughtered at the experimental processing plant (UE1295, Nouzilly, France). Before killing by ventral neck cutting, the birds were stunned in a waterbath (125 Hz AC, 80 mA/bird, 5 s). After evisceration, whole carcasses were air chilled (airflow of 7 m3) and stored at 2°C until the next day. Glycolytic potential (GP) and lipid content of P. major were determined on 12 birds per line selected at random. Tissue for RNA extraction and protein solubilization were collected 15 min postmortem from 9 chickens per line (issued from the 12 individuals assayed for GP and lipid measurements) and immediately frozen in liquid nitrogen.
Muscle and Meat Characteristics
Muscle glycogen, glucose-6-phosphate, free glucose, and lactate were measured in P. major from 12 individuals per line according to Dalrymple and Hamm (1973), from 1 g of frozen tissue. Glycolytic potential takes into account the main intermediates of glycogen degradation in live and postmortem muscle and therefore represents an estimate of the resting glycogen content (Monin and Sellier, 1985); it was calculated as follows:
and was expressed as micromoles of lactate equivalent per gram of fresh tissue. On the same individuals, the P. major total lipids were extracted quantitatively by homogenizing a sample of minced tissue in chloroform/methanol 2:1 (vol/vol) and then quantified gravimetrically (Folch et al., 1957).
All carcasses were processed 1 d after slaughter. Abdominal fat and pectoral muscles (P. major and P. minor) were removed and weighed. Abdominal fat and breast yields were calculated and expressed as percentage of BW. The ultimate pH (pHu) of the P. major muscle of all carcasses was measured at 24 h postmortem by direct insertion of an electrode (pH meter Model 506, Crison Instruments, Barcelona, Spain) into the muscle. At the same time, breast color was measured on the rostral, ventral side of the P. major muscle by using a Miniscan Spectrocolorimeter (Hunter Lab, Reston, VA). Color was measured by the CIELAB trichromatic system as lightness (L*), redness (a*), and yellowness (b*) values. The water-holding capacity of meat was estimated by measuring drip loss of the raw meat after storage. The breast P. major muscle was weighed 24 h postmortem and immediately placed in a plastic bag, hung from a hook, and stored at 2°C for 48 h. After hanging, the sample was wiped with absorbent paper and weighed again. The difference in weight corresponded to the drip loss and was expressed as the percentage of the initial muscle weight.
RNA Isolation and Reverse Transcription PCR
Total RNA was extracted using a commercial kit (RNA Now, Ozyme, Saint Quentin Yvelynes, France) according to Chomczynski and Sacchi (1987). Concentrations of RNA were measured by spectrophotometry (optical density at 260 nm) using a NanoDrop ND-1000 (NanoDrop Technologies LLC, Wilmington, DE) and their integrity was checked by electrophoresis. After DNase treatment (using Ambion’s DNA-Free 1906 kit obtained from Clinisciences, Montrouge, France), total RNA (5 µg) was reverse transcribed using RNase H– MMLV reverse transcriptase (Superscript II, Invitrogen, Illkirch, France) and random primers (Promega, Charbonnières les Bains, France).
Determination of mRNA Levels by Real-Time Reverse Transcription PCR
After reverse transcription, the cDNA of PRKA subunits (1, 2, β1, β2, 1, 2, and 3), GYS, PYG, GSK3, and PHK subunits (, β, , and ) were amplified in real-time using an ABI PRISM 7000 apparatus (Applied Biosystems, Courtaboeuf, France) with the SYBR Green I qPCR Master Mix Plus (Eurogentec, Angers, France). The 18S ribosomal RNA (18S rRNA) chosen as a reference was determined with the TaqMan Universal qPCR Master Mix Kit and a predeveloped Taqman assay reagent (Applied Biosystems). For each gene, specific primers were designed from the corresponding chicken sequence to be intron-spanning to avoid coamplification of genomic DNA (Table 1) and were obtained from Eurogentec. The cycling conditions consisted of a denaturation step at 95°C for 10 min, followed by a 2-step amplification program (15 s at 95°C, followed by 1 min at 62°C or 64°C for PRKA β2 and 1) repeated 40 times. At the end, a dissociation program consisting of 15 s at 95°C, 20 s at 62°C or 64°C, and 15 s at 95°C was performed. The amplification products for all cDNA were checked by electrophoresis and exhibited the expected size (Table 1). Their sequences were also checked. For each individual sample (n = 9 per line), the cDNA of genes under study were amplified in triplicate in the same run. Each PCR run included a no-template control and triplicates of control; that is, a pool of all cDNA and unknown samples. To confirm the precision and the reproducibility of real-time PCR, intraassay variations were evaluated and found to be between 0.08 and 1.18% (CV) according to the gene. The calculation of absolute mRNA levels was based on the PCR efficiency and the threshold cycle deviation of an unknown cDNA vs. the control cDNA according to the equation proposed by Pfaffl (2001) and as described in Guernec et al. (2003). Absolute mRNA levels were corrected for 18S rRNA to give relative levels.
