Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 57.550
Filtrar
1.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445537

RESUMO

Protein Blocks (PBs) are a widely used structural alphabet describing local protein backbone conformation in terms of 16 possible conformational states, adopted by five consecutive amino acids. The representation of complex protein 3D structures as 1D PB sequences was previously successfully applied to protein structure alignment and protein structure prediction. In the current study, we present a new model, PYTHIA (predicting any conformation at high accuracy), for the prediction of the protein local conformations in terms of PBs directly from the amino acid sequence. PYTHIA is based on a deep residual inception-inside-inception neural network with convolutional block attention modules, predicting 1 of 16 PB classes from evolutionary information combined to physicochemical properties of individual amino acids. PYTHIA clearly outperforms the LOCUSTRA reference method for all PB classes and demonstrates great performance for PB prediction on particularly challenging proteins from the CASP14 free modelling category.


Assuntos
Algoritmos , Aprendizado Profundo , Redes Neurais de Computação , Conformação Proteica , Proteínas/química , Análise de Sequência de Proteína/métodos , Software , Bases de Dados de Proteínas , Humanos , Modelos Moleculares
2.
Nat Commun ; 12(1): 5015, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408139

RESUMO

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/química , Proteômica
3.
Nat Commun ; 12(1): 5011, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408149

RESUMO

Sequence-based contact prediction has shown considerable promise in assisting non-homologous structure modeling, but it often requires many homologous sequences and a sufficient number of correct contacts to achieve correct folds. Here, we developed a method, C-QUARK, that integrates multiple deep-learning and coevolution-based contact-maps to guide the replica-exchange Monte Carlo fragment assembly simulations. The method was tested on 247 non-redundant proteins, where C-QUARK could fold 75% of the cases with TM-scores (template-modeling scores) ≥0.5, which was 2.6 times more than that achieved by QUARK. For the 59 cases that had either low contact accuracy or few homologous sequences, C-QUARK correctly folded 6 times more proteins than other contact-based folding methods. C-QUARK was also tested on 64 free-modeling targets from the 13th CASP (critical assessment of protein structure prediction) experiment and had an average GDT_TS (global distance test) score that was 5% higher than the best CASP predictors. These data demonstrate, in a robust manner, the progress in modeling non-homologous protein structures using low-accuracy and sparse contact-map predictions.


Assuntos
Biologia Computacional/métodos , Proteínas/química , Bases de Dados de Proteínas , Modelos Moleculares , Método de Monte Carlo , Conformação Proteica , Dobramento de Proteína , Proteínas/genética , Software
4.
J Chem Phys ; 155(5): 054102, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34364321

RESUMO

Markov state models (MSMs) have become one of the preferred methods for the analysis and interpretation of molecular dynamics (MD) simulations of conformational transitions in biopolymers. While there is great variation in terms of implementation, a well-defined workflow involving multiple steps is often adopted. Typically, molecular coordinates are first subjected to dimensionality reduction and then clustered into small "microstates," which are subsequently lumped into "macrostates" using the information from the slowest eigenmodes. However, the microstate dynamics is often non-Markovian, and long lag times are required to converge the relevant slow dynamics in the MSM. Here, we propose a variation on this typical workflow, taking advantage of hierarchical density-based clustering. When applied to simulation data, this type of clustering separates high population regions of conformational space from others that are rarely visited. In this way, density-based clustering naturally implements assignment of the data based on transitions between metastable states, resulting in a core-set MSM. As a result, the state definition becomes more consistent with the assumption of Markovianity, and the timescales of the slow dynamics of the system are recovered more effectively. We present results of this simplified workflow for a model potential and MD simulations of the alanine dipeptide and the FiP35 WW domain.


