As craniossinostoses são malformações craniofaciais caracterizadas pelo fechamento precoce de uma ou mais suturas cranianas. Elas são doenças congênitas e são causadas por mutações em diversos genes devido ao grande número de vias envolvidas na formação e manutenção das suturas cranianas. Embora mutações em 53 genes já tenham sido descritas o conhecimento da genética e da patofisiologia das craniossinostoses ainda é incompleto. Nesse trabalho tivemos como objetivo a identificação de novas mutações associadas às craniossinostoses bem como o aprofundamento do conhecimento sobre a atuação dessas mutações em células humanas por meio de estudos funcionais. Para identificarmos novas mutações utilizamos metodologias de sequenciamento em larga escala conhecidas como sequenciamento de noiva geração (NGS). Identificamos a mutação causal em uma paciente proveniente de um casamento consanguíneo portadora da síndrome de Raine (p.P496L em FAM20C). Também delimitamos a poucas mutações candidatas outros onze casos atípicos de craniossinostose. Por fim estudamos os efeitos de diferentes FGFs sobre o comportamento de células com a mutação mais comum causadora da S. de Apert, p.S252W em FGFR2. Descobrimos que os FGFs10 e 19 têm ações distintas sobre o perfil transcricional e sobre a taxa de proliferação de células mutantes. Também descobrimos que as células tronco mesenquimais e as células fibroblastóides têm comportamentos distintos ao serem tratadas com FGF19. Os resultados aqui apresentados serão de grande serventia para o melhor delineamento da biologia das suturas cranianas e da patofisiologia das craniossinostoses / Craniosynostosis are craniofacial malformations defined by early closure of the cranial sutures. They are congenital diseases caused by mutations in several genes due to the diversity of pathways involved in the development and maintenance of the cranial sutures. Even though 53 genes have already been linked to various forms of craniosynostosis, the knowledge about the genetics and pathophysiology is incomplete. In this work we aimed to identify new mutations associated with craniosynostosis as well as to further the knowledge of how those mutations act in human cells. To identify new variants associated with craniosynostosis we used large scale sequencing techniques known as next generation sequencing (NGS). We were able to identify the causal mutation in one patient from a consanguineous marriage with Raine syndrome (p.P496L in FAM20C). We also were able to elect candidate mutations in other eleven cases of atypical craniosynostosis. Lastly, we studied the effects of different FGFs over the behavior of human cells harboring the most common Apert syndrome mutation, p.S252W in FGFR2. We discovered that FGFs 10 and 19 have different effects over the transcriptional profile and proliferation rate of mutant cells. We also found that FGF19 have opposite effects in mesenchymal stem cells and fibroblastoid cells osteogenic differentiation. The results shown here will be of great service to better understand the biology of cranial suture and the pathophysiology of craniosynostosis

Les cellules NK, membres de l’immunité innée, sont impliquées dans le contrôle des infections virales et notamment l’infection à cytomégalovirus (CMV). Généralement bien tolérée chez l’individu immunocompétent, l’infection à CMV demeure associée à une forte morbidité chez les individus dont le système immunitaire est compromis (patients transplantés, coinfectés par le VIH) ou immature (fœtus et nouveaux nés). Lors de l’infection par le CMV, une relation étroite s’établit entre le système immunitaire et le virus. Celle-ci génère en effet une forte mobilisation, associée à un profond remodelage, de différents compartiments immuns. Au cours de ce travail de thèse, nous nous sommes intéressées à l’équilibre qui s’instaure entre le virus et le système immunitaire. Dans une première approche menée in vitro, nous avons exploré le rôle de différentes sous-populations NK dont les cellules NK NKG2C+, caractéristiques de l’infection à CMV, en réponse à des cellules endothéliales, isolées à partir de donneurs de rein, infectées par le CMV. Ensuite, une cohorte rare d’adultes immunocompétents souffrant d’une primo-infection symptomatique à CMV nous a permis d’étudier la course naturelle de l’infection à CMV. Cette approche ex vivo a constitué d’une part en l’analyse du polymorphisme de la réponse de l’hôte au virus, par l’étude phénotypique et transcriptomique non seulement de la réponse NK mais aussi d’autres effecteurs lymphocytaires. D’autre part, nous avons exploré l’impact du polymorphisme génétique viral, par le séquençage par NGS d’isolats cliniques du CMV, sur le pouvoir pathogène du virus. L’ensemble