CajaDB: uma plataforma para dados moleculares de Sagui comum (Callithrix jacchus) e análises de transcriptoma

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(1)MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. CajaDB: UMA PLATAFORMA PARA DADOS MOLECULARES DE SAGUI COMUM (Callithrix jacchus) E ANÁLISES DE TRANSCRIPTOMA. VIVIANE BRITO NOGUEIRA. Natal-RN 2017.

(2) VIVIANE BRITO NOGUEIRA. CajaDB: UMA PLATAFORMA PARA DADOS MOLECULARES DE SAGUI COMUM (Callithrix jacchus) E ANÁLISES DE TRANSCRIPTOMA. Dissertação apresentada ao Programa de PósGraduação em Ciências da Saúde da Universidade Federal do Rio Grande do Norte como requisito para a obtenção do título de Mestre em Ciências da Saúde.. Orientadora: Profª. Drª. Maria Bernardete Cordeiro de Sousa. Co-orientador: Prof. Dr. Sandro José de Souza.. Natal-RN 2017 ii.

(3) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial do Centro Ciências da Saúde - CCS Nogueira, Viviane Brito. CajaDB: uma plataforma para dados moleculares de Sagui comum (Callithrix jacchus) e análises de transcriptoma / Viviane Brito Nogueira. - 2018. 54f.: il. Dissertação (Mestrado em Ciências da Saúde) - Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde. Natal, RN, 2018. Orientador: Profa. Dra. Maria Bernardete Cordeiro de Sousa. Coorientador: Prof. Dr. Sandro José de Souza.. 1. Bioinformática - Dissertação. 2. Transcriptoma Dissertação. 3. Diferenças sexuais - Dissertação. I. Sousa, Maria Bernardete Cordeiro de. II. Souza, Sandro José de. III. Título. RN/UF/BSCCS. CDU 575.111:616.8. Bibliotecária: Adriana Alves da Silva Alves Dias CRB 15 474 iii.

(4) MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE. Coordenador do Programa de Pós-Graduação em Ciências da Saúde: Prof. Dr. Erivaldo Sócrates Tabosa do Egito. Natal-RN 2017 iv.

(5) VIVIANE BRITO NOGUEIRA. CajaDB: UMA PLATAFORMA PARA DADOS MOLECULARES DE SAGUI COMUM (Callithrix jacchus) E ANÁLISES DE TRANSCRIPTOMA. Banca Examinadora ___________________________________________________ Profª. Drª. Maria Bernardete Cordeiro de Sousa Presidente da Banca/Orientadora (UFRN) __________________________________________________ Prof. Dr. João Paulo Matos Santos Lima (UFRN) Membro Interno _________________________________________________ Profa. Drª. Fabiola da Silva Albuquerque (UFPB) Membro Externo _________________________________________________ Profa. Drª. Selma Maria Bezerra Jerônimo (UFRN) Membro Interno _________________________________________________ Profa. Drª. Carla Soraia Soares de Castro (UFPB) Membro Externo. Aprovada em:. v. /. /2017..

(6) DEDICATÓRIA. Dedico esta dissertação à mulher mais incrível que eu conheço: minha mãe.. vi.

(7) AGRADECIMENTOS. A minha família: Agradeço por terem sido fonte de cuidado, inspiração, apoio e força. Agradeço, acima de tudo, pela compreensão inabalável durante esses anos de ausência física. Vocês são minha maior preciosidade. Todos os títulos são insignificantes perto de vocês. A minha orientadora: Agradeço pelas inúmeras conversas, cuidado, paciência e ética. Um dia quero poder facilitar a caminhada científica de alunos da mesma forma que a senhora faz comigo. Aos companheiros nordestinos (ou em solo Potiguar): É difícil mensurar a importância de pessoas que nos apoiam longe de casa (lê-se: minha tribo). Difícil mensurar o quanto a convivência com vocês transformou minhas perspectivas. Eu aprendi muito com cada um.... Agradeço cada abraço, cada desabafo e cada vez que gastaram seu tempo para me apoiar técnica e/ou emocionalmente durante o desenvolvimento deste trabalho.. vii.