Assuntos
Dipeptídeos/química , Cadeias de Markov , Simulação de Dinâmica Molecular/estatística & dados numéricos , Proteínas/química , Análise por Conglomerados , Conformação Proteica , Domínios WW
5.
Phys Chem Chem Phys ; 23(33): 17856-17865, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34378547

RESUMO

Using a model ß-hairpin protein as a representative example of simple two-state folders whose kinetics are uncomplicated by the presence of on- and off-pathway intermediates, it is studied how the search for the protein's native state among native-like states affects the folding kinetics. It is revealed that the first-passage time (FPT) distributions are essentially single-exponential not only for the times to overcome the free energy barrier between the unfolded and native-like states but also for the times to find the native state among the native-like ones. The FPT distributions of this type are observed through all studied two-state-like regimes of protein folding, varying from a regime close to two-state folding to a regime close to downhill folding. If the protein explores native-like states for a time much longer than the time to overcome the free energy barrier, which is characteristic of high temperatures, the resulting FPT distribution to reach the native state remains close to exponential but the mean FPT (MFPT) is determined not by the height of the free energy barrier but by the time to explore native-like states. In particular, the mean time to overcome the free energy barrier is in reasonable agreement with the Kramers rate formula and generally far shorter than the overall MFPT to reach the native state. The observed increase of the overall MFPT, as a result of longer exploration of native-like states, may lead to an overestimate of the height of the free energy barrier between the unfolded and folded states when it is calculated from the overall MFPT.


Assuntos
Proteínas/química , Termodinâmica , Simulação de Dinâmica Molecular , Dobramento de Proteína , Estrutura Secundária de Proteína
6.
Nat Commun ; 12(1): 4849, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381032

RESUMO

Although various artificial protein nanoarchitectures have been constructed, controlling the transformation between different protein assemblies has largely been unexplored. Here, we describe an approach to realize the self-assembly transformation of dimeric building blocks by adjusting their geometric arrangement. Thermotoga maritima ferritin (TmFtn) naturally occurs as a dimer; twelve of these dimers interact with each other in a head-to-side manner to generate 24-meric hollow protein nanocage in the presence of Ca2+ or PEG. By tuning two contiguous dimeric proteins to interact in a fully or partially side-by-side fashion through protein interface redesign, we can render the self-assembly transformation of such dimeric building blocks from the protein nanocage to filament, nanorod and nanoribbon in response to multiple external stimuli. We show similar dimeric protein building blocks can generate three kinds of protein materials in a manner that highly resembles natural pentamer building blocks from viral capsids that form different protein assemblies.


Assuntos
Nanoestruturas/química , Proteínas/química , Cálcio/química , Ferritinas/química , Nanoestruturas/ultraestrutura , Nanotecnologia , Polietilenoglicóis/química , Multimerização Proteica , Thermotoga maritima
7.
Adv Protein Chem Struct Biol ; 127: 217-248, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34340768

RESUMO

Protein structure characterization is fundamental to understand protein properties, such as folding process and protein resistance to thermal stress, up to unveiling organism pathologies (e.g., prion disease). In this chapter, we provide an overview on how the spectral properties of the networks reconstructed from the Protein Contact Map (PCM) can be used to generate informative observables. As a specific case study, we apply two different network approaches to an example protein dataset, for the aim of discriminating protein folding state, and for the reconstruction of protein 3D structure.


Assuntos
Bases de Dados de Proteínas , Dobramento de Proteína , Mapas de Interação de Proteínas , Proteínas/química , Proteínas/metabolismo , Animais , Humanos , Domínios Proteicos , Estabilidade Proteica
8.
Phys Chem Chem Phys ; 23(31): 16488-16500, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34342317

RESUMO

Protein-protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein-protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.


Assuntos
Proteínas/química , Cinética , Fenômenos Ópticos , Ligação Proteica , Proteínas/metabolismo
9.
J Chem Phys ; 155(4): 044107, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34340388

RESUMO

Protein dynamics play an essential role in function regulation. In recent years, many experimental and theoretical studies have shown that changes in protein fluctuations in the backbone and side chains fulfill a pivotal role associated with amino acid mutations, chemical modifications, and ligand binding. The dynamic correlations between protein side chains have not been sufficiently studied, and no reliable analysis method has been available so far. Therefore, we developed a method to evaluate the dynamic correlation between protein side chains using mutual information and molecular dynamics simulations. To eliminate the structural superposition errors dealing with conventional analysis methods, and to accurately extract the intrinsic fluctuation properties of the side chains, we employed distance principal component analysis (distPCA). The motion of the side chain was then projected onto the eigenvector space obtained by distPCA, and the mutual information between the projected motions was calculated. The proposed method was then applied to a small protein "eglin c" and the mutants. The results show that even a single mutation significantly changed the dynamic correlations and also suggest that the dynamic change is deeply related to the stability. Those results indicate that our developed method could be useful for analyzing the molecular mechanism of allosteric communication in proteins.


