Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Chemphyschem ; 21(10): 1060-1069, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32301564

RESUMO

We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfonate] attached at position 81 of Cc (SL-Cc). A dissociation constant KD of 20±4×10-6  M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.

2.
Sci Rep ; 8(1): 6898, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720594

RESUMO

We present a quantitative study of different molecular iron forms found in the temporal cortex of Alzheimer (AD) patients. Applying the methodology we developed in our previous work, we quantify the concentrations of non-heme Fe(III) by Electron Paramagnetic Resonance (EPR), magnetite/maghemite and ferrihydrite by SQUID magnetometry, together with the MRI transverse relaxation rate [Formula: see text], to obtain a systematic view of molecular iron in the temporal cortex. Significantly higher values of [Formula: see text], a larger concentration of ferrihydrite, and a larger magnetic moment of magnetite/maghemite particles are found in the brain of AD patients. Moreover, we found correlations between the concentration of the iron detected by EPR, the concentration of the ferrihydrite mineral and the average iron loading of ferritin. We discuss these findings in the framework of iron dis-homeostasis, which has been proposed to occur in the brain of AD patients.


Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Ferro/metabolismo , Lobo Temporal/metabolismo , Lobo Temporal/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/diagnóstico por imagem , Estudos de Casos e Controles , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Compostos Férricos/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Lobo Temporal/diagnóstico por imagem
3.
PLoS One ; 13(1): e0191197, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29351320

RESUMO

The interaction of the complementary K (Ac-(KIAALKE)3-GW-NH2) and E (Ac-(EIAALEK)3-GY-NH2) peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009) is investigated by electron paramagnetic resonance (EPR). By frozen solution continuous-wave EPR and double electron-electron resonance (DEER), the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm) and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.


Assuntos
Proteínas de Fusão de Membrana/química , Sequência de Aminoácidos , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Fusão de Membrana/fisiologia , Proteínas de Fusão de Membrana/fisiologia , Modelos Moleculares , Oligopeptídeos/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Marcadores de Spin , Temperatura
4.
Psychiatr Prax ; 45(1): 16-22, 2018 01.
Artigo em Alemão | MEDLINE | ID: mdl-28371953

RESUMO

OBJECTIVE: The project "Stigma Management - StigMa" aims on the evaluation of an adaptive therapy program for patients with psychiatric illness to help them in managing internalized stigma and self-stigmatization. METHODS: The patients for this pilot-study were recruited in day-hospitals of pro mente tirol. 26 patients participated in 11 group sessions, following 6 modules: "Education", "Activation of Resources", "Social Network", "Self-Esteem", "Social competence in public places" and "My personal stigma management". The control group consisted of 20 patients who did not participate in StigMa. Pre-post-evaluation was done by the Internalized Stigma of Mental Illness-Scale 1. RESULTS: No significant interaction effects could be observed, although in the treatment group, the burden of perceived discrimination was significantly less pronounced after training than before it. The program, however, was evaluated as being extremely positive by the participants. CONCLUSIONS: The program StigMa will be adapted in accordance with the suggestions of the participants and reevaluated taking into consideration methodological optimization.


Assuntos
Estigma Social , Estereotipagem , Alemanha , Humanos , Educação de Pacientes como Assunto , Projetos Piloto , Autoimagem
5.
J Phys Condens Matter ; 29(41): 415801, 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-28872048

RESUMO

Muon spin rotation is employed to investigate the spin dynamics of ferritin proteins isolated from the brain of an Alzheimer's disease (AD) patient and of a healthy control, using a sample of horse-spleen ferritin as a reference. A model based on the Néel theory of superparamagnetism is developed in order to interpret the spin relaxation rate of the muons stopped by the core of the protein. Using this model, our preliminary observations show that ferritins from the healthy control are filled with a mineral compatible with ferrihydrite, while ferritins from the AD patient contain a crystalline phase with a larger magnetocrystalline anisotropy, possibly compatible with magnetite or maghemite.