de ces travaux devrait contribuer à la meilleure compréhension du rôle des cellules NK dans le contrôle de l’infection à CMV. / NK cells are innate lymphocyte effectors involved in the control of viral infections and particularly cytomegalovirus (CMV) infection. Usually well tolerated in immunocompetent individuals, CMV infection remains life life-threatening in immunosuppressed patients, as transplant recipients or HIV-infected patients, or for fetuses in case of congenital infection. Upon primary infection, CMV establishes a close relationship with the immune system. CMV infection is known to drive an important immune response and to deeply imprint several immune compartments. In this present work, we focused on the host-virus balance that takes place upon infection. Through a first in vitro approach, we investigated the role of different NK cell subpopulations, including NKG2C+ NK cells which represent one of the hallmarks of CMV infection, in response to CMVinfected endothelial cells isolated from kidney donors. Then, an ex vivo approach was conducted in a cohort of immunocompetent adults diagnosed with symptomatic primary CMV infection. On one hand, our aim was to explore the host immune response polymorphism, through phenotypic and transcriptomic analyses of lymphocyte responses. On the other hand, we investigated the viral genome polymorphism, through NGS sequencing of clinical CMV isolates, which could modulate the viral pathogenicity. Taken together, these findings should contribute to the better understanding of the role of NK cells during the course of CMV infection.

As craniossinostoses são malformações craniofaciais caracterizadas pelo fechamento precoce de uma ou mais suturas cranianas. Elas são doenças congênitas e são causadas por mutações em diversos genes devido ao grande número de vias envolvidas na formação e manutenção das suturas cranianas. Embora mutações em 53 genes já tenham sido descritas o conhecimento da genética e da patofisiologia das craniossinostoses ainda é incompleto. Nesse trabalho tivemos como objetivo a identificação de novas mutações associadas às craniossinostoses bem como o aprofundamento do conhecimento sobre a atuação dessas mutações em células humanas por meio de estudos funcionais. Para identificarmos novas mutações utilizamos metodologias de sequenciamento em larga escala conhecidas como sequenciamento de noiva geração (NGS). Identificamos a mutação causal em uma paciente proveniente de um casamento consanguíneo portadora da síndrome de Raine (p.P496L em FAM20C). Também delimitamos a poucas mutações candidatas outros onze casos atípicos de craniossinostose. Por fim estudamos os efeitos de diferentes FGFs sobre o comportamento de células com a mutação mais comum causadora da S. de Apert, p.S252W em FGFR2. Descobrimos que os FGFs10 e 19 têm ações distintas sobre o perfil transcricional e sobre a taxa de proliferação de células mutantes. Também descobrimos que as células tronco mesenquimais e as células fibroblastóides têm comportamentos distintos ao serem tratadas com FGF19. Os resultados aqui apresentados serão de grande serventia para o melhor delineamento da biologia das suturas cranianas e da patofisiologia das craniossinostoses / Craniosynostosis are craniofacial malformations defined by early closure of the cranial sutures. They are congenital diseases caused by mutations in several genes due to the diversity of pathways involved in the development and maintenance of the cranial sutures. Even though 53 genes have already been linked to various forms of craniosynostosis, the knowledge about the genetics and pathophysiology is incomplete. In this work we aimed to identify new mutations associated with craniosynostosis as well as to further the knowledge of how those mutations act in human cells. To identify new variants associated with craniosynostosis we used large scale sequencing techniques known as next generation sequencing (NGS). We were able to identify the causal mutation in one patient from a consanguineous marriage with Raine syndrome (p.P496L in FAM20C). We also were able to elect candidate mutations in other eleven cases of atypical craniosynostosis. Lastly, we studied the effects of different FGFs over the behavior of human cells harboring the most common Apert syndrome mutation, p.S252W in FGFR2. We discovered that FGFs 10 and 19 have different effects over the transcriptional profile and proliferation rate of mutant cells. We also found that FGF19 have opposite effects in mesenchymal stem cells and fibroblastoid cells osteogenic differentiation. The results shown here will be of great service to better understand the biology of cranial suture and the pathophysiology of craniosynostosis