Determination of AMPK Expression and Phosphorylation
All chemicals were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France) unless otherwise noted. Frozen tissues were ground in liquid nitrogen. Powdered muscles (300 mg) were homogenized on ice with a homogenizer (IKA T25 Ultra-Turrax, Fontenay-sous-Bois, France) in a buffer containing 150 mmol/L NaCl, 10 mmol/L Tris (hydroxymethyl) amino-methane (pH 7.4), 1 mmol/L EDTA, 1 mmol/L ethylene glycol-bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 1% triton X-100, 0.5% NP-40, protease inhibitors (complete protease inhibitor cocktail, Roche, Meylan, France), and phosphatase inhibitors (100 mmol/L sodium fluoride, 10 mmol/L sodium phosphate, and 2 mmol/L sodium orthovanadate). Homogenates were centrifuged at 1,000 x g for 30 min at 4°C and supernatants were ultracentrifuged for 45 min at 150,000 x g. Supernatants were aliquoted and stored at –80°C. Protein concentrations were determined by the Bradford (1976) method using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Marnes-la-Coquette, France). Muscle lysates (40 µg of protein) were subjected to 10% (wt/vol) SDS-PAGE under reducing conditions and electrotransferred as described previously (Duchêne et al., 2008). The membranes were then incubated overnight at 4°C with appropriate antibodies (see below) at a final dilution of 1:1,000 in Tris-buffered saline (2 mM Tris-HCl, pH 8, 15 mM NaCl, pH 7.6) containing 0.1% Tween-20 and 5% nonfat dry milk powder. Rabbit polyclonal antibodies to AMPK1 and to phospho-AMPK Thr172 were obtained from Upstate Biotechnology Inc. (Lake Placid, NY) and from New England Biolabs Inc. (Beverly, MA), respectively. Mouse monoclonal anti-human vinculin was purchased from Sigma-Aldrich (St. Louis, MO). After washing, the membranes were incubated with an Alexa Fluor secondary antibody (Molecular Probes, In-terchim, Montluçon, France). Bands were visualized by infrared fluorescence by the Odyssey Imaging System (Li-Cor Inc. Biotechnology, Lincoln, NE) and quantified by Odyssey infrared imaging system software (application software, version 1.2).
Statistical Analysis
All data were analyzed using SAS (SAS Inst. Inc., Cary, NC). The accepted type I error was 5%. The effects of line were analyzed by a 1-way ANOVA (GLM procedure). Comparisons of means for each significant effect were performed by Student-Newman-Keul’s test using the SNK statement of the GLM procedure. To assess the relationships between muscle and meat traits, Pearson correlation coefficients were analyzed with the CORR procedure.
