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RAPID COMMUNICATION
Association of MMP-8 polymorphisms with tendinopathy of the primary posterior tibial tendon: a pilot study
Alexandre Leme Godoy-SantosI,
Corresponding author
alexandrelemegodoy@gmail.com

Tel.: 55 11 30696888
, Rafael TrevisanI, Túlio Diniz FernandesI, Maria Cristina L.G. dos SantosII
I Instituto de Ortopedia e Traumatologia – Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo/SP, Brazil.
II Departamento de Biologia Celular - Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba/PR, Brazil.
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="cesec10" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle10">INTRODUCTION</span><p id="para10" class="elsevierStylePara elsevierViewall">Tendon disorders are common and are treated on a daily basis in orthopedic foot and ankle practices&#46; Some tendons are particularly vulnerable to primary degenerative changes&#44; such as the patellar&#44; Achilles&#44; rotator cuff&#44; biceps&#44; posterior tibial&#44; and fibular tendons&#46;<a class="elsevierStyleCrossRef" href="#bib1">1</a></p><p id="para20" class="elsevierStylePara elsevierViewall">Significant advances in histopathology and research imaging techniques have contributed to an improved understanding of the pathophysiology of tendon degeneration&#46;<a class="elsevierStyleCrossRef" href="#bib2">2</a> However&#44; both mechanical factors and vascular and neurological disorders have limitations in explaining the etiology of many cases&#46;<a class="elsevierStyleCrossRefs" href="#bib1">1&#44;3&#44;4</a> It is known that intense physical activity may predispose an individual to tendinopathy&#44; but some individuals have a predisposition with no clinically recognized cause&#46;</p><p id="para30" class="elsevierStylePara elsevierViewall">The literature suggests that individual characteristics&#44; including genetic inheritance&#44; may influence the likelihood of developing tendinopathy&#46; Thus&#44; there is a group of individuals with a genetic background that causes increased susceptibility to diseases of the tendon&#46;</p><p id="para40" class="elsevierStylePara elsevierViewall">Matrix metalloproteinases &#40;MMPs&#41; are a pivotal family of zinc-dependent enzymes responsible for the degradation of extracellular matrix components&#44; including basement membrane collagen&#44; interstitial collagen&#44; fibronectin and various proteoglycans&#44; during normal remodeling&#44; repair processes&#44; development and inflammation&#46; MMPs are expressed in response to specific stimuli by resident connective tissue cells and by the major inflammatory cell types that invade tissues during remodeling events&#44; including tendinopathy&#46;</p><p id="para50" class="elsevierStylePara elsevierViewall">MMP-8&#44; or collagenase-2&#44; was initially discovered in neutrophils&#44; in which it was thought to be exclusively produced&#44; but this enzyme was subsequently shown to be expressed by a variety of other cell types&#44; including endothelial cells&#44; smooth muscle cells&#44; macrophages&#44; polymorphonuclear leukocytes&#44; gingival fibroblasts&#44; keratinocytes&#44; chondrocytes&#44; odontoblasts<a class="elsevierStyleCrossRefs" href="#bib5">5&#8211;10</a> and oral<a class="elsevierStyleCrossRef" href="#bib11">11</a> cancer cells&#46;</p><p id="para60" class="elsevierStylePara elsevierViewall">It has been suggested that MMP-8 degrades type I collagen&#44; thereby contributing to tissue degradation and remodeling&#46;<a class="elsevierStyleCrossRef" href="#bib12">12</a> MMP-8 is an important mediator of tissue destruction in several inflammatory diseases and is related to cardiovascular disease&#44;<a class="elsevierStyleCrossRef" href="#bib9">9</a> bronchiectasis&#44;<a class="elsevierStyleCrossRef" href="#bib13">13</a> pulmonary insufficiency&#44;<a class="elsevierStyleCrossRef" href="#bib14">14</a> periodontitis&#44;<a class="elsevierStyleCrossRefs" href="#bib15">15&#8211;17</a> melanoma&#44;<a class="elsevierStyleCrossRef" href="#bib18">18</a> cancer of the head and neck&#44;<a class="elsevierStyleCrossRef" href="#bib19">19</a> and diabetic wound healing&#46;<a class="elsevierStyleCrossRef" href="#bib20">20</a></p><p id="para70" class="elsevierStylePara elsevierViewall">The MMP-8 gene&#44; located on chromosome 11&#44; contains functional polymorphisms in the promoter region&#44; including the substitution of a cytosine by a thymine at position -799 &#40;rs11225395&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib21">21</a> Alterations in this gene have been associated with chronic dilatation of the bronchi13 and breast cancer&#46;<a class="elsevierStyleCrossRef" href="#bib22">22</a></p><p id="para80" class="elsevierStylePara elsevierViewall">The discovery of genetic markers of tendinopathy risk could allow for the identification of susceptible individuals and&#44; thus&#44; early therapeutic interventions&#46; MMPs play key roles in tissue destruction and may have important roles in the pathogenesis of tendinopathy&#46; Therefore&#44; we hypothesized that the -799C&#47;T polymorphism in MMP-8 was associated with