Bibliotecas de produtos naturais são fontes de moléculas com ações farmacológicas, com diversas aplicações biotecnológicas. Estes compostos apresentam alta especificidade para o alvo, resultante de longo processo de seleção natural, sendo interessantes como ferramentas de estudo e princípios ativos. Porém, apesar da riqueza de estruturas presentes em tais secreções, as dificuldades em obter moléculas puras em grandes quantidades, o alto custo e o tempo de purificação, tornam-se barreiras para seu uso. Assim, cada vez mais, as técnicas ômicas são usadas como uma alternativa para produção destas toxinas obtidas em maior escala. A transcriptômica, técnica que consiste na produção de biblioteca de cDNA a partir do RNA obtido da de organismo, tecido ou célula de interesse, é altamente relevante, já que identifica o material proteico que é realmente transcrito a partir do RNA obtido em determinado tempo e situação. Sapos ainda são animais pouco estudados, quando comparados a outros animais peçonhentos e venenosos, e um dos fatores responsáveis por isso é o baixo rendimento na purificação de toxinas de seu veneno. A fim de se identificar componentes presentes nas secreções do sapo R. schneideri, foi, primeiramente, realizada extração de RNA poli A+ das secreções recém obtidas das glândulas mucosas e das secreções armazenadas por 2 anos no laboratório. A secreção armazenada há dois anos revelou qualidade e quantidade mais apropriadas para o estudo e foi, portanto, usada como material de partida para a construção do transcriptoma por metodologia tradicional. Este transcriptoma resultou em 6 clones com boa qualidade, sendo que um deles, Rs02, apresentou similaridade com a região do pró peptídeo da odorranaina. Uma vez que este transcriptoma não revelou resultados satisfatórios devido à sua baixa eficiência e visando maximizar o conhecimento sobre a secreção, um novo transcriptoma foi construído usando sequenciamento de nova geração, com sequenciador Illumina e RNA total extraído do tegumento contendo glândulas mucosas de um espécime como material de partida. O novo transcriptoma resultou em aproximadamente 131 milhões de reads brutos. Os reads foram filtrados de modo que apenas aqueles com boa qualidade (Q>20) fossem submetidos à montagem de novo. Esta etapa resultou em aproximadamente 130 milhões de reads, com média de 68 pb. Os reads foram então agrupados em contigs usando o programa SOAPdenovo2-Trans e submetidos a diversas abordagens de anotação funcional. Foram encontrados cDNAs codificantes de diversos peptídeos e proteínas com potencial aplicação biotecnológica. Além da abordagem ômica, ensaios de caracterização bioquímica, como atividade enzimática, cromatografia e eletroforese, auxiliaram a detecção de protease sem relatos prévios em secreções de sapo, uma fosfolipase A2, bem como lectina e galectina. Adicionalmente, através do transcriptoma foram identificadas cobatoxinas, mucinas e ficolinas. Portanto, este trabalho foi pioneiro no entendimento molecular do veneno da glândula mucosa de R. schneideri por métodos de vanguarda e análises bioquímicas. / Natural products libraries are known as medicine molecules sources once these molecules have a high target specificity inherited from the long natural evolutionary process. Thereby, animal, plant and microorganisms\' secretions are very important to biotechnological applications. However, despite the large number of molecules that can be found in these secretions, the difficulty on obtainment enough purification yield, high cost and long purification time, besides the small secretion amount provided by the studied organism, are factors that make this kind of study even harsher. These are the reasons why omic studies are becoming an alternative to produce these toxins in a larger scale, which allow new researches. Transcriptome is a technique that consists on the production of cDNA libraries from the secretion or gland RNA, using the transcriptase reverse enzyme, which results in a holistic poison understanding. Toads are animals that are still not widely studied, if we compare them with other venomous animals, mainly because of the insufficient purification yield. Thus, the mucous gland transcriptome from Rhinella schneideri poison, a toad that is widely found in Brazilian territory, has a great relevance on the elucidation and possibility to use several kind of molecules, especially because this gland produces unknown molecules. Thereof, aiming to unravel this poison molecules, we first compared the RNA yield from fresh and two years stored mucous gland secretion. The stored secretion has shown more suitable RNA, which was used to construct a Sanger sequencing transcriptome. It resulted in 6 clones and one of them had a good