(8) RESUMO CajaDB: UMA PLATAFORMA PARA DADOS MOLECULARES DE SAGUI COMUM (Callithrix jacchus) E ANÁLISES DE TRANSCRIPTOMA:. O sagui comum (Callithrix jacchus), um pequeno primata de novo mundo, tem sido amplamente empregado como modelo biológico, não apenas para decifrar disfunções em transtornos neuropsiquiátricos como também para compreender circuitos neurais envolvidos no comportamento social humano. A este respeito, a disponibilidade de dados de expressão gênica advindos de tecnologias nextgeneration sequencing (NGS) representam uma oportunidade para novos estudos aprofundados na genética e na epigenética desta espécie. Uma das fronteiras na neurociência é manusear esses dados em larga escala a fim de conectar vias moleculares ao comportamento do sistema nervoso. Para tornar esses dados mais acessíveis para a comunidade científica sem formação em bioinformática, foi criado o CajaDB, um banco de dados que fornece uma interface web para dados de genômica, expressão gênica e splicing alternativo, incluindo ferramentas para análises biológicas. Com os dados processados para esta plataforma foram realizadas duas análises distintas: (1) Expressão diferencial de genes nos hemisférios direito e esquerdo, uma vez que lateralização é um aspecto crucial do funcionamento da arquitetura cerebral para habilidades cognitivas, onde foram encontrados 49 genes diferencialmente expressos, sendo 24 para o hemisfério esquerdo e 25 para o hemisfério direito; (2) Expressão diferencial de genes entre machos e fêmeas, com foco em córtex frontal e comparação com dados equivalentes de humanos. Neste último caso, foi verificado que genes com expressão enviesada para machos são conservados e enriquecidos para funções de manutenção celular. Já genes com expressão enviesada para fêmea foram relacionados a funções de plasticidade neural, envolvidos com remodelamento dos circuitos sinápticos, cascatas de estresse e comportamento visual. Com base em conhecimentos sobre dimorfismo comportamental entre sexos de saguis, é sugerido que estas expressões diferenciais podem estar relacionadas a determinadas circuitarias neurais associadas às estratégias adaptativas de sobrevivência e reprodução para cada sexo. Diante do exposto, espera-se que os dados disponíveis no banco de dados associados às ferramentas biológicas disponíveis facilitem a geração de hipóteses e a interpretação de resultados sobre o funcionamento cerebral nesta espécie que é um modelo biológico largamente utilizado, abrindo perspectivas de investigação e desenvolvimento de novos tratamentos para doenças neuropsiquiátricas no futuro. CajaDB está disponível em cajadb.neuro.ufrn.br. PALAVRAS- CHAVE: Bioinformática – Transcriptômica – Diferenças sexuais Lateralidade.. viii.

(9) ABSTRACT CajaDB: A DATABASE OF COMMON MARMOSETS (Callithrix jacchus) AND TRANSCRIPTOMICS ANALYSIS:. Common marmoset (Callithrix jacchus), a small New World monkey, has been widely used as a biological model not only to elucidate brain dysfunction in neuropsychiatric disorders, but also for deciphering neural circuits involved in human social behaviors. In this regard, the availability of gene expression data derived from next-generation sequencing (NGS) technologies represents an opportunity for deeper studies on the genetic and epigenetic architecture of this species. One of the frontiers in neuroscience field requires handling omics large-scale data sets for connecting molecular pathways to nervous system behavior. To make these omics datasets more accessible for the scientific community without a solid bioinformatics background, we have created CajaDB, a database that provides a friendly interface for genomic, expression and alternative splicing data, including tools for biological analyses. Using the processed data two analysis were conducted: (1) Differential expression between right and left hemispheres, once lateralization is a crucial aspect of the functional brain architecture for cognitive abilities. It was found 49 differentially expressed genes, where 24 genes had left hemisphere bias and 25 genes had right hemisphere bias. (2) Sex-biased gene expression with focus in frontal comparing to humans. It was found that genes whose expression is male biased are conserved between marmosets and humans and enriched with "housekeeping" functions. On the other hand, female-biased genes are more related to neural plasticity functions involved in remodeling of synaptic circuits, stress cascades and visual behavior. Based on knowledge of dimorphic social behavior of male and female common marmosets we discuss that these differences might be linked to neuronal circuitry underlying the expression of the adaptive strategies in each sex and related to survival and reproductive behavior traits. Hence, it is expected that data available in the webpage associated with available biological tools will facilitate generation of hypotheses and interpretation of results on brain functioning, facilitating improvements in neurological diseases treatment in the future. CajaDB is available at cajadb.neuro.ufrn.br. KEY WORDS: Bioinformatics – Transcriptomics – Sexual differences - Laterality.. ix.

