The mechanisms of centriole biogenesis

Résumé : Le centrosome contient une paire de centrioles entourée par du matériel péricentriolaire (PCM) et cet ensemble constitue le centre organisateur des microtubules de la majorité des cellules animales. Tout comme l'ADN, 1'unique centrosome présent au début du cycle cellulaire est dupliqué une et une seule fois pour former deux centrosomes qui vont orchestrer la mise en place du fuseau mitotique. La duplication du centrosome doit être soumise à une régulation précise car la présence d'un seul ou de plus de deux centrosomes peut entraîner la formation d'un fuseau mitotique aberrant, la mauvaise ségrégation des chromosomes et l'aneuploïdie. Bien que la duplication des centrioles soit un phénomène clé pour la duplication du centrosome lui-même, les mécanismes impliqués dans la formation des centrioles sont peu connus et constituent une importante question de biologie cellulaire. Dans cette thèse, nous nous sommes concentrés sur l'analyse de HsSAS-6. Nous avons trouvé que cette protéine est nécessaire pour la formation d'un centriole et qu'elle est localisée spécifiquement à la base des nouveaux centrioles formés. Les niveaux de HsSAS-6 oscillent pendant le cycle cellulaire : la protéine est absente en G1, commence à s'accumuler au niveau du centriole et dans le cytoplasme dès le début de la phase S de synthèse et disparaît abruptement pendant l'anaphase, où probablement APC/CCdlh1 la dirige vers une dégradation par le protéasome 26S. Il est important de noter que la surexpression de HsSAS-6 entraîne la formation de multiples centrioles au lieu d'un seul, ce qui indique que les niveaux de HsSAS-6 déterminent le nombre de centrioles formés. En plus de HsSAS-6, nous avons aussi étudié la lignée mutante sas-2 de C. elegans qui quelques fois assemble un fuseau multi-polaire dans l'embryon à une cellule. Nous avons montré que ce phénotype est la conséquence de la présence de multiples centrioles dans les cellules du sperme. Enfin, nous avons aussi préparé une palette de vecteurs compatibles avec le système Gateway pour permettre la génération rapide de lignées cellulaires humaines exprimant des protéines de manière inductible. De plus, nous avons commencé à développer une méthode pour évaluer la duplication des centrioles par le biais d'une plateforme de criblage d'une librairie de siRNA humains. Dans l'ensemble, notre travail a pu apporter une nouvelle compréhension du processus de duplication des centrioles et a contribué au développement de nouveaux outils de recherche de ce processus. Summary : Centrosomes contain a pair of centrioles surrounded by pericentriolar material (PCM) and serve as the main microtubule organizing centers (MTOCs) of most animal cells. Just like the DNA, the single centrosome present early in the cell cycle duplicates once and only once to give rise to two centrosomes which will then direct assembly of a bipolar spindle. Centrosome duplication must be precisely regulated because the presence of either one or more than two centrosomes can lead to the assembly of an aberrant spindle, chromosome missegregation and aneuploidy. Although duplication of centrioles is key for that of the entire centrosome, the mechanisms underlying centriole formation are poorly understood and represent an important question in cell biology. In this thesis, we focused on the analysis of HsSAS-6. We found that this protein is required for centriole formation and that it is localized specifically at the base of newly forming centrioles. The levels of HsSAS-6 oscillate across the cell cycle. The protein is absent during G1, starts to accumulate at the centriole and in the cytoplasm at the onset of S phase and disappears abruptly during anaphase when it is targeted for 26S proteasome dependent degradation probably by the APC/CCdh1. Importantly, overexpression of HsSAS-6 leads to the formation of multiple centrioles instead of just one, indicating that levels of HsSAS-6 determine the number of centrioles at each cell cycle. Besides HsSAS-6 that is the main focus of this thesis, we have also investigated the C. elegans mutant strain sas-2, which sometimes assembles a multipolar spindle in the one cell stage embryo. We have shown that this phenotype derives from the presence of multiple centrioles in sperm cells. Moreover, we prepared a set of Gateway compatible vectors for fast generation of human cell lines with inducible protein expression. Finally, we started to develop an assay for centriole duplication that can be used in a high throughput setting for screening of human siRNA libraries. Taken together, our work brought novel insights into the process of centriole duplication and lead to the development of new tools for further investigation of this process.

Securin and separase modulate membrane traffic by affecting endosomal acidification.

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function.