hit with odorranain pro-peptide region. In order to increase the knowledge about the secretion, we turned to Next Generation Sequencing transcriptome using Illumina technologies and one specimen integument as raw material. The new transcriptome resulted in approximately 131 million raw reads. These reads were filtered so that only those with good quality (Q>20) were used to perform the assembling. The latter step resulted in approximately 130 million reads with 68 bp average length. The reads were grouped into contigs using the assembler. Then, the resulting contigs were submitted to different functional annotation approaches. We unraveled cDNAs encoding peptides and protein with biotechnological application. Besides the omic approach, assays for biochemical characterization including chromatography, electrophoresis and proteolytic assays complemented the identification of a protease for the first time in toad secretions, phospholipase A2, as much as lectins and galectins. Thus, the transcriptome also allowed the identification of cobatoxins, mucins and ficolins. Therefore, this is the first work about the molecular composition of Rhinella schneideri mucous gland poison through avant-garde methodology and biochemical anaylsis.

This thesis contributes to the knowledge of the species diversity of the Crenicichla lacustris sp. group in the La Plata River basin with description of three new species. Speciation mechanisms within two different species flocks from the middle Paraná/Iguazu and Uruguay Rivers were studied with a phylogenomic approach applying a novel genotyping method based on a Double-Digest Restriction site Adjacent DNA (ddRAD) sequencing. Our results support a repeated origin of morphological species being evolved several times sympatrically and independently in different drainages. A considerable role of hybridization/introgression as an evolutionary force was also proposed. The thesis further uncovers biogeographic aspects of the southern part of Brazilian shield and adjacent coastal rivers.

Cette étude a pour objet la mise en évidence de traces d'ADN bactérien pathogène dans des échantillons animaux et humains anciens, et ainsi améliorer les connaissances sur l'évolution des maladies au cours du temps. En parallèle, nous avons étudié les phénomènes de dégradation de l'ADN dans le sol sur des cadavres de souris enterrées après avoir été contaminées par des bactéries non pathogènes. Cette étude des processus taphonomiques s'est étalée sur trois ans et a permis de montrer une disparition rapide des bactéries simulantes, remplacé par l'ADN des bactéries du sol, qui colonisent rapidement la dépouille et dégradent tant l'ADN endogène (murin) qu'exogène (bactérien). Cette disparition rapide explique la grande difficulté à mettre en évidence des pathogènes dans des échantillons anciens, à de rares exceptions près. Notre étude n'a pas permis de détecter d'agents pathogènes particuliers dans les échantillons que nous avons étudié, mais nous avons mis en évidence l'intérêt d'analyser certains types de restes pour accéder à une information génétique préservée. Le tartre dentaire indique est un bon indicateur de la flore buccale de l'hôte et les kystes calcifiés assurent une bonne préservation de l'ADN endogène, moins soumis à contamination et digestion par les bactéries de l'environnement. Les kystes présentent en règle générale une teneur en ADN endogène supérieure à tous les autres tissus étudiés. / The aim of this study was the identification of pathogenic bacterial DNA traces in ancient animal and human samples, and thus improve knowledge of past diseases that affect humankind over time. In parallel, we studied the DNA degradation phenomena in the soil on the buried corpses of mice after being contaminated by non-pathogenic bacteria. This study of taphonomic processes was spread over three years and has shown a rapid disappearance of simulant bacteria, replaced with the DNA of soil bacteria that colonize the body quickly after burial and degrade both the endogenous DNA (murine) that exogenous (bacteria). This quick degradation can explain the high difficulty to detect and identify bacterial pathogens in old samples, with very few exceptions. Despite the fact in our study we were not able to detect specific pathogens in the samples we have studied, we have shown the interest to analyze certain types of remnants to access preserved and informative genetic data. Dental calculus is a good indicator of the oral flora of the host and calcified cysts ensure good preservation of the endogenous DNA, less subject to contamination and digestion by bacteria from the environment. Cysts generally have an endogenous DNA content higher than all other tissues examined.