(10) LISTA DE ABREVIATURAS. GO - Gene Ontology FPKM - Fragments per Kilobase of Transcript per Million Mapped Reads KEGG - Kyoto Encyclopedia of Genes and Genomes RNA-seq – Sequenciamento de RNA CajaDB – Callithrix jacchus database FDR - False Discovery Rate NGS - Next-generation Sequencing (high-throughput sequencing) GWAS - Estudos de associação genome-wide. x.

(11) SUMÁRIO. 1. INTRODUÇÃO. 12. 2. JUSTIFICATIVA. 14. 3. OBJETIVOS. 16. 3.1 Objetivo 1. 16. 3.2 Objetivo 2. 16. 4. MÉTODOS. 17. 5. ARTIGOS PRODUZIDOS. 20. 5.1 Artigo 1. 20. 5.2 Artigo 2. 36. 6. COMENTÁRIOS, CRÍTICAS E CONCLUSÕES. 49. 7. REFERÊNCIAS. 51. xi.

(12) 12. 1 INTRODUÇÃO Doenças neuropsiquiátricas geram custos altíssimos para a sociedade. A esquizofrenia, por exemplo, é uma doença crônica, a qual dispõe de medicamentos apenas para tratamento paliativo. A etiologia do autismo, ainda que explorada amplamente, não traz perspectiva de melhoria iminente dos sintomas ou cura. São conhecidas cerca de 600 doenças do sistema nervoso, o que indica uma alta probabilidade de toda a população sofrer com alguma dessas doenças em algum momento de sua vida (1). Devido ao prejuízo social pela não compreensão do funcionamento cerebral e de suas disfunções em caso de transtornos, há crescente necessidade de estudos nesta área. A extrema heterogeneidade celular, a complexidade dos circuitos neurais e a confiabilidade de materiais “post-mortem” para estudos em humanos levaram a neurociência a adotar os estudos de genômica comparativa/funcional e bancos de dados de larga-escala. Adicionalmente, a grande quantidade de dados gerados na era das ômicas requer a integração da bioinformática com laboratórios de neurociência moderna. Essa integração é um desafio e será cada vez mais crítica na medida em que tecnologias mais poderosas atinjam novas ordens de magnitude, como sequenciamento high-throughput. A previsão de aplicação das ômicas no estudo de doenças neurológicas e psiquiátricas, tanto em sistemas-modelo quanto em pacientes, é ampla e irá acelerar significativamente os avanços no desenvolvimento terapêutico dentro da neurociência (2). Neste contexto, a existência de animais que possam ser utilizados para auxiliar na pesquisa científica, como ocorre com sagui (Callithrix jacchus), um pequeno macaco do Novo Mundo, abrem grandes alternativas de investigação. Eles têm sido amplamente utilizados como modelo biológico na neurociência não só na tentativa de elucidar a disfunção cerebral em distúrbios neuropsiquiátricos, mas também para decifrar circuitos neurais envolvidos em comportamentos sociais humanos. Como os humanos, saguis formam uma sociedade com vínculos sociais baseados em cuidado cooperativo, que é incomum em primatas (3). Seu tamanho pequeno, fácil manipulação em ambiente laboratorial, alta taxa de reprodução e possibilidade de engenharia genética (4) o tornam um modelo interessante para estudos comportamentais e moleculares..

(13) 13. O comportamento social engloba ações que os indivíduos realizam para sobreviver e reproduzir. A relação entre comportamentos sociais e expressão gênica tem sido objeto de discussões intensas, uma vez que essa relação é muito complexa e envolve muitas esferas do conhecimento (5,6). A organização morfológica e funcional do cérebro depende de influências genéticas e ambientais, e há reconhecimento de que a informação social pode alterar o funcionamento cerebral e a expressão gênica (7–9). Além disso, os hemisférios cerebrais esquerdo e direito são funcional e anatomicamente assimétricos, envolvidos no desempenho cognitivo e em aspectos subjetivos do comportamento, respectivamente. Isto foi amplamente descrito em humanos (10,11) e primatas não humanos (12,13), incluindo os saguis (14–17). À medida que a lateralidade hemisférica é um traço conservado do sistema nervoso em vertebrados (18), as abordagens de genética em modelos experimentais, incluindo o sagui, podem fornecer novos conhecimentos sobre os complexos caminhos neurais poligênicos provavelmente envolvidos na expressão e lateralização da linguagem encontradas em seres humanos. Adicionalmente, os níveis de transcrição diferencial entre indivíduos do sexo masculino e feminino da mesma espécie são conhecidos como expressão gênica enviesada por sexo (sex-biased gene expression) (19). Esse fenômeno pode regular as diferenças sexuais que afetam muitas características biológicas, incluindo prevalência e/ou prognóstico de doenças, morfologia, neuroquímica e comportamento. Elucidar as bases moleculares de tais diferenças é, portanto, notavelmente importante tanto para a neurobiologia básica quanto para a neuropatofisiologia. No entanto, as diferenças entre sexos ainda são relativamente pouco exploradas na neurociência tendo escassos estudos publicados, a maioria sem contextualização molecular (20,21). Devido à grande possibilidade de estudos utilizando o sagui como modelo biológico sob perspectiva molecular, há necessidade de melhoria no acesso aos dados desta espécie, especialmente para cientistas que não possuem formação para manipulação de dados de bioinformática em larga escala..