Proteostasis of aging and stem cells

Estudi realitzat a partir d’una estada a la the Salk Institute, Estats Units, entre 2010 i 2012. L'estabilitat del genoma és essencial per a la supervivència de les cèl • lules mare, però, l'estabilitat del proteoma pot tenir un paper igualment important en la identitat de cèl • lules mare i la seva funció. La nostra hipòtesi és que les cèl • lules mare tenen la capacitat de proteostasis augmentada en comparació amb els seus homòlegs diferenciats i ens varem preguntar si l'activitat del proteasoma és diferent a les cèl • lules mare embrionàries humanes (hESCs). En particular, els nostres resultats mostren que les poblacions de cèl• lules mare presenten una activitat del proteasoma que es correlaciona amb majors nivells de la subunitat 19S del proteasoma PSMD11/RPN-6 i un corresponent augment del ensamblatge del 26S/30S proteasoma. L'expressió ectòpica de PSMD11 és suficient per augmentar l'activitat del proteasoma. Sorprenentment, varem trobar que la llarga vida del GLP-1 C. elegans mutant té també un augment dramàtic en l'activitat del proteasoma associat a nivells augmentats en l'expressió de RPN-6. El factor de transcripció DAF-16 és essencial per l'augment de la longevitat de GLP-1 i els cucs mutants que trobem DAF-16 necessari per a l'augment d'expressió de RPN-6 i, per tant, per l'activació de l'activitat del proteasoma en GLP-1 mutant animals. Una possibilitat interessant és que els gens que regulen la vida i la resistència a l'estrès en C. elegans poden també regular la funció hESCs de mamífer, cèl • lules que son considerades immortals. Aquests resultats ens van portar a la conclusió de que FOXO4, un factor de transcripció sensible a la insulina/IGF-1, regula l'activitat del proteasoma en hESCs, el que suggereix un paper per FOXO4 en la funció d’aquestes cèl • lules. En efecte, FOXO4 es necessari per a la diferenciació en llinatges neuronals de les hESCs. Els nostres resultats estableixen una nova regulació de laproteostasis en hESCs que uneix la longevitat i la resistència a l'estrès en invertebrats amb la funció i identitat de les hESCs.

NCoR1 is a conserved physiological modulator of muscle mass and oxidative function.

Transcriptional coregulators control the activity of many transcription factors and are thought to have wide-ranging effects on gene expression patterns. We show here that muscle-specific loss of nuclear receptor corepressor 1 (NCoR1) in mice leads to enhanced exercise endurance due to an increase of both muscle mass and of mitochondrial number and activity. The activation of selected transcription factors that control muscle function, such as MEF2, PPARβ/δ, and ERRs, underpins these phenotypic alterations. NCoR1 levels are decreased in conditions that require fat oxidation, resetting transcriptional programs to boost oxidative metabolism. Knockdown of gei-8, the sole C. elegans NCoR homolog, also robustly increased muscle mitochondria and respiration, suggesting conservation of NCoR1 function. Collectively, our data suggest that NCoR1 plays an adaptive role in muscle physiology and that interference with NCoR1 action could be used to improve muscle function.

Fusion of the human gene for the polyubiquitination co-effector UEV-1 with Kua, a newly identified gene

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination.[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).

A general definition and nomenclature for alternative splicing events

Understanding the molecular mechanisms responsible for the regulation of the transcriptome present in eukaryotic cells isone of the most challenging tasks in the postgenomic era. In this regard, alternative splicing (AS) is a key phenomenoncontributing to the production of different mature transcripts from the same primary RNA sequence. As a plethora ofdifferent transcript forms is available in databases, a first step to uncover the biology that drives AS is to identify thedifferent types of reflected splicing variation. In this work, we present a general definition of the AS event along with anotation system that involves the relative positions of the splice sites. This nomenclature univocally and dynamically assignsa specific ‘‘AS code’’ to every possible pattern of splicing variation. On the basis of this definition and the correspondingcodes, we have developed a computational tool (AStalavista) that automatically characterizes the complete landscape of ASevents in a given transcript annotation of a genome, thus providing a platform to investigate the transcriptome diversityacross genes, chromosomes, and species. Our analysis reveals that a substantial part—in human more than a quarter—ofthe observed splicing variations are ignored in common classification pipelines. We have used AStalavista to investigate andto compare the AS landscape of different reference annotation sets in human and in other metazoan species and found thatproportions of AS events change substantially depending on the annotation protocol, species-specific attributes, andcoding constraints acting on the transcripts. The AStalavista system therefore provides a general framework to conductspecific studies investigating the occurrence, impact, and regulation of AS.

Estimation of protein coding density in a corpus of DNA sequence data

A number of experimental methods have been reported for estimating the number of genes in a genome, or the closely related coding density of a genome, defined as the fraction of base pairs in codons. Recently, DNA sequence data representative of the genome as a whole have become available for several organisms, making the problem of estimating coding density amenable to sequence analytic methods. Estimates of coding density for a single genome vary widely, so that methods with characterized error bounds have become increasingly desirable. We present a method to estimate the protein coding density in a corpus of DNA sequence data, in which a ‘coding statistic’ is calculated for a large number of windows of the sequence under study, and the distribution of the statistic is decomposed into two normal distributions, assumed to be the distributions of the coding statistic in the coding and noncoding fractions of the sequence windows. The accuracy of the method is evaluated using known data and application is made to the yeast chromosome III sequence and to C.elegans cosmid sequences. It can also be applied to fragmentary data, for example a collection of short sequences determined in the course of STS mapping.

Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome?

Selenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly.

Assessment of transcript reconstruction methods for RNA-seq.

We evaluated 25 protocol variants of 14 independent computational methods for exon identification, transcript reconstruction and expression-level quantification from RNA-seq data. Our results show that most algorithms are able to identify discrete transcript components with high success rates but that assembly of complete isoform structures poses a major challenge even when all constituent elements are identified. Expression-level estimates also varied widely across methods, even when based on similar transcript models. Consequently, the complexity of higher eukaryotic genomes imposes severe limitations on transcript recall and splice product discrimination that are likely to remain limiting factors for the analysis of current-generation RNA-seq data.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Pollicipes caboverdensis sp. nov. (Crustacea: Cirripedia: Scalpelliformes), an intertidal barnacle from the Cape Verde Islands

Recently, genetic evidence supported the existence of a new species of the genus Pollicipes from the Cape Verde Islands, previously considered a population of P. pollicipes. However, P. pollicipes was not sampled at its southern limit of distribution (Dakar, Senegal), which is geographically separated from the Cape Verde Islands by about 500 km. Herein we describe Pollicipes caboverdensis sp. nov. from the Cape Verde Islands and compare its morphology with the other three species of Pollicipes: P. pollicipes, P. elegans and P. polymerus. Pollicipes pollicipes was sampled at both the middle (Portugal) and southern limit (Dakar, Senegal) of its geographical distribution. The genetic divergence among and within these two regions and Cape Verde was calculated through the analysis of partial mtDNA CO1 gene sequences. Pollicipes caboverdensis sp. nov. has a single whorl of capitular plates below the subrostrum, peduncular scales pointing up toward the capitulum and multi-articulate caudal appendages (all characters shared with P. pollicipesand P. elegans), reddish-orange capitular plates (large specimens), a single rostral median latus between the median latus and the rostrolatus (both characters shared with P. elegans), and uniquely possesses peduncular scales that are approximately the same width as height. The genetic distance between the Cape Verde population and the Senegal and Portugal populations is 13–14%, whilst between Senegal and Portugal it is < 1%.

An external site controls closing of the epithelial Na+ channel ENaC.

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid-sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the 'degenerin site' activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (betaS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a approximately 3-fold increase in the open probability. This effect is due to an 8-fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N-terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of betaS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state- and size-dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.

O gênero Xylocopa Latreille no Rio Grande do Sul, Brasil (Hymenoptera, Anthophoridae)

The genus Xylocopa Latreille in Rio Grande do Sul, Brazil (Hymenoptera, Anthophoridae). A survey of the genus Xylocopa Latreille, 1802 is given for Rio Grande do Sul, the southernmost State of Brazil. Data are based on several studies on the bee fauna of southern Brazil and on unpublished observations. A key is provided to the species (males and females) and information on distribution, nesting habits and relation to flowers. Rio Grande do Sul is strikingly rich in species of Xylocopa because of the diversity of habitats and its geographic position in the transition of tropical/subtropical to temperate climate. Nineteen species, classified into ten subgenera, have been recorded in Rio Grande do Sul. Here we maintain the subgenera Ioxylocopa, Megaxylocopa and Xylocospila, which were put into synonymy recently by Minckley (1998). The species are: Xylocopa (Dasyxylocopa) bimaculata Friese, 1903; Xylocopa (Ioxylocopa) chrysopoda Schrottky, 1902; Xylocopa (Megaxylocopa) frontalis (Olivier, 1789); Xylocopa (Nanoxylocopa) ciliata Burmeister, 1876; Xylocopa (Neoxylocopa) augusti Lepeletier, 1841; Xylocopa (N.) brasilianorum (Linnaeus, 1767); Xylocopa (N.) haematospila Moure, 1951; Xylocopa (N.) hirsutissima Maidl, 1912; Xylocopa (N.) nigrocincta Smith, 1854; Xylocopa (N.) ordinaria Smith, 1874; Xylocopa (N.) suspecta Moure & Camargo, 1988; Xylocopa (N.) tacanensis Moure, 1949; Xylocopa (Schonnherria) macrops Lepeletier, 1841; Xylocopa (S.) simillima Smith, 1854; Xylocopa (S.) splendidula Lepeletier, 1841; Xylocopa (S.) varians Smith, 1874; Xylocopa (Stenoxylocopa) artifex Smith, 1874; Xylocopa (Xylocopoda) elegans Hurd & Moure, 1963; Xylocopa (Xylocopsis) funesta Maidl, 1912; Xylocopa (Xylocospila) bambusae Schrottky, 1902. Xylocopa tacanensis is for the first time recorded in Brasil.