Les facteurs de transcription ETS sont divisés en sous-familles en fonction de leurs similitudes en matière de séquence protéique, de séquences de liaison à l'ADN et d’interactions avec différents cofacteurs. Ils sont régulés par des signaux extracellulaires et contribuent à divers processus cellulaires, dont la prolifération cellulaire et la transformation tumorale. Les gènes de la famille ETS sont fréquemment ciblés par des processus oncogéniques que ce soit des translocations chromosomiques ou des gains du nombre de leurs copies. Le gène PU.1/SPI1 est également ciblé par des mutations ponctuelles inactivatrices dans les hémopathies myéloïdes humaines. Nous avons étudié une mutation somatique récurrente du gène PU.1/SPI1 (c.676C>G, p.Q226E), identifiée chez environ 6% des patients atteints d’une macroglobulinémie de Waldenström (MW), un syndrome lymphoprolifératif B chronique rare. La mutation modifie les caractéristiques de liaison à l'ADN de la protéine mutante, passant des séquences classiques reconnues par SPI1 à des séquences reconnues par d’autres protéines ETS comme ETS1, et d’une liaison à des régions enhancer à une liaison à des régions promotrices. La liaison accrue du mutant de SPI1 aux régions promotrices active des programmes transcriptionnels impliquant des voies de signalisation intracellulaire généralement favorisées par d'autres membres de la famille ETS. Les conséquences fonctionnelles de cette mutation sont une augmentation de la prolifération cellulaire et une diminution de la différenciation lymphoïde B terminale dans une lignée cellulaire modèle et des échantillons primaires de MW. Nous décrivons ici un mécanisme de subversion oncogénique de la fonction d’un facteur de transcription suite à la modification subtile de la spécificité de liaison à l'ADN de la protéine mutante, menant à un arrêt de différenciation. La démonstration qu'une mutation somatique ponctuelle peut modifier l'équilibre de liaison d’un facteur de transcription à l’échelle du génome fournit un paradigme mécanistique sur la façon dont les mutations faux sens dans les gènes codant pour des facteurs de transcription pourraient être oncogéniques dans les tumeurs humaines. / The ETS-domain transcription factors are divided into subfamilies based on protein similarities, DNA binding sequences and interaction with cofactors. They are regulated by extracellular clues and contribute to a variety of cellular processes, including proliferation and transformation. ETS genes are targeted by oncogenic processes through chromosomal translocations and copy number gains. The PU.1/SPI1 gene is also targeted by inactivating point mutations in human myeloid malignancies. We investigated a recurrent somatic missense mutation (Q226E) of the PU.1/SPI1 gene in Waldenström macroglobulinemia, a human B-cell lymphoproliferative disorder. The mutation changes DNA binding of the mutant protein from classical SPI1 to ETS1-like sequences, shifting the balance from binding to promoter regions from enhancers. Increased binding by mutant SPI1 at promoters activates gene expression of intracellular signaling pathways typically promoted by other ETS factor family members. The functional consequences are decreased terminal B-cell differentiation in a model cell line and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration DNA binding specificity leading to differentiation arrest. The demonstration that a somatic point mutation subtly changes the balance of genome binding provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.