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RESULTS
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Characterization of Carcass, Muscle, and Meat Quality Traits in Lean and Fat Lines
The carcass and P. major muscle trait means and corresponding SD for each line are presented in Table 2. At 9 wk of age, BW was greater (P 
0.001) and breast yield less (P 0.001) in the fat than in the lean line. The abdominal fat weight and yield were, respectively, 2.9- and 2.8-fold greater (P 0.001) in fat birds than in lean birds. The lipid content of the P. major muscle was the same in both lines. In contrast, correlated responses to divergent selection for abdominal fatness were observed on breast meat traits. The fat chickens exhibited greater P. major muscle glycogen content (P 0.05) and GP (P 0.01) than the lean chickens. Moreover, the GP of P. major muscle was highly positively correlated with abdominal fat yield and negatively with breast yield (Table 3). The GP was also negatively correlated to pHu (Table 3). Therefore, in relation to its greater glycogen reserve at the time of death, the P. major muscle of fat birds was characterized by a lower pHu. In the same way, because of the correlations between muscle pHu and meat quality traits (Table 3), the color of breast meat was lighter (greater L*, P 0.001) and less red (lower a*, P 0.001) in fat chickens, which also exhibited greater drip loss (P 0.05). In addition, breast meat of fat chickens was characterized by lower values of yellowness (b*) in meat color (P 0.001). No difference in muscle lactate content at 15 min postmortem was observed between the fat and lean birds processed for quality measurements (data not shown). However, we noticed that lactate tended to be greater in lean than in fat birds assayed for AMPK protein (65.8 ± 5.2 and 54.1 ± 7.9, respectively; P 0.09, n = 6).
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Table 2. Body weight, yields, and pectoralis major muscle and meat quality traits of the lean and fat lines at 9 wk of age1
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AMPK Characterization and Regulation in the P. major Muscle of Lean and Fat Lines
All the PRKA genes coding for the different isoforms of the AMPK subunits , β, and were expressed in the P. major muscle of both lines. However, differences in expression levels existed among the PRKA genes. According to the cycle threshold at which the fluorescence reached a given value (0.02 in this study) for each gene, the 2, β2, and 1 or 3 subunit isoforms appeared preferentially expressed in the P. major compared with the 1, β1, and 2 subunits, respectively (Table 4). For comparison of the lines, 18S rRNA was chosen as reference gene because it was not different between lines. Further results based on the ratios between PRKA mRNA and 18S rRNA in the P. major muscle are presented in Figure 2. Although the relative levels of mRNA expression for PRKA genes showed only limited variations, PRKA 2 and 1 mRNA levels were significantly less (–25% for each; P 0.05), whereas PRKA β1 and 2 mRNA levels were greater (+50% and +30%, respectively; P 0.05) in lean chickens than in fat chickens.
By Western blot, the AMPK1 (63 kDa) protein was shown to be present in the P. major muscle of both lines. A quantitative analysis revealed no significant difference in AMPK1 protein content between the fat and lean lines (Figure 3). Conversely, the phosphorylation level of the -subunit of AMPK on the Thr172 residue was 3-fold greater in the muscle of lean birds compared with that of fat birds (Figure 3).
mRNA Expression of GYS, PYG, GSK3, and PHK in the P. major Muscle of Lean and Fat Lines
Results on the mRNA levels of GYS, PYG, GSK3, and PHK genes in the P. major muscle are presented in Figures 4 and 5. The expression of GYS and PYG was upregulated (P 0.05) in the lean line (Figure 4). The expression of the GSK3 was not altered by line (Figure 5). Among the PHK subunits, the regulatory d subunit was upregulated (P 0.01) in the P. major muscle of the lean line. There was also a trend for the regulatory and the catalytic subunits of the PHK to be upregulated (P 0.06) in lean birds. By contrast, the regulatory β subunit of the PHK mRNA levels was not affected by the line.
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Figure 4. Relative mRNA levels (lean/fat, %) for glycogen synthase (GYS) and glycogen phosphorylase (PYG) in pectoralis major muscle. The relative expression of each gene (i.e., corrected for 18S ribosomal RNA) was determined by real-time reverse transcription PCR as described in the Materials and Methods section. The value for the fat line was fixed to 100 (dotted line). The data (means ± SE, n = 9) are expressed as lean/fat (%). *P 0.05, difference between lines.