Fauna de Dissomphalus Ashmead (Hymenoptera, Bethylidae) da Mata Atlântica Brasileira, com descrição de 23 espécies novas

Foram realizadas coletas padronizadas em 18 pontos ao longo da Mata Atlântica Brasileira no escopo do Programa BIOTA/FAPESP usando-se varredura de vegetação, e armadilhas Malaise e Möricke. Foi coletado um total de 2.811 exemplares de Dissomphalus. Foram reconhecidas 30 espécies descritas, a saber: Dissomphalus conicus Azevedo, 2003, D. h-ramus Redighieri & Azevedo, 2004, D. laminaris Redighieri & Azevedo, 2004, D. manus Azevedo, 2003, D. umbilicus Azevedo, 2003, D. verrucosus Redighieri & Azevedo, 2004, D. alticlypeatus Azevedo, 2003, D. bicerutus Azevedo, 2003, D. gilvipes Evans, 1979, D. krombeini Azevedo, 1999, D. gordus Azevedo, 2003, D. undatus Azevedo, 2003, D. cristatus Redighieri & Azevedo, 2004, D. laticephalus Azevedo, 2003, D. lobicephalus Azevedo, 2003, D. completus Azevedo, 1999, D. gigantus Azevedo, 1999, D. scamatus Azevedo, 1999, D. napo Evans, 1979, D. punctatus (Kieffer, 1910), D. infissus Evans, 1969, D. plaumanni Evans, 1964, D. concavatus Azevedo, 1999, D. rectilineus Azevedo, 1999, D. bifurcatus Azevedo, 1999, D. extrarramis Azevedo, 1999, D. strictus Azevedo, 1999, D. connubialis Evans, 1966, D. microstictus Evans, 1969, D. scopatus Redighieri & Azevedo, 2004. Além disso, foram descritas e ilustradas 23 espécies novas: Dissomphalus inclinatus sp. nov., D. divisus sp. nov., D. distans sp. nov., D. crassus sp. nov., D. filiformis sp. nov., D. inflexus sp. nov., D. spissus sp. nov., D. firmus sp. nov., D. setosus sp. nov., D. tubulatus sp. nov., D. differens sp. nov., D. lamellatus sp. nov., D. fimbriatus sp. nov., D. magnus sp. nov., D. trilobatus sp. nov., D. amplifoveatus sp. nov., D. personatus sp. nov., D. excellens sp. nov., D. peculiaris sp. nov., D. bahiensis sp. nov., D. amplexus sp. nov., D. elegans sp. nov. e D. amplus sp. nov.. Foram propostos 2 grupos novos de espécies, brasiliensis com duas espécies e setosus com oito espécies. Dissomphalus connubialis Evans, 1966 foi revalidado a partir de D. brasiliensis Kieffer, 1910. Dissomphalus bispinulatus Evans, 1969 foi considerado sinônimo junior de D. brasiliensis. Foi proposto para o gênero uma chave de espécies Neotropicais baseada em machos. Algumas espécies como Dissomphalus rectilineus, D. plaumanni, D. connubialis e D. gigantus são amplamente distribuídos ao longo deste bioma. Por outro lado, espécies como Dissomphalus completus, D. bifurcatus, D. napo, D. gilvipes, D. microstictus, D. brasiliensis, D. scamatus, D. strictus, D. undatus, D. alticlypeatus, D. laticephalus, D. verrucosus, D. extrarramis, D. concavatus, D. krombeini, D. gordus, D. lobicephalus e 13 espécies novas são restritas a regiões específicas, apresentando congruência com os subcentros deste bioma.

Revisão taxonômica, análise cladística e descrição de espécies novas de Aglaenita Spinola (Hemiptera, Cicadellidae, Neocoelidiinae)

Aglaenita e as quatro espécies conhecidas do gênero são redescritas. Nove espécies novas são descritas. Foi realizada uma análise cladística baseada em 22 caracteres morfológicos e 15 táxons terminais, utilizando duas espécies de Biza como grupo externo. A análise mostrou que Aglaenita é um gênero monofilético e o relacionamento entre as espécies foi: (A. spatulata sp. nov. ((A. dubia, A. elegans) ((A. bicornis sp. nov., A. spinipenis sp. nov.) (A. bifurcata sp. nov. (A. affinis sp. nov. ((A. bipunctata (A. similis, A. tridentata sp. nov.)) (A. longicornis sp. nov. (A. coariensis sp. nov., A. unciformis sp. nov.)))))))). As treze espécies são ilustradas, uma chave para identificação das mesmas e sua distribuição geográfica conhecida são fornecidas. A fêmea de A. elegans é descrita pela primeira vez.