Orientador: Cesar Martins / Abstract: One of the biggest challenges in chromosome biology is to understand the occurrence and complex genetics of extra, non-essential karyotype elements, commonly known as supernumerary B chromosomes (Bs). Bs are present in diverse species of eukaryotes and their molecular characterization remains elusive for years. A distinguished feature that makes them different from the normal chromosomes (called A chromosomes) is their way of inheritance in irregular fashion. Over the last decades, their genetic composition, function and evolution have remained an unresolved query, although a few successful attempts have been made to address these phenomena. The non-Mendelian inheritance and unpairing/non-recombining abilities make the B chromosomes immensely interesting for genomics studies, thus arising different questions about their genetic composition, survival, maintenance and role inside the cell. This study aims to uncover these phenomena in different species. Here, we sequenced the genomes of three model organisms including fish species Astyanax mexicanus and Astyanax correntinus, and grasshopper Abracris flavolineata with (B+) and without Bs (B-) to identify the B-localized sequences, called B chromosome blocks (“B-blocks”). We established approaches for this analysis that comprised of steps such as comparative genomics analysis and annotation of B chromosomal genes and DNA repeat types. The next generation sequencing (NGS) analyses identified thousands of genes fragments as well as... (Complete abstract click electronic access below) / Doutor

Recent improvements in sequencing technologies have caused various interesting problems to arouse. Having millions of read sequences as the final product of sequencing genome at a lower cost compared to micro array era, has encouraged scientists to enhance previous methods in various areas of bioinformatics. Genotyping and generating genetic maps to study inherited genotypes in order to analyze specific traits in a population is one of the fields of bioinformatics that involves generating different genetic markers and identify polymorphisms in different individuals of a population. Presence/absence markers are the main focus of this thesis. This is one type of Restriction site Associate DNA (RAD) markers which is present in some samples and absent in others and is the sign of variation in the cut site of a restriction enzyme. However, the counts of markers in an experiment are highly correlated and calling true absence and presence is not a straightforward task which means any marker with zero count is not necessarily absent in the sample under study. This is also the case for non-zero count markers which are not necessarily present. A good model that can fit the data is able to make true calls. We propose two different contexts for designing such models as a solution to this problem and investigate their performance. On the other hand, utilizing features of next generation sequencing technology in an even more efficient way, requires the ability to multiplex high number of samples in a single experiment run. In that case, appropriate barcoding, that is robust to various sources of noise in the machine, becomes paramount. Designing such barcodes in an efficient way is a challenging task which is addressed in detail as another problem of this thesis. We make two contributions. One, we propose an algorithm for barcoding multiplexed RADSeq samples. Two, we propose an algorithm for the statistical selection of presence/absence markers on the basis of RADSeq data on two related individuals. Operating characteristics of our methods are explored using both simulated and real data.

Submitted by Dominick Jesus (dominickdejesus@hotmail.com) on 2016-01-05T19:39:18Z No. of bitstreams: 2 Dissertação_Jessica Motta de Sousa.pdf: 16274582 bytes, checksum: 070535e90da95d5052fdada5bc749d48 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-01-05T19:39:18Z (GMT). No. of bitstreams: 2 Dissertação_Jessica Motta de Sousa.pdf: 16274582 bytes, checksum: 070535e90da95d5052fdada5bc749d48 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-06-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Several hypotheses have been formulated to explain Amazonian biodiversity patterns, whose biotic diversification has been seen as a result of historically complex scenarios covering a wide range of temporal and spatial scales. Anurofauna has the potential to enhance our understanding of the biogeographic patterns of diversification and processes of speciation, since it serves as a model for inferring historical events. However, the challenge for those seeking to elucidate the processes of diversification of Amazonian frogs is that large portion of its diversity is cryptic, which result in an inaccuracy of limits and distributions of species, which drastically alters our perception of structuring of biodiversity and obscures biogeographic patterns. One of the components of the Amazon anurofauna is the species Hypsiboas cinerascens, which was used as a model to investigate and contribute to the knowledge of the patterns of genetic distribution of anurofauna in the Amazon. Given its wide geographic distribution, some authors have indicated the existence of a species complex with molecular data (mitochondrial gene sequences and genomic