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DISCUSSION
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The fat and lean lines were obtained by divergent selection within a population originally derived from meat-type lines (Leclercq et al., 1980). At 9 wk of age, birds from the fat line exhibited almost 3 times greater abdominal fatness than birds from the lean line. The fat line was also characterized by decreased breast yield than the lean line but similar intramuscular fat content. A previous study carried out on the same 2 lines (Ricard et al., 1983) showed that the different fat deposits responded differently to selection for abdominal fat content. Although intermuscular and subcutaneous fat diverged, no differences in fat content of thigh muscles were observed between the 2 lines. Despite this lack of differences between P. major fat content, the present study highlighted differences in glycogen content of the P. major muscle between fat and lean birds. The P. major muscle from the fat birds was characterized by a greater amount of glycogen at the time of death. Moreover, the muscle glycogen content appeared positively correlated to abdominal fatness and negatively correlated to breast yield. Plasma glucose concentrations have been shown to be less in fat than in lean birds (Leclercq et al., 1988). Moreover, high negative phenotypic correlations have been reported between plasma glucose concentrations and abdominal fat content (Lefebvre et al., 1988). Together these observations suggest a greater glucose uptake that could lead to greater muscle glycogen content in fat than in lean birds. A relationship between muscle glycogen content and carcass fatness was already suggested in meat-type chicken by a strong genetic negative correlation (–0.54) reported between abdominal fatness and breast meat pHu (Le Bihan-Duval et al., 2001). On the other hand, a negative genetic correlation (–0.52) was reported between muscle fiber diameter and glycogen content at death in chicken breast muscle (Berri et al., 2007). Therefore, in this species, selection for meat yield and leanness would have led to decreased muscle glycogen content and greater pHu, whereas in pigs, muscle glycogen content is positively correlated with lean meat content and negatively to backfat thickness (Larzul et al., 1999). Variations in muscle glycogen content affect breast meat quality traits through their effect on pHu. Because of its lower pHu, the breast meat from fat birds was paler, less red, and more exudative than the meat from lean birds. Therefore, in relation to the greater amount of glycogen in muscle at death, the breast meat from fat birds would likely be less adapted to further processing than meat from lean birds. The present results are consistent with several studies that suggested that genetic selection for body composition could have changed several chicken characteristics including muscle biochemistry and ultimately meat characteristics. Indeed, selection for increased breast meat yield and reduced carcass fatness was associated with slower rate of postmortem muscle acidification, greater ultimate pH, and breast meat that was lighter in color and exhibited a lower drip loss (Le Bihan-Duval et al., 1999; Berri et al., 2001). Chicken lines diverging on growth rate also exhibit large differences in meat quality traits, with the heavier and fattier birds being characterized by a greater rate of postmortem acidification, lower ultimate pH, and lighter breast meat color than the smaller and leaner birds (Nadaf et al., 2007).
To identify the molecular mechanisms underlying the variations in muscle and meat characteristics evidenced by the present study, we first focused on the determination of the AMPK at both the gene and protein levels. As already reported in chicken skeletal muscle (Proszkowiec-Weglarz et al., 2006), the gene expression of all 7 isoforms of the 3 subunits of AMPK were detected in the chicken P. major muscle. The 2, β2, 1, and 3 isoforms were expressed at greater levels than the 1, β1, and 2 isoforms. In the present study, the 1 and 3 isoforms were expressed at similar levels, whereas a greater expression of 3 compared with 1 was reported earlier (Proszkowiec-Weglarz et al., 2006). Difference in type and age of chickens and in PCR techniques used in the 2 studies could explain the discrepancies between results. In both human and rodent white skeletal muscles, 3 has been shown to be the most abundant isoform, suggesting a key role for this isoform in this particular type of muscle (Mahlapuu et al., 2004). According to our results, the high abundance of both 1 and 3 at the transcriptional level could suggest an important role for both in chicken white glycolytic muscle.