tendinopathy of the primary posterior tibial tendon and could be a risk factor for this condition&#46;</p></span><span id="cesec20" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle20">MATERIALS AND METHODS</span><span id="cesec30" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle30">Subject selection</span><p id="para90" class="elsevierStylePara elsevierViewall">A total of 64 subjects were recruited from the patient pool at the Foot and Ankle Group&#44; Department of Orthopedics&#44; at the Traumatology Hospital of the University of S&#227;o Paulo&#44; Brazil&#46; After a brief explanation of the study&#44; as recommended by the the hospital&#39;s Research Ethics Committee&#44; subjects were identified according to age&#44; sex&#44; diagnosis&#44; pathology&#44; medical history&#44; postoperative complications&#44; medication use&#44; personal history of systemic diseases&#44; and family history of infectious and inflammatory diseases&#46; The subjects were divided into two groups&#44; with 66&#37; female and a mean age of 51 years&#58;</p><p id="para100" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Test group</span> - 14 patients undergoing surgical procedures who had pathological diagnoses of degenerative lesions of the posterior tibial tendon&#46;</p><p id="para110" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">Control group</span> - 50 patients with posterior tibial tendon integrity and with no signs of degeneration based on magnetic resonance images produced to investigate complaints about the ankle and foot&#46;</p><p id="para120" class="elsevierStylePara elsevierViewall">All the subjects were in good general health and did not display any of the following exclusion criteria&#58; rheumatic diseases&#44; immunological diseases&#44; diabetes&#44; hepatitis or prior or current infections in the topography of the foot and ankle&#46;</p></span><span id="cesec40" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle40">DNA sampling and extraction</span><p id="para130" class="elsevierStylePara elsevierViewall">Buccal epithelial cells were sampled as described by Trevilatto and Line &#40;2000&#41;&#44;<a class="elsevierStyleCrossRef" href="#bib23">23</a> and DNA extraction was performed as by Aidar and Line &#40;2007&#41;&#46;<a class="elsevierStyleCrossRef" href="#bib24">24</a> The DNA concentration was estimated by measuring the absorbance ratio at 260&#47;280 nm&#46;</p></span><span id="cesec50" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle50">Polymerase chain reaction and restriction endonuclease digestion</span><p id="para140" class="elsevierStylePara elsevierViewall">The MMP-8 genotypes were determined using the polymerase chain reaction - restricted fragment length polymorphism &#40;PCR-RFLP&#41; assay&#46; The PCR primers used for amplifying the MMP-8 polymorphism were the forward primer 5&#8242;-CAGAGACTCAAGTGGGA-3&#8242; and the reverse primer 5&#8242;-TTTCATTTGTGGAGGGGC-3&#8242;&#46; The PCR reactions were performed in a total volume of 10 &#956;l that contained 400 ng of genomic DNA&#44; 5 &#956;l of SYBR GREEN JUMPSTART TAQ READY MIX &#40;Amersham Pharmacia-Biotech&#44; Uppsala&#44; Sweden&#41; and 200 nmol of each primer&#46; A 6-&#956;l aliquot of each PCR product was then digested with 1 unit of SfcI enzyme at 37&#176;C overnight&#46;</p></span><span id="cesec60" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle60">Gel electrophoresis</span><p id="para150" class="elsevierStylePara elsevierViewall">The entire digest was electrophoresed on a 10&#37; vertical non-denaturing polyacrylamide gel at 20 mA&#46; The gel was stained with ethidium bromide&#46;</p></span><span id="cesec70" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle70">Statistical analysis</span><p id="para160" class="elsevierStylePara elsevierViewall">The significance of the differences in the observed frequencies of polymorphisms between both groups was assessed using the chi-squared test&#44; with a <span class="elsevierStyleItalic">p</span>&#60;0&#46;05 indicating statistical significance&#46;</p></span></span><span id="cesec80" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle80">RESULTS</span><p id="para170" class="elsevierStylePara elsevierViewall">The primers used for PCR efficiently amplified the MMP-8 locus of interest&#44; and the <span class="elsevierStyleItalic">Sfc</span>I enzyme digestion cleaved the PCR products into two fragments when the polymorphic site contained the C allele &#40;but not T&#41;&#46; Electrophoresis produced DNA bands of 32 and 74 bp for C alleles and a band of 106 bp for T alleles&#44; whereas the heterozygote displayed a combination of both alleles &#40;32&#44; 74&#44; and 106 bp&#41;&#46;</p><p id="para180" class="elsevierStylePara elsevierViewall">There was a significant difference in the allele frequencies of the control and test groups &#40;<span class="elsevierStyleItalic">p</span> &#61; 0&#46;0006&#41;&#46; In the control group&#44; the C allele was observed with a frequency of 71&#37;&#44; whereas in the test group&#44; the T allele was present at a frequency of 64&#46;3&#37;&#46; The C&#47;C genotype was found in 66&#37; of the