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1.
Biomol NMR Assign ; 16(2): 349-355, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36050579

RESUMO

Miro2 and Miro1 are mitochondrial-associated proteins critical for regulating mitochondrial movement within the cell. Both Miro1 and Miro2 have roles in promoting neuron function, but recently Miro2 has been shown to have additional roles in response to nutrient starvation in tumor cells. Miro1 and 2 consist of two small GTPase domains flanking a pair of EF-hands. The N-terminal GTPase (nGTPase) domain is responsible for initiating mitochondrial trafficking and interactions with GCN1 in prostate cancer. The crystal structure of Miro1 nGTPase bound to GTP has been solved. However, no structural data is available for the nGTPase domain of Miro2. To better understand the similarities and differences in the functions of Miro1 and Miro2, we have initiated structural studies of Miro2. Here we report the backbone NMR chemical shift assignments of a 22 KDa construct of the nGTPase domain of Miro2 bound to GTP that includes residues 1-180 of the full-length protein. We affirm that the overall secondary structure of this complex closely resembles that of Miro1 nGTPase bound to GTP. Minor variations in the overall structures can be attributed to crystal packing interactions in the structure of Miro1. These NMR studies will form the foundation for future work identifying the specific interaction sites between Miro2 and its cellular binding partners.


Assuntos
Proteínas Mitocondriais , Proteínas rho de Ligação ao GTP , Guanosina Trifosfato/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(37): e2123092119, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36067314

RESUMO

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.


Assuntos
Bacteriófago T7 , Proteínas de Escherichia coli , Escherichia coli , GTP Fosfo-Hidrolases , Guanosina Trifosfato , Proteínas Virais , Bacteriófago T7/fisiologia , Microscopia Crioeletrônica , Replicação do DNA , DNA Viral/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , Proteínas de Escherichia coli/química , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Conformação Proteica , Proteínas Virais/química , Replicação Viral
3.
Endocrinology ; 158(10): 3592-3604, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28977602

RESUMO

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.


Assuntos
Adamantano/análogos & derivados , Aorta/efeitos dos fármacos , Diástole/efeitos dos fármacos , Dipeptídeos/farmacologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Coração/efeitos dos fármacos , Rigidez Vascular/efeitos dos fármacos , Adamantano/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiotensina II/toxicidade , Animais , Pressão Sanguínea/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Antígenos CD8/efeitos dos fármacos , Antígenos CD8/metabolismo , Cardiomegalia/induzido quimicamente , Dipeptidil Peptidase 4/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Ecocardiografia , Fibrose/induzido quimicamente , Expressão Gênica/efeitos dos fármacos , Coração/fisiopatologia , Inflamação , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Vasoconstritores/toxicidade
4.
J Theor Biol ; 420: 232-240, 2017 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-28322875

RESUMO

Understanding if and how mutants reach fixation in populations is an important question in evolutionary biology. We study the impact of population growth has on the success of mutants. To systematically understand the effects of growth we decouple competition from reproduction; competition follows a birth-death process and is governed by an evolutionary game, while growth is determined by an externally controlled branching rate. In stochastic simulations we find non-monotonic behaviour of the fixation probability of mutants as the speed of growth is varied; the right amount of growth can lead to a higher success rate. These results are observed in both coordination and coexistence game scenarios, and we find that the 'one-third law' for coordination games can break down in the presence of growth. We also propose a simplified description in terms of stochastic differential equations to approximate the individual-based model.


Assuntos
Evolução Biológica , Teoria dos Jogos , Modelos Biológicos , Mutação , Crescimento Demográfico , Animais , Comportamento Competitivo , Humanos , Probabilidade , Reprodução , Processos Estocásticos
5.
Nucleic Acids Res ; 45(4): 1958-1970, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-27956495

RESUMO

DNA polymerase ß (pol ß) requires nuclear localization to fulfil its DNA repair function. Although its small size has been interpreted to imply the absence of a need for active nuclear import, sequence and structural analysis suggests that a monopartite nuclear localization signal (NLS) may reside in the N-terminal lyase domain. Binding of this domain to Importin α1 (Impα1) was confirmed by gel filtration and NMR studies. Affinity was quantified by fluorescence polarization analysis of a fluorescein-tagged peptide corresponding to pol ß residues 2-13. These studies indicate high affinity binding, characterized by a low micromolar Kd, that is selective for the murine Importin α1 (mImpα1) minor site, with the Kd strengthening to ∼140 nM for the full lyase domain (residues 2-87). A further reduction in Kd obtains in binding studies with human Importin α5 (hImpα5), which in some cases has been demonstrated to bind small domains connected to the NLS. The role of this NLS was confirmed by fluorescent imaging of wild-type and NLS-mutated pol ß(R4S,K5S) in mouse embryonic fibroblasts lacking endogenous pol ß. Together these data demonstrate that pol ß contains a specific NLS sequence in the N-terminal lyase domain that promotes transport of the protein independent of its interaction partners. Active nuclear uptake allows development of a nuclear/cytosolic concentration gradient against a background of passive diffusion.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/genética , Sinais de Localização Nuclear/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte , Linhagem Celular , DNA Polimerase beta/metabolismo , Humanos , Espaço Intracelular , Espectroscopia de Ressonância Magnética , Camundongos , Mutação , Sinais de Localização Nuclear/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , alfa Carioferinas/metabolismo
6.
Sci Rep ; 5: 13405, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26304019