Selection for or against fatness affected the mRNA levels of the catalytic 2 and the 2 regulatory β1 and 1 AMPK subunits, which, respectively, serve a scaffolding and a AMP-binding function (Wong and Lodish, 2006). The AMP-dependence of the AMPK complex is markedly affected by the identity of the isoform present, with 2-containing complexes having a greater AMP-related response than those containing 1 or 3 (Cheung et al., 2000). The downregulation of 1 mRNA level in the muscle of lean chickens would lead to a greater proportion of 2, likely implying a greater response of the kinase complex to AMP, which is coherent with the greater AMPK activation observed in this genotype. However, because we have not yet investigated protein levels of the different AMPK subunits, it seems premature to relate differences in transcriptional level to those observed for muscle glycogen content between chicken lines. Regarding the 1 AMPK subunit, whose gene expression differed between the fat and lean line, it is worthy to note that in pork the gene encoding for it (PRKAG1) is mapped close to a region containing a QTL influencing fatness traits, suggesting possible involvement in the determination of body composition in this species (Demeure et al., 2004). The expression of several genes coding for enzymes involved in the synthesis and degradation of glycogen (Figure 1) were assessed. The upregulation of both GYS and PYG expression in the muscle of the lean birds would suggest a faster glycogen turnover in this line. Although no difference in gene expression of the GSK3 was observed in the present study, there was a general trend for the subunits of PHK to be overexpressed in the muscle of the lean line, although differences between lines were only significant for the regulatory d subunit. Whether or not the greater expression of PHK is consistent with the decreased muscle glycogen content of the lean line remains to be answered.
In the present study, AMPK activation (assessed through phosphorylation level on Thr172 residue) was markedly greater in the P. major of lean birds than in that of fat birds. There is evidence that AMPK activation results in decreased glycogen stores by activating glycogen phosphorylase and inhibiting glycogen synthase (Carling and Hardie, 1989; Longnus et al., 2003; Jørgensen et al., 2004; Miyamoto et al., 2007). Therefore, the greater AMPK activation observed in lean birds is consistent with their decreased muscle glycogen content and suggests a potential role for AMPK in modulating chicken postmortem muscle metabolism. Moreover, data on lactate measured 15 min postmortem suggest a faster glycolysis after death in birds that exhibited a greater AMPK activation. The control of postmortem glycolysis by AMPK was first evidenced in pig muscle through the discovery of a mutation of the AMPK3 subunit responsible for a 70% increase in muscle glycogen and the production of meat characterized by low ultimate pH, reduced water-holding capacity, and poor processing ability (Milan et al., 2000; Andersson, 2003). The involvement of AMPK in modulating postmortem muscle metabolism was recently confirmed in several studies on mouse (Shen and Du, 2005; Shen et al., 2005, 2008) and pig (Shen et al., 2006a,b, 2007). These studies demonstrated that AMPK activation by diet, exercise, drugs, or stress can affect both the early rate and the extent of postmortem pH decrease, and is therefore a potential molecular target for the control of meat quality. Our observation confirms, in chickens, that AMPK can be a target to control muscle postmortem metabolism and meat quality. Therefore, one can consider modulating AMPK activation through nutrition or other breeding practices while taking into account general animal metabolism such as protein synthesis, which may be inhibited upon AMPK activation through mammalian target of rapamycin signaling (Rolfe and Brown, 1997; Hayashi and Proud, 2007).
In conclusion, the mRNA encoding for the 2 enzymes regulating glycogen synthesis (GYS) and degradation (PYG) are both upregulated in the lean genotype showing the low muscle glycogen content. Although this remains to be directly measured, the greater activity of the AMPK complex in the lean genotype (as evidenced by pAMPK) could trigger a greater rate of glycogen degradation by activating PYG activity and a decreased rate of synthesis by inactivating GYS activity. The modest overexpression of PHK , β, and d, if confirmed at the protein level, could further enhance this mechanism.
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Footnotes
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1 This research program was supported by a grant from the ANR-GENANIMAL Program (France). V. Sibut was supported by a grant from CIFRE (France). The authors thank the staff from the breeding facilities (INRA, UE1295 Pôle d’Expérimentation Avicole de Tours, Nouzilly, France) and laboratories (INRA, UR83 Recherches Avicoles, Nouzilly and UR370 QuaPA, Saint-Genès-Champanelle, France) for their technical assistance. They also thank J. Dupont (INRA, UMR6175 PRC, Nouzilly, France) for interesting discussions and for providing antibodies to AMPK subunits. 
2 Corresponding author: berri{at}tours.inra.fr
Received for publication March 26, 2008.
Accepted for publication June 24, 2008.
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Abstract
INTRODUCTION