6.
Isr J Chem ; 57(7-8): 762-770, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28919642

RESUMO

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction. Here, we study the effect of phosphorylation at positions 87 and 129, mimicked by the mutations S87A, S129A (nonphosphorylated) and S87D, S129D (phosphorylated) on membrane binding. Local binding is detected by spin-label continuous-wave electron paramagnetic resonance. For S87A/D, six positions (27, 56, 63, 69, 76, and 90) are probed; and for S129A/D, three (27, 56, and 69). Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected by the phosphorylation state of S129. For phosphorylation at S87, local unbinding of αS from the membrane is observed. We speculate that modulating the local membrane affinity by phosphorylation could tune the way αS interacts with different membranes; for example, tuning its membrane fusion activity.

7.
J Phys Chem B ; 121(17): 4379-4387, 2017 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-28422504

RESUMO

We address the interpretation, via an integrated computational approach, of the experimental continuous-wave electron paramagnetic resonance (cw-EPR) spectra of a complete set of conformationally highly restricted, stable 310-helical peptides from hexa- to nonamers, each bis-labeled with nitroxide radical-containing TOAC (4-amino-1-oxyl-2,2,6,6-tetramethylpiperidine-4-carboxylic acid) residues. The usefulness of TOAC for this type of analysis has been shown already to be due to its cyclic piperidine side chain, which is rigidly connected to the peptide backbone α-carbon. The TOAC α-amino acids are separated by two, three, four, and five intervening residues. This set of compounds has allowed us to modulate both the radical···radical distance and the relative orientation parameters. To further validate our conclusion, a comparative analysis has been carried out on three singly TOAC-labeled peptides of similar main-chain length.


Assuntos
Óxidos N-Cíclicos/química , Óxidos de Nitrogênio/química , Peptídeos/química , Teoria Quântica , Espectroscopia de Ressonância de Spin Eletrônica , Marcadores de Spin
8.
Sci Rep ; 6: 38916, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27941952

RESUMO

We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer's patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer's Disease which are characterized by abnormal iron accumulation in the brain.


Assuntos
Doença de Alzheimer/metabolismo , Química Encefálica , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Compostos Férricos/química , Ferro/química , Magnetometria/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Fenômenos Magnéticos , Nanopartículas de Magnetita
9.
Front Physiol ; 7: 409, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27708587

RESUMO

Understanding the functioning of ion channels, as well as utilizing their properties for biochemical applications requires control over channel activity. Herein we report a reversible control over the functioning of a mechanosensitive ion channel by interfering with its interaction with the lipid bilayer. The mechanosensitive channel of large conductance from Escherichia coli is reconstituted into liposomes and activated to its different sub-open states by titrating lysophosphatidylcholine (LPC) into the lipid bilayer. Activated channels are closed back by the removal of LPC out of the membrane by bovine serum albumin (BSA). Electron paramagnetic resonance spectra showed the LPC-dose-dependent gradual opening of the channel pore in the form of incrementally increasing spin label mobility and decreasing spin-spin interaction. A method to reversibly open and close mechanosensitive channels to distinct sub-open conformations during their journey from the closed to the fully open state enables detailed structural studies to follow the conformational changes during channel functioning. The ability of BSA to revert the action of LPC opens new perspectives for the functional studies of other membrane proteins that are known to be activated by LPC.