(14) 14. 2 JUSTIFICATIVA O uso de modelos animais é necessário para o avanço do entendimento de processos biomédicos, evolutivos e comportamentais sempre que a experimentação humana não for possível. Primatas possuem estruturas cerebrais especializadas que dão origem a capacidades cognitivas e de percepção mais complexas. Pode-se citar a expansão do córtex frontal, parte do qual está implicado com os distúrbios mentais e não tem homólogo em outros mamíferos (1). Sagui (Callithrix jacchus) é um primata neotropical com alto potencial para uso como modelo de estudo devido ao baixo custo de manutenção, um padrão de relações sociais complexas e baseada na formação de pares e cuidado cooperativo. Adicionalmente, os saguis têm sido extensivamente estudados em termos de reprodução, desenvolvimento, comportamento e toxicologia (22,23). Com os avanços significativos nas tecnologias de sequenciamento de alto rendimento e, consequentemente, a expansão exponencial de dados biológicos, a bioinformática encontra dificuldades no armazenamento e análise dos dados gerados (24). Dessa maneira, há necessidade de organização de dados a fim de extrair novos conhecimentos científicos. O conceito de KDD (do inglês Knowledge Discovery in Database) é abordado como uma forma de buscar conhecimento a partir de bases de dados (25). Até os estudos mais refinados de genômica funcional essencialmente incluem a representação de dados em forma de lista. Esse tipo de organização de dados não representa nem mesmo uma pequena fração do potencial de informação existente nos dados (2). Uma plataforma web, expondo dados moleculares de sagui de maneira interativa, inerentemente culmina em uma melhor compreensão dos dados, maior possibilidade de elucidar mecanismos de funcionamento cerebral e em maior perspectiva de melhoria para o tratamento de doenças neuropsiquiátricas. Nesse contexto, para facilitar o acesso e a análise dos dados das ômicas de saguis, desenvolveu-se uma aplicação com interface amigável - o CajaDB - que pode ser utilizado pela comunidade científica sem sólida formação em bioinformática. O CajaDB está disponível em cajadb.neuro.ufrn.br. Adicionalmente, não há estudos de lateralização cerebral em saguis incluindo uma contextualização molecular relatados na literatura. Dessa maneira, para ilustrar o uso do CajaDB, estudou-se genes.

(15) 15. diferencialmente expressos nos hemisférios cerebrais esquerdo e direito. Por fim, objetivou-se analisar a expressão gênica enviesada por sexo e splicing alternativo entre os tecidos. Focou-se em córtex frontal de saguis de machos e fêmeas, uma vez que esta região do cérebro é responsável por muitas funções comportamentais superiores. Para fornecer o valor translacional desta espécie, comparou-se os dados de saguis com os dados de expressão de humanos em córtex frontal..

(16) 16. 3 OBJETIVOS. 3.1 Objetivo 1: Estruturar um banco de dados moleculares de sagui (Callithrix jacchus). . Processar dados de expressão gênica (RNA-seq) de diferentes tecidos de C. jacchus;. . Estruturar um banco de dados de informações de genômica, expressão gênica e splicing alternativo com ferramentas interativas para visualização e torná-los públicos, em forma de plataforma web;. . Visualizar dados de expressão gênica diferencial entre os hemisférios direito e esquerdo e utilizar as ferramentas biológicas disponíveis para ilustrar o uso da plataforma web.. 3.2 Objetivo 2: Analisar expressão gênica enviesada entre sexos de saguis (C. jacchus). . Processar expressão diferencial e splicing entre machos e fêmeas nos tecidos: fígado, coração, córtex frontal, cerebelo, rim e gônadas.. . Comparar dados de expressão diferencial entre sexo de saguis com dados de humanos (Homo sapiens).. . Buscar correlatos comportamentais para a expressão gênica diferencial em macho e fêmea..

(17) 17. 4 MÉTODOS 4.1 Amostras de tecidos e fonte de dados Genoma (versão CalJac3) (23) e transcriptoma de referência de sagui (C. jacchus) foram baixados do UCSC genome browser. Dados públicos de sequenciamento de RNA (RNAseq) apresentando alta cobertura de três projetos diferentes foram baixados do SRA/NCBI, como segue: (a) Projeto 1 (26) - córtex frontal (SRR975175 e SRR975174), cerebelo (SRR975177 e SRR975176), coração (SRR975178 e SRR975179), rim (SRR975180 e SRR975181), fígado (SRR975182 e SRR975183) - todos tanto com dados de macho como de fêmea; (b) Projeto 2 (27) – medula (SRR1758976), hemisfério cerebral esquerdo (SRR1758977), pituitária (SRR1758978), hemisfério cerebral direito (SRR1758979), cólon (SRR1758980), coração (SRR1758982), rim (SRR1758983), fígado (SRR1758984), pulmão (SRR1758985), linfonodo (SRR1758986), músculo esquelético (SRR1758987) e baço (SRR1758988) - todos os dados de fêmea; (c) Projeto 3 (28) – bexiga (SRR866208), hipocampo (SRR866209) e músculo esquelético (SRR866213), com dados de macho, além de córtex cerebral (SRR867043) e cerebelo (SRR867044), com dados de fêmea. Esses somam 25 tecidos não redundantes onde sexos foram tratados separadamente. 4.2 Processamento de dados Todas as reads de RNA-seq foram mapeadas usando TopHat v2.1.0 (Bowtie2 – 2.2.5), (29,30) ao genoma de referência. Cufflinks 2.2.1 (31) foi usado para calcular a expressão em valores de FPKM (fragments per kilobase of transcript per million mapped reads) para todos os genes do genoma, pela fórmula a seguir:. Onde: Xi significa counts, Ii significa bases e N significa número total de reads sequenciadas. Vários pacotes do BioConductor (32) foram utilizados para análises..

(18) 18. 4.3 Ferramentas e análises do CajaDB 4.3.1 Classificação de genes de sagui em relação aos níveis de transcrição Para a classificação da especificidade de expressão dos genes por tecidos, utilizou-se um cutoff de 0,5 FPKM. Cada um dos 16.206 genes foi classificado, descrito por Fagerberg (33), em uma das oito categorias com base nos níveis de FPKM em 25 tecidos: (a) Não detectado - menor que 1 FPKM em todos os 25 tecidos; (b) Tecido específico - nível de FPKM 50 vezes maior em um tecido em comparação com todos os outros tecidos; (c) Tecido enriquecido - nível cinco vezes maior de FPKM em um tecido em comparação com todos os outros tecidos; (d) Grupo enriquecido - nível de FPKM médio cinco vezes maior nível em um grupo de 2-7 tecidos em comparação com todos os outros tecidos; (e) Misto baixo - detectado em 1-24 tecidos e pelo menos um tecido possui FPKM < 10; (f) Misto alto - detectado em 1-24 tecidos e todos os tecidos detectados possuem FPKM > 10; (g) Expresso em todos os baixos detectados em 25 tecidos e pelo menos um tecido <10 FPKM; e (h) Expresso em todos os detectores elevados em 25 tecidos e em todos os tecidos> 10 FPKM. 4.3.2 Classificação de genes associados a transtornos neuropsiquiátricos Para listar os genes de sagui associados a transtornos neuropsiquiátricos, foi aplicada uma análise de genômica comparativa com foco na semelhança entre a anotação de genomas humanos e de saguis. A seção "doenças do sistema nervoso" no catálogo de estudos de associação genome-wide (GWAS) (34) possui 756 genes candidatos, que foram utilizados como referência. 4.3.3 Aplicação para visualização interativa dos dados O CajaDB, disponível em cajadb.neuro.ufrn.br, tem uma arquitetura modular. O módulo Home possui informações para orientar a exploração de dados. O módulo do genoma possui uma ferramenta de visualização do genoma: diagrama interativo com genes destacados associados à doença neuropsiquiátrica. O módulo Expression possui um heatmap interativo (Clustergrammer de MaayanLab) com ferramentas de filtragem, organização e geração de imagem. É possível estimar a ontologia de um conjunto de genes pela ferramenta EnrichR (35). Também no módulo de expressão, existe uma.

(19) 19. ferramenta para visualizar a rede proteína-proteína-rede através do StringDB, uma aplicação com interações proteína-proteína conhecidas e previstas (36). No módulo Splicing, dados de isoformas canônicas e alternativas combinadas a eventos alternativos de splicing estão disponíveis para um gene específico, para todos os genes em todos os tecidos. 4.4 Análises utilizando dados de transcriptomica de sagui (C. jacchus) 4.4.1 Expressão diferencial na lateralidade cerebral Cuffdiff (37) foi utilizado para estimar a expressão diferencial entre os dados do cérebro do hemisfério esquerdo e direito de Peng (27) a nível de transcrição. Os valores de P foram ajustados pela taxa de descoberta falsa (False Discovery Rate – FDR) (38). 4.4.2 Expressão diferencial entre sexos Expressão diferencial entre sexos de sagui e de tecidos humanos foi definida como a diferença normalizada entre a expressão em machos e fêmeas: Δ = (m-f) / (m + f), onde Δ = -1 significa apenas expressão em fêmea, Δ = 0 significa expressão imparcial, e Δ = 1 significa apenas expressão em macho (39). Os genes do sexo feminino e masculino foram definidos como o intervalo Δ de [-1.0-0.5] e [0.5-1.0], respectivamente. Para identificar as categorias do Gene Ontology (GO) Consortium (2015) e as rotas de Kyoto Encyclopedia of Genes and Genomes (KEGG) enriquecidas para subconjuntos particulares de genes enviesados, foi utilizado o teste hipergeométrico de sobre representação (p <0,01, enrichGO e enriquecimento de KEGG em R). Para identificar e visualizar eventos alternativos de splicing (exon skipping, alt. 5′ border, alt. 3′ border e intron retention), foi utilizado o Splicing Express (40). Os eventos de splicing alternativo enviesado para um dos sexos foram definidos como uma variante de splicing apresentando uma expressão relativa maior ou igual a 75% da expressão total no dado tecido de macho e fêmea..

(20) 20. 4.5 Análises estatísticas Todas as análises estatísticas foram realizadas utilizando R (R Development Core Team, 2009) 3.3 (http: // www.R-project.org). Os valores de P foram ajustados por FDR (38). Os procedimentos de enriquecimento de genes enviesados foram testados por simulações de Monte Carlo (1.000 conjuntos de amostragem aleatória). Durante cada simulação, um conjunto aleatório foi gerado com o mesmo tamanho do conjunto investigado (para genes enviesados para fêmea e macho). A significância (p-mcarlo) foi definida como o número de genes em uma determinada categoria pelo número de amostragem aleatória (1000). 5. ARTIGOS PRODUZIDOS 5.1 Artigo 1: Sex-biased gene expression in the frontal cortex of common marmosets (Callithrix jacchus) and prospective behavioral correlates. Submetido a Brain and Behavior (Fator de impacto 2.157)..

(21) 21. Sex-biased gene expression in the frontal cortex of common marmosets (Callithrix jacchus) and prospective behavioral correlates Viviane Brito Nogueira, Masters' student in Health Sciences Graduate Program, Federal University of Rio Grande do Norte. Danilo Oliveira Imparato, Bioinformatics Multidisciplinary Environment, Federal University of Rio Grande do Norte. Sandro José de Souza, Brain Institute, Bioinformatics Multidisciplinary Environment; Federal University of Rio Grande do Norte. Maria Bernardete Cordeiro de Sousa, Brain Institute and Health Sciences Graduate Program, Federal University of Rio Grande do Norte. Corresponding Author: Maria Bernardete Cordeiro de Sousa Brain Institute Federal University of Rio Grande do Norte Av. Nascimento de Castro, 2155 - Morro Branco, Natal - RN, 59056-450 Tel: +55 (84) 3215-4592 E-mail: mbcsousa@neuro.ufrn.br. Abstract Introduction The common marmoset (Callithrix jacchus), a small New World monkey, has been widely used as a biological model in neuroscience not only in an attempt to elucidate brain dysfunction in neuropsychiatric disorders but also for deciphering neural circuits involved in human social behaviors. In this regard, the availability of gene expression data derived from next-generation sequencing (NGS) technologies represents an opportunity for deeper studies on the genetic and epigenetic architecture of this species. Methods Gene expression and alternative splicing profiles across tissues were analyzed in male and female marmosets. To provide the translational value of this species we focused in sex-biased gene expression from the frontal cortex of male and female in common marmosets and compared to humans (Homo sapiens). Results In this study, we found that genes whose expression is male biased are conserved between marmosets and humans and enriched