RESUMO

We have characterized the nuclear localization signal (NLS) of XRCC1 structurally using X-ray crystallography and functionally using fluorescence imaging. Crystallography and binding studies confirm the bipartite nature of the XRCC1 NLS interaction with Importin α (Impα) in which the major and minor binding motifs are separated by >20 residues, and resolve previous inconsistent determinations. Binding studies of peptides corresponding to the bipartite NLS, as well as its major and minor binding motifs, to both wild-type and mutated forms of Impα reveal pronounced cooperative binding behavior that is generated by the proximity effect of the tethered major and minor motifs of the NLS. The cooperativity stems from the increased local concentration of the second motif near its cognate binding site that is a consequence of the stepwise binding behavior of the bipartite NLS. We predict that the stepwise dissociation of the NLS from Impα facilitates unloading by providing a partially complexed intermediate that is available for competitive binding by Nup50 or the Importin ß binding domain. This behavior provides a basis for meeting the intrinsically conflicting high affinity and high flux requirements of an efficient nuclear transport system.


Assuntos
Núcleo Celular/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Camundongos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Distribuição Tecidual , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , alfa Carioferinas/química , alfa Carioferinas/metabolismo
7.
Structure ; 22(12): 1754-1763, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25456813

RESUMO

XRCC1, a scaffold protein involved in DNA repair, contains an N-terminal domain (X1NTD) that interacts specifically with DNA polymerase ß. It was recently discovered that X1NTD contains a disulfide switch that allows it to adopt either of two metamorphic structures. In the present study, we demonstrate that formation of an N-terminal proline carbimate adduct resulting from the nonenzymatic reaction of Pro2 with CO2 is essential for stabilizing the oxidized structure, X1NTDox. The kinetic response of X1NTDred to H2O2, monitored by NMR, was determined to be very slow, consistent with involvement of the buried, kinetically trapped Cys12 residue, but was significantly accelerated by addition of protein disulfide isomerase or by Cu(2+). NMR analysis of a sample containing the pol ß polymerase domain, and both the reduced and oxidized forms of X1NTD, indicates that the oxidized form binds to the enzyme 25-fold more tightly than the reduced form.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Linhagem Celular , Escherichia coli , Oxirredução , Ligação Proteica , Dobramento de Proteína , Proteína 1 Complementadora Cruzada de Reparo de Raio-X
8.
J Immunol ; 187(5): 2405-17, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21821796

RESUMO

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80(+) macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1-dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.


Assuntos
Tolerância Imunológica/imunologia , Macrófagos/imunologia , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Baço/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos/imunologia , Apoptose/imunologia , Antígeno B7-1/biossíntese , Antígeno B7-1/imunologia , Antígeno B7-H1 , Separação Celular , Encefalomielite Autoimune Experimental/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Interleucina-10/biossíntese , Interleucina-10/imunologia , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Baço/citologia
9.
J Immunol ; 180(11): 7385-93, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18490738

RESUMO

CD43 is a highly glycosylated transmembrane protein that regulates T cell activation. CD43(-/-) T cells are hyperproliferative and the cytoplasmic tail of CD43 has been found to be sufficient to reconstitute wild-type proliferation levels, suggesting an intracellular mechanism. In this study, we report that upon TCR ligation CD43(-/-) T cells demonstrated no increase in tyrosine phosphorylation but a decreased calcium flux. Interestingly, CD43(-/-) T cells preferentially differentiated into Th2 cells in vitro, and CD43(-/-) T cells show increased GATA-3 translocation into the nucleus. In vivo, CD43(-/-) mice exhibited increased inflammation in two separate models of Th2-mediated allergic airway disease. In contrast, in Th1-mediated diabetes, nonobese diabetic CD43(-/-) mice did not significantly differ from wild-type mice in disease onset or progression. Th1-induced experimental autoimmune encephalomyelitis to MOG(35-55) was also normal in the CD43(-/-) mice. Nonetheless, the CD43(-/-) mice produced more IL-5 when restimulated with MOG(35-55) in vitro and demonstrated decreased delayed-type hypersensitivity responses. Together, these data demonstrate that although CD43(-/-) T cells preferentially differentiate into Th2 cells, this response is not sufficient to protect against Th1-mediated autoimmune responses.