data) providing of important tools for the delimitation of evolutionary lineages and their distributional limits, thus clarifying current taxonomy, and for the identification of cryptic species, and thus biogeographic patterns. Given the above, we used sequences of mitochondrial genes 16S RNA and cytochrome b together with the new8 generation sequences (ddRAD-tags) to study the distribution patterns of genetic diversity of H. cinerascens in the Amazon and so testing for cryptic lineages, and inferring biogeographic patterns of the lineages found. Through genetic distance analyses and formation of biological groups with 16S rRNA, concatenated phylogeny of the mitochondrial 16S rRNA and Cytochrome B genes, phylogenomic analyses of the ddRAD-tags, and estimating the time of divergence of both genomes, we identified the possible existence of nine evolutionary lineages in H. cinerascens that originated in the Miocene to the Pliocene: Japurá-Peru, Manaus-Juruti-Guyana, Matupiri-Purus, Santarém-Alta Floresta, Tefé-Jutaí, Morrinho-Rondônia, Uacari, French Guiana and Negro-Trombetas. The filogenomic analyzes confirmed the lineages found in the mtDNA, but with some discrepancies between the topologies. Due to the robustness of the dating of gDNA, we use it to infer the biogeographical history of the group. We suggested that the transcontinental formation of the Amazon River in the last 10 Ma may have been the precursor event for the diversification of the lineages, but due to the complexity of the relationships between groups and the lack of sampling throughout the complete distribution of the H. cinerascens species complex, possibly different historical and ecological factors events influenced their distributions, which can not be identified accurately with our data. The Negro-Trombetas lineage has a distinct biogeographic history of the other lineages of the complex, which may be associated with open forest environments in the region of Guyana. In the future a taxonomic revision of the group should be carried out to verify the existence of new species. / Diversas hipóteses foram formuladas para explicar os padrões de biodiversidade amazônica, cuja diversificação biótica tem sido vista como um produto que envolve cenários historicamente complexos e que abrangem uma ampla gama de escalas temporais e espaciais. A anurofauna possui o potencial de aprimorar o entendimento dos processos biogeográficos nos padrões de especiação e diversificação, já que serve como modelo para inferir eventos históricos. Porém, o desafio para quem busca elucidar o processo de diversificação de anuros amazônicos é que grande parcela de sua diversidade é críptica, que tem por consequência uma imprecisão de limites e distribuições das espécies, o que altera drasticamente a nossa percepção da estrutura da biodiversidade e oculta padrões biogeográficos. Um dos componentes da anurofauna amazônica é a espécie Hypsiboas cinerascens, que foi utilizada como modelo para investigar e contribuir com o conhecimento sobre os padrões de distribuição genética da anurofauna na Amazônia. Considerando sua ampla distribuição geográfica, alguns autores indicam a existência de um complexo de espécies e os dados moleculares (sequências de genes mitocondriais e dados genômicos) são importantes ferramentas para a delimitação de linhagens evolutivas e seus limites de distribuição, de forma a clarificar a taxonomia vigente, bem como para a identificação de espécies crípticas, e consequentemente a mostrar padrões biogeográficos. Conforme o exposto, utilizamos o sequenciamento dos genes mitocondriais 16S rRNA e Citocromo b juntamente com o sequenciamento de nova geração (ddRAD-tags), para estudar os padrões de distribuição da diversidade genética de H. cinerascens na Amazônia e assim testar a presença de linhagens crípticas, inferindo padrões biogeográficos sobre as linhagens encontradas. Por meio de análises de distâncias genéticas e formação de grupos biológicos com o 16S rRNA, filogenia concatenada dos genes mitocondriais 16S rRNA e Citocromo B, filogenômica dos ddrad-tags, e estimação do tempo de divergência de ambos genomas, definimos a possível existência de 9 linhagens evolutivas em H.. cinerascens que se originaram do Mioceno ao Plioceno: Japurá- Peru, Manaus-Juruti-Guiana, Matupiri-Purus, Santarém-Alta Floresta, Tefé-Jutaí, Morrinho- Rondônia, Uacari, Guiana Francesa e Negro-Trombetas. As análises filogenômicas confirmaram as linhagens encontradas com o mtDNA, porém com algumas discordâncias entre as topologias. Devido a maior robustez da datação do gDNA, a utilizamos para inferir a história biogegráfica do grupo. Sugerimos que a formação transcontinental do Rio Amazonas nos últimos 10 Ma pode ter sido o evento precursor da diversificação de linhagens, porém devido a complexidade das relações entre os grupos e a falta de amostragem da completa distribuição da espécie, possivelmente diferentes eventos históricos e fatores ecológicos influenciaram as suas distribuições, nos quais não podem ser definidos com exatidão com os nossos dados. A linhagem Negro-Trombetas possui uma história biogeográfica distinta das outras linhagens do complexo, que pode estar associada a ambientes florestais mais abertos na região das Guianas. Futuramente deve ser realizada a revisão taxonômica do grupo para verificar a existência de novas espécies.