control group&#44; whereas the T&#47;T genotype was observed in 50&#37; of the test group &#40;<span class="elsevierStyleItalic">p</span> &#61; 0&#46;0036&#41;&#46; The frequencies of the different alleles and genotypes of the MMP-8 gene are shown in <a class="elsevierStyleCrossRef" href="#tbl1">Table 1</a>&#46;</p><elsevierMultimedia ident="tbl1"></elsevierMultimedia></span><span id="cesec90" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle90">DISCUSSION</span><p id="para190" class="elsevierStylePara elsevierViewall">Posterior tibial tendon dysfunction has been associated with obesity&#44; hypertension&#44; and diabetes&#46;<a class="elsevierStyleCrossRef" href="#bib25">25</a> There has also been an association with decreased vascular supply&#46;<a class="elsevierStyleCrossRefs" href="#bib26">26&#44;27</a> However&#44; some patients have a predisposition for this condition without a clinically recognized cause&#44; which suggests that genetic inheritance may play an important role in tendinopathy&#46;</p><p id="para200" class="elsevierStylePara elsevierViewall">Some studies have already shown the influence of genetic polymorphisms on tendinopathy&#46; Mokone and collaborators<a class="elsevierStyleCrossRef" href="#bib28">28</a> demonstrated that a polymorphism in the alpha 1 type V collagen gene &#40;COL5A1&#41; was associated with Achilles tendon pathologies&#44; with the A2 allele appearing to play a protective role&#46; The same group also demonstrated the role of tenascin-C in Achilles tendon pathologies&#46;<a class="elsevierStyleCrossRef" href="#bib29">29</a> By analyzing a guanine-thymine &#40;GT&#41;n repeat polymorphism in the tenascin-C gene&#44; the authors showed that individuals with 12 or 14 GT repeats had a 6-fold higher risk of developing lesions in the Achilles tendon&#46; A G&#47;T polymorphism in the alpha-1 chain of type I collagen has been associated with anterior cruciate ligament rupture<a class="elsevierStyleCrossRef" href="#bib30">30</a> but not with Achilles tendon injuries&#46;<a class="elsevierStyleCrossRef" href="#bib31">31</a> Raleigh and collaborators<a class="elsevierStyleCrossRef" href="#bib32">32</a> found that variants within the MMP-3 gene were associated with Achilles tendinopathy but not Achilles tendon rupture&#46; The presence of cholesterol deposits in the tendons &#40;tendon xanthomas&#41; of familial hypercholesterolemia patients has been associated with polymorphisms in the reverse cholesterol transport and low-density lipoprotein oxidation pathways&#46;<a class="elsevierStyleCrossRef" href="#bib33">33</a></p><p id="para210" class="elsevierStylePara elsevierViewall">In the present pilot study&#44; the -799C&#47;T polymorphism in the promoter region of the MMP-8 gene was associated with tendinopathy of the primary posterior tibial tendon&#46; The C allele was observed in the majority of the control group&#44; whereas the T allele was more frequent in the test group&#46; Patients bearing the T allele were more likely to have lesions in the posterior tibial tendon&#46; It is possible that this allele can provide the molecular basis for a more intense degradation of the extracellular matrix&#44; which could lead to an increased susceptibility to lesions in the posterior tibial tendon&#46; The clinical significance of these results&#44; however&#44; must be interpreted with caution because the data were derived from only 64 patients &#40;including only 14 in the test group&#41;&#44; which provided a moderate power to detect a statistical relationship between the polymorphism and the disease&#46;</p><p id="para220" class="elsevierStylePara elsevierViewall">Indeed&#44; genetic polymorphisms probably influence the degeneration of tendons through the cumulative effect of multiple polymorphisms that involve complex interactions between multiple genes&#46;<a class="elsevierStyleCrossRef" href="#bib34">34</a> Understanding the importance of each polymorphic allele is necessary for assessing the contribution of each polymorphism to the disease phenotype&#46;<a class="elsevierStyleCrossRef" href="#bib35">35</a></p><p id="para230" class="elsevierStylePara elsevierViewall">Further investigations should determine the roles of several genetic polymorphisms in maintaining homeostasis of the tendon and in tendon pathologies&#46; Because MMP-8 affects the degradation of a large number of extracellular proteins and influences the degradation and remodeling of injured tissues&#44; the study of this gene is important for a better understanding of the process of tendon degeneration&#46; The determination of the genetic patterns of patients with tendinopathy could enable the identification of individuals at higher risk of developing this disease and those with impaired regeneration capabilities&#46; Thus&#44; any identified genetic markers may contribute to the appropriate preoperative selection&#44; the preparation of strategies for prevention&#44; and individualized therapy to modulate the effects of the genetic markers and increase the success rate of treatments&#46; Thus&#44; it is important that a larger test group be studied in the future to more conclusively identify the influence of the -799C&#47;T MMP-8 polymorphism on the disease phenotype&#46;</p></span><span id="cesec100" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="cestitle100">CONCLUSION</span><p id="para240" class="elsevierStylePara elsevierViewall">The utilized primers were effective for the PCR-RFLP analysis of the -799C&#47;T polymorphism in the MMP-8 gene&#46; The preliminary results indicate that this polymorphism may be a risk factor for tendinopathy and could be used as a genetic marker for the primary lesions of the posterior tibial tendon&#46;</p></span></span>"
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                  \t\t\t\t  " align="justify" valign="top" scope="col">MMP-8&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " colspan="2" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Control Group</th><th class="td" title="\n
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                  \t\t\t\t  " colspan="2" align="center" valign="top" scope="col" style="border-bottom: 2px solid black">Test Group</th><th class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top" scope="col" style="border-bottom: 2px solid black"><span class="elsevierStyleItalic">p</span>-value&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t" scope="col">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="center" valign="top" scope="col">n&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="center" valign="top" scope="col">&#37;&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="center" valign="top" scope="col">n&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="center" valign="top" scope="col">&#37;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top" scope="col">&#40;chi-squared&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td" title="\n
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                  \t\t\t\t  " colspan="2" align="center" valign="top">n &#61; 100</td><td class="td" title="\n
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                  \t\t\t\t  " colspan="2" align="center" valign="top">n &#61; 28</td><td class="td" title="\n
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                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="justify" valign="top">C&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">71&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">71&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">10&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">35&#46;7&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top"><span class="elsevierStyleItalic">p</span> &#61; 0&#46;0006&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="justify" valign="top">T&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">29&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">29&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">18&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">64&#46;3&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
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                  \t\t\t\t  " align="justify" valign="top">Genotype&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " colspan="2" align="center" valign="top">n &#61; 50</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " colspan="2" align="center" valign="top">n &#61; 14</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top">&#40;Fisher&#39;s exact test&#41;&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="justify" valign="top">C&#47;C&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">33&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">66&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top">03&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top">21&#46;4&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="justify" valign="top">T&#47;T&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">12&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top">24&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">07&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">50&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top">p &#61; 0&#46;0036&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td></tr><tr title="table-row"><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="justify" valign="top" style="border-bottom: 2px solid black">C&#47;T&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="center" valign="top" style="border-bottom: 2px solid black">05&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t  " align="center" valign="top" style="border-bottom: 2px solid black">10&#46;0&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top" style="border-bottom: 2px solid black">04&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
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                  \t\t\t\t  " align="center" valign="top" style="border-bottom: 2px solid black">28&#46;6&nbsp;\t\t\t\t\t\t\n
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Article information
ISSN: 18075932
Original language: English
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es en pt

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