10.
J Biol Phys ; 42(3): 299-315, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26984615

RESUMO

The amyloid ß (A ß) peptide is important in the context of Alzheimer's disease, since it is one of the major components of the fibrils that constitute amyloid plaques. Agents that can influence fibril formation are important, and of those, membrane mimics are particularly relevant, because the hydrophobic part of A ß suggests a possible membrane activity of the peptide. We employed spin-label EPR to investigate the aggregation process of A ß1-40 in the presence of the sodium dodecyl sulfate (SDS) detergent as a membrane-mimicking agent. In this work, the effect of SDS on A ß is studied using two positions of spin label, the N-terminus and position 26. By comparing the two label positions, the effect of local mobility of the spin label is eliminated, revealing A ß aggregation in the SDS concentration regime below the critical micelle concentration (CMC). We demonstrate that, at low SDS concentrations, the N-terminus of A ß participates in the solubilization, most likely by being located at the particle-water interface. At higher SDS concentrations, an SDS-solubilized state that is a precursor to the one A ß/micelle state above the CMC of SDS prevails. We propose that A ß is membrane active and that aggregates include SDS. This study reveals the unique potential of EPR in studying A ß aggregation in the presence of detergent.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Detergentes/metabolismo , Membranas Artificiais , Fragmentos de Peptídeos/metabolismo , Dodecilsulfato de Sódio/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Fragmentos de Peptídeos/química , Ligação Proteica , Rotação , Dodecilsulfato de Sódio/farmacologia , Marcadores de Spin
11.
Phys Chem Chem Phys ; 18(8): 5729-42, 2016 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-26356049

RESUMO

Paramagnetic NMR is a useful technique to study proteins and protein complexes and the use of paramagnetic relaxation enhancement (PRE) for this purpose has become wide-spread. PREs are commonly generated using paramagnetic spin labels (SLs) that contain an unpaired electron in the form of a nitroxide radical, with 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydropyrrol-3-ylmethyl methane thiosulfonate (MTSL) being the most popular tag. The inherent flexibility of the SL causes sampling of several conformations in solution, which can be problematic as over- or underestimation of the spatial distribution of the unpaired electron in structural calculations will lead to errors in the distance restraints. We investigated the effect of this mobility on the accuracy of protein-protein docking calculations using intermolecular PRE data by comparing MTSL and the less mobile 3-methanesulfonilthiomethyl-4-(pyridin-3-yl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl (pyMTSL) on the dynamic complex of cytochrome c and cytochrome c peroxidase. No significant differences were found between the two SLs. Docking was performed using either single or multiple conformers and either fixed or flexible SLs. It was found that mobility of the SLs is the limiting factor for obtaining accurate solutions. Optimization of SL conformer orientations using intra-molecular PRE improves the accuracy of docking.


Assuntos
Proteínas/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica
12.
PLoS One ; 10(11): e0142795, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26588454

RESUMO

Binding of human α-Synuclein, a protein associated with Parkinson's disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50-100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria.


Assuntos
Mitocôndrias/química , Doença de Parkinson/metabolismo , Lipossomas Unilamelares/química , alfa-Sinucleína/química , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Doença de Parkinson/patologia , Conformação Proteica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Lipossomas Unilamelares/metabolismo , alfa-Sinucleína/metabolismo
13.
J Chem Phys ; 142(23): 234201, 2015 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-26093552

RESUMO

We develop a theoretical description of Dynamic Nuclear Polarization (DNP) in solids under Magic Angle Spinning (MAS) to describe the magnetic field dependence of the DNP effect. The treatment is based on an efficient scheme for numerical solution of the Liouville-von Neumann equation, which explicitly takes into account the variation of magnetic interactions during the sample spinning. The dependence of the cross-effect MAS-DNP on various parameters, such as the hyperfine interaction, electron-electron dipolar interaction, microwave field strength, and electron spin relaxation rates, is analyzed. Electron spin relaxation rates are determined by electron paramagnetic resonance measurements, and calculations are compared to experimental data. Our results suggest that the observed nuclear magnetic resonance signal enhancements provided by MAS-DNP can be explained by discriminating between "bulk" and "core" nuclei and by taking into account the slow DNP build-up rate for the bulk nuclei.

14.
J Phys Chem B ; 119(43): 13507-14, 2015 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-26101942

RESUMO

Protein folding is one of the important challenges in biochemistry. Understanding the folding process requires mapping of protein structure as it folds. Here we test the potential of distance determination between paramagnetic spin-labels by a pulsed electron paramagnetic resonance method. We use double electron-electron spin resonance (DEER) to study the denaturant-dependent equilibrium folding of flavodoxin. This flavoprotein is spin-labeled with MTSL ((1-oxy-,2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate) at positions 69 and 131. We find that nativelike spin-label separation dominates the distance distributions up to 0.8 M guanidine hydrochloride. At 2.3 M denaturant, the distance distributions show an additional component, which we attribute to a folding intermediate. Upon further increase of denaturant concentration, the protein expands and evidence for a larger number of conformations than in the native state is found. We thus demonstrate that DEER is a versatile technique to expand the arsenal of methods for investigating how proteins fold.