Assuntos
Proteínas/química , Regulação Alostérica , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Conformação Proteica
10.
J Chem Phys ; 155(4): 040903, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34340394

RESUMO

Proteins are complex, heterogeneous macromolecules that exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires experimental tools to characterize them with high spatial and temporal precision. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution. Two-dimensional (2D) IR methods that provide richer information are becoming more routine but remain challenging to apply to proteins. Spectral congestion typically prevents selective investigation of native vibrations; however, the problem can be overcome by site-specific introduction of amino acid side chains that have vibrational groups with frequencies in the "transparent window" of protein spectra. This Perspective provides an overview of the history and recent progress in the development of transparent window 2D IR of proteins.


Assuntos
Proteínas/química , Espectrofotometria Infravermelho/métodos , Monóxido de Carbono/química , Cianetos/química , Ligantes , Metais/química , Simulação de Dinâmica Molecular , Conformação Proteica
11.
PLoS Comput Biol ; 17(8): e1009284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34347784

RESUMO

Modeling the impact of amino acid mutations on protein-protein interaction plays a crucial role in protein engineering and drug design. In this study, we develop GeoPPI, a novel structure-based deep-learning framework to predict the change of binding affinity upon mutations. Based on the three-dimensional structure of a protein, GeoPPI first learns a geometric representation that encodes topology features of the protein structure via a self-supervised learning scheme. These representations are then used as features for training gradient-boosting trees to predict the changes of protein-protein binding affinity upon mutations. We find that GeoPPI is able to learn meaningful features that characterize interactions between atoms in protein structures. In addition, through extensive experiments, we show that GeoPPI achieves new state-of-the-art performance in predicting the binding affinity changes upon both single- and multi-point mutations on six benchmark datasets. Moreover, we show that GeoPPI can accurately estimate the difference of binding affinities between a few recently identified SARS-CoV-2 antibodies and the receptor-binding domain (RBD) of the S protein. These results demonstrate the potential of GeoPPI as a powerful and useful computational tool in protein design and engineering. Our code and datasets are available at: https://github.com/Liuxg16/GeoPPI.


Assuntos
Substituição de Aminoácidos , Modelos Químicos , Proteínas/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas/química , Proteínas/genética
12.
J Phys Chem Lett ; 12(32): 7659-7664, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34351767

RESUMO

From stem cell freeze-drying to organ storage, considerable recent efforts have been directed toward the development of new preservation technologies. A prominent protein stabilizing strategy involves vitrification in glassy matrices, most notably those formed of sugars such as the biologically relevant preservative trehalose. Here, we compare the folding thermodynamics of a model miniprotein in solution and in the glassy state of the sugars trehalose and glucose. Using synchrotron radiation circular dichroism (SRCD), we find that the same native structure persists in solution and glass. However, upon transition to the glass, a completely different, conformationally restricted unfolded state replaces the disordered denatured state found in solution, potentially inhibiting misfolding. Concomitantly, a large exothermic contribution is observed in glass, exposing the stabilizing effect of interactions with the sugar matrix on the native state. Our results shed light on the mechanism of protein stabilization in sugar glass and should aid in future preservation technologies.


Assuntos
Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteínas/metabolismo , Trealose/química , Sequência de Aminoácidos , Dobramento de Proteína/efeitos dos fármacos , Proteínas/química , Termodinâmica , Vitrificação
13.
Molecules ; 26(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34443469

RESUMO

The classical genetic code maps nucleotide triplets to amino acids. The associated sequence composition is complex, representing many elaborations during evolution of form and function. Other genomic elements code for the expression and processing of RNA transcripts. However, over 50% of the human genome consists of widely dispersed repetitive sequences. Among these are simple sequence repeats (SSRs), representing a class of flipons, that under physiological conditions, form alternative nucleic acid conformations such as Z-DNA, G4 quartets, I-motifs, and triplexes. Proteins that bind in a structure-specific manner enable the seeding of condensates with the potential to regulate a wide range of biological processes. SSRs also encode the low complexity peptide repeats to patch condensates together, increasing the number of combinations possible. In situations where SSRs are transcribed, SSR-specific, single-stranded binding proteins may further impact condensate formation. Jointly, flipons and patches speed evolution by enhancing the functionality of condensates. Here, the focus is on the selection of SSR flipons and peptide patches that solve for survival under a wide range of environmental contexts, generating complexity with simple parts.


Assuntos
DNA Forma Z/química , DNA Forma Z/genética , Evolução Molecular , Conformação de Ácido Nucleico , Proteínas/química , Proteínas/genética , Animais , Códon , DNA Forma Z/metabolismo , Genética , Humanos , Repetições de Microssatélites/genética , Proteínas/metabolismo
14.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360558

RESUMO

Experimental screening of large sets of compounds against macromolecular targets is a key strategy to identify novel bioactivities. However, large-scale screening requires substantial experimental resources and is time-consuming and challenging. Therefore, small to medium-sized compound libraries with a high chance of producing genuine hits on an arbitrary protein of interest would be of great value to fields related to early drug discovery, in particular biochemical and cell research. Here, we present a computational approach that incorporates drug-likeness, predicted bioactivities, biological space coverage, and target novelty, to generate optimized compound libraries with maximized chances of producing genuine hits for a wide range of proteins. The computational approach evaluates drug-likeness with a set of established rules, predicts bioactivities with a validated, similarity-based approach, and optimizes the composition of small sets of compounds towards maximum target coverage and novelty. We found that, in comparison to the random selection of compounds for a library, our approach generates substantially improved compound sets. Quantified as the "fitness" of compound libraries, the calculated improvements ranged from +60% (for a library of 15,000 compounds) to +184% (for a library of 1000 compounds). The best of the optimized compound libraries prepared in this work are available for download as a dataset bundle ("BonMOLière").


Assuntos
Algoritmos , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/normas , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala/métodos , Humanos
15.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360575

RESUMO

Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Proteínas/química , Proteínas/fisiologia , Animais , Humanos
16.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361104

RESUMO

Most non-communicable diseases are associated with dysfunction of proteins or protein complexes. The relationship between sequence and structure has been analyzed for a long time, and the analysis of the sequences organization in domains and motifs remains an actual research area. Here, we propose a mathematical method for revealing the hierarchical organization of protein sequences. The method is based on the pentapeptide as a unit of protein sequences. Employing the frequency of occurrence of pentapeptides in sequences of natural proteins and a special mathematical approach, this method revealed a hierarchical structure in the protein sequence. The method was applied to 24,647 non-homologous protein sequences with sizes ranging from 50 to 400 residues from the NRDB90 database. Statistical analysis of the branching points of the graphs revealed 11 characteristic values of y (the width of the inscribed function), showing the relationship of these multiple fragments of the sequences. Several examples illustrate how fragments of the protein spatial structure correspond to the elements of the hierarchical structure of the protein sequence. This methodology provides a promising basis for a mathematically-based classification of the elements of the spatial organization of proteins. Elements of the hierarchical structure of different levels of the hierarchy can be used to solve biotechnological and medical problems.


Assuntos
Algoritmos , Bases de Dados de Proteínas , Conformação Proteica , Proteínas/química , Humanos , Modelos Moleculares
17.
Ultrason Sonochem ; 76: 105653, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34198127

RESUMO

The review focus on the effect of ultrasound on protein functionality. The presence of transient ultrasonic mechanical waves induce various sonochemical and sonomechanical effects on a protein. Sonochemical effects include the breakage of chains and/or the modification of side groups of aminoacids. Sonomechanical modifications by enhanced molecular agitation, might lead to the transient or permanent modification of the 3D structure of the folded protein. Since the biological function of proteins depends on the maintenance of its 3D folded structure, both sonochemical and sonomechanical effects might affect its properties. A protein might maintain its 3D structure and functionality after minor sonochemical effects, however, the enhanced mass transfer by sonomechanical effects might expose internal hydrophobic residues of the protein, making protein unfolding to an irreversible denatured state. Ultrasound enhanced mass transport effects are unique pathways to change the 3D folded structure of proteins which lead to a new functionality of proteins as support shield materials during the formation microspheres. Enzymes are proteins and their reactions should be conducted in a reactor set-up where enzymes are protected from sonic waves to maximize their catalytic efficiency. In this review, focused examples on protein dispersions/emulsions and enzyme catalysis are given.


Assuntos
Proteínas/metabolismo , Ondas Ultrassônicas , Dobramento de Proteína , Proteínas/química
19.
Ultrason Sonochem ; 77: 105668, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34298307

RESUMO

To extend the shelf life and retain bioactive proteins in milk, this study utilized microfiltration (MF) combined with ultrasonication to treat skim milk and investigated its efficiency in removing bacteria and retaining bioactive proteins compared with HTST pasteurization and microfiltration alone. Results showed that microfiltration combined with ultrasonication at 1296 J/mL could completely remove the bacteria in skim milk. Ultrasonication further extended the shelf life (4 °C) of microfiltered skim milk, which could reach at least 40 days when MF was combined with ˃1296 J/mL ultrasonication. In addition, ELISA showed that HTST pasteurization significantly decreased the levels of IgG by ~30%, IgA by ~ 50%, IgM by ~60%, and lactoferrin by ~40%, whereas the activity of the enzymes lactoperoxidase and xanthine oxidase were also decreased by ~ 20%. Compared with HTST, MF alone or combined with ultrasonication retained these bioactive proteins to a larger degree. On the other hand, proteomics indicated both damage to casein micelle and fat globule structures in milk when ultrasonication at >1296 J/mL was applied, as shown by increases in caseins and milk fat globular proteins. Simultaneously, this ultrasound intensity also decreased levels of bioactive proteins, such as complement factors. Taken together, this study provided new insights that may help to implement this novel combination of non-thermal technologies for the dairy industry aimed at improving milk quality and functionality.


Assuntos
Filtração , Armazenamento de Alimentos , Leite/química , Proteínas/química , Sonicação , Animais , Temperatura
20.
Nucleic Acids Res ; 49(15): 8573-8591, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34329467

RESUMO

R-loops, which consist of a DNA/RNA hybrid and a displaced single-stranded DNA (ssDNA), are increasingly recognized as critical regulators of chromatin biology. R-loops are particularly enriched at gene promoters, where they play important roles in regulating gene expression. However, the molecular mechanisms that control promoter-associated R-loops remain unclear. The epigenetic 'reader' Tudor domain-containing protein 3 (TDRD3), which recognizes methylarginine marks on histones and on the C-terminal domain of RNA polymerase II, was previously shown to recruit DNA topoisomerase 3B (TOP3B) to relax negatively supercoiled DNA and prevent R-loop formation. Here, we further characterize the function of TDRD3 in R-loop metabolism and introduce the DExH-box helicase 9 (DHX9) as a novel interaction partner of the TDRD3/TOP3B complex. TDRD3 directly interacts with DHX9 via its Tudor domain. This interaction is important for recruiting DHX9 to target gene promoters, where it resolves R-loops in a helicase activity-dependent manner to facilitate gene expression. Additionally, TDRD3 also stimulates the helicase activity of DHX9. This stimulation relies on the OB-fold of TDRD3, which likely binds the ssDNA in the R-loop structure. Thus, DHX9 functions together with TOP3B to suppress promoter-associated R-loops. Collectively, these findings reveal new functions of TDRD3 and provide important mechanistic insights into the regulation of R-loop metabolism.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas/metabolismo , Estruturas R-Loop , Cromatina , DNA Topoisomerases Tipo I/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Transcrição Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...