Assuntos
Inflamação/imunologia , Leucossialina/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Autoimunidade/imunologia , Cálcio/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Proliferação de Células , Células Cultivadas , Fator de Transcrição GATA3/metabolismo , Imunoglobulina E/sangue , Inflamação/metabolismo , Interleucina-4/metabolismo , Interleucina-5/imunologia , Interleucina-5/metabolismo , Leucossialina/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/metabolismo
10.
J Autoimmun ; 27(4): 218-31, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17289470

RESUMO

The probability that epitope spreading occurs in multiple sclerosis (MS) and the fact that patients have been shown to respond to multiple myelin epitopes concurrently makes the use of peptide-specific tolerance therapies targeting single epitopes problematic. To attempt to overcome this limitation, we have employed cocktails of peptides in the ECDI coupled-APC tolerance system in mice to determine if T cell responses to multiple autoepitopes can be targeted simultaneously. Preventative tolerance induced with splenocytes coupled with a peptide cocktail of four distinct encephalitogenic epitopes (PLP(139-151), PLP(178-191), MBP(84-104), and MOG(92-106)) inhibited initiation of active EAE induced with each individual peptide and by a mixture of the four peptides by preventing activation of autoreactive Th1 cells and subsequent infiltration of inflammatory cells into the CNS. Most relevant to treatment of clinical MS, therapeutic tolerance initiated by splenocytes coupled with the peptide cocktail administered at the peak of acute disease prevented clinical relapses due to epitope spreading and ameliorated a diverse disease induced with a mixture of the four peptides. Interestingly, therapeutic tolerance appeared to be mediated by a mechanism distinct from preventative tolerance, i.e. by significantly increasing the levels of production of the anti-inflammatory cytokines TGF-beta and/or IL-10 in both the periphery and the CNS.


Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Tolerância Imunológica , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Bainha de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Feminino , Imunização , Camundongos , Dados de Sequência Molecular , Linfócitos T/imunologia
11.
Proc Natl Acad Sci U S A ; 102(27): 9595-600, 2005 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-15983366

RESUMO

The ability of different forms of myelin peptides to induce tolerance for the treatment of preestablished murine experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, was evaluated. i.v. administration of myelin peptide-pulsed, ethylene carbodiimide-fixed syngeneic splenocytes, but not soluble myelin peptide monomers or oligomers, proved exceedingly effective at treating preestablished EAE, resulting in amelioration of disease progression. In addition to the lack of therapeutic efficacy of soluble peptide and peptide oligomer, administering them i.v. after the onset of clinical symptoms in many but not all peptide-induced EAE models led to a rapid-onset anaphylactic reaction characterized by respiratory distress, erythema, decreased body temperature, unresponsiveness, and, often, death. By using anti-IgE antibody treatments and mice with targeted mutations of the FcgammaRIII alpha-chain or the common gamma-chain of FcepsilonRI and FcgammaRI/III, we demonstrate that IgE crosslinking of FcepsilonRI appears to be necessary and sufficient for myelin peptide-induced anaphylaxis. The implications of these findings to myelin peptide/protein tolerance strategies for the treatment of multiple sclerosis are discussed.


Assuntos
Anafilaxia/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Imunoglobulina E/metabolismo , Proteína Básica da Mielina/uso terapêutico , Fragmentos de Peptídeos/efeitos adversos , Animais , Temperatura Corporal , Ensaio de Imunoadsorção Enzimática , Feminino , Histamina/sangue , Imunoglobulina E/imunologia , Imunoglobulinas/sangue , Camundongos , Camundongos Knockout , Proteína Básica da Mielina/administração & dosagem , Fragmentos de Peptídeos/uso terapêutico , Receptores de IgE/metabolismo
12.
J Clin Invest ; 109(2): 233-41, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11805135

RESUMO

Experimental autoimmune encephalomyelitis (EAE) is a Th1-mediated demyelinating disease of the CNS with similarities to multiple sclerosis. We and others have shown that a short-term course of anti-CD154 mAb treatment to block CD154-CD40 interactions can be used to prevent or even treat ongoing PLP139-151-induced relapsing EAE. However, little is known of the long-term effects of CD154 blockade on the development of antigen-specific T cell function. Here, we show that short-term treatment with anti-CD154 at the time of PLP139-151/CFA immunization inhibits clinical disease for up to 100 days after immunization. At this point, comparable numbers of Th1 cells are observed in anti-CD154 and control Ig-treated mice, as assessed by antigen-specific ELISPOT assays. Thus, the long-term Th1/Th2 balance is largely unaffected. Inflammatory responses are diminished in anti-CD154-treated mice, as indicated by reduced in vivo delayed-type hypersensitivity and reduced levels of splenic IFN-gamma secretion in vitro. However, upon adoptive transfer of T cells isolated from the spleens of anti-CD154-treated mice, these cells contributed as effectively to clinical disease as those obtained from control-treated mice. Thus, anti-CD154 therapy leads to long-term therapeutic efficacy without exerting a long-term influence on Th1 development.


Assuntos
Antígenos CD40/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas de Membrana , Receptores Virais/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/uso terapêutico , Divisão Celular/efeitos dos fármacos , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Hipersensibilidade Tardia/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Receptores de Retorno de Linfócitos/imunologia , Linfócitos T/imunologia , Fatores de Tempo
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