Assuntos
Flavodoxina/química , Dobramento de Proteína , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares
15.
Angew Chem Int Ed Engl ; 53(26): 6814-8, 2014 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-24842721

RESUMO

Photochemical activation of [(PNNH)Rh(N3)] (PNNH = 6-di-(tert-butyl)phosphinomethyl-2,2'-bipyridine) complex 2 produced the paramagnetic (S = 1/2), [(PNN)Rh=N(·)-Rh(PNN)] complex 3 (PNN(-) = methylene-deprotonated PNNH), which could be crystallographically characterized. Spectroscopic investigation of 3 indicates a predominant nitridyl radical ((·)N(2-)) character, which was confirmed computationally. Complex 3 reacts selectively with CO, producing two equivalents of [(PNN)Rh(I)(CO)] complex 4, presumably by nitridyl radical N,N-coupling.


Assuntos
Complexos de Coordenação/química , Radicais Livres/química , Irídio/química , Piridinas/química , Ródio/química , Monóxido de Carbono , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Magnetismo , Conformação Molecular
16.
Biophys J ; 106(6): 1349-58, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24655510

RESUMO

The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Cloroplastos/química , Simulação de Dinâmica Molecular , Ribonucleoproteínas/química , Sequência de Aminoácidos , Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Ribonucleoproteínas/genética
17.
Biopolymers ; 102(3): 244-51, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24488683

RESUMO

For 3D-structure determination in biophysical systems EPR is rapidly gaining ground. Proteins labeled specifically with two nitroxide spin labels can be prepared, and several EPR methods are available for distance determination, which makes it possible to determine distance constraints. However, such methods require frozen solutions, potentially causing non-physiological states of the sample. Here, we target spin- spin interaction in liquid solution at room temperature using rigid model compounds. A series of 310 -helical peptides, based on α-aminoisobutyric acid (Aib), is synthesized with pairs of spin labels separated by three, four, and five amino acids. To avoid flexibility, the noncoded nitroxyl-containing α-amino acid TOAC that is rigidly connected with the peptide backbone, is used. The EPR spectra of the peptides show a decreasing amount of coupling between the two spin labels within this series. We suggest through-bond interaction as the dominating mechanism for exchange interaction (J) and find a stronger J-coupling than in the corresponding Ala-based TOAC-peptides investigated previously (Hanson, et al., J Am Chem Soc 1996, 118, 7618-7625). We speculate that stronger coupling in Aib- vs Ala- peptides is due to intrinsically stronger through-bond interaction in the Aib-based peptides.


Assuntos
Óxidos N-Cíclicos/química , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Peptídeos/química , Marcadores de Spin , Simulação por Computador , Estrutura Secundária de Proteína
18.
Chembiochem ; 13(9): 1312-8, 2012 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-22619165

RESUMO

Cytochrome f (Cyt f) and plastocyanin (Pc) form a highly transient complex as part of the photosynthetic redox chain. The complex from Nostoc sp. PCC 7119 was studied by NMR relaxation spectroscopy with the aim of determining the orientation of Pc relative to Cyt f. Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc. The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.


Assuntos
Citocromos f/metabolismo , Nostoc , Plastocianina/metabolismo , Citocromos f/química , Mesilatos/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Plastocianina/química , Ligação Proteica , Conformação Proteica
19.
Biochemistry ; 51(19): 3960-2, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22494024

RESUMO

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.


Assuntos
Membrana Celular/metabolismo , alfa-Sinucleína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Corpos de Lewy/metabolismo , Membranas Artificiais , Mutação , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , alfa-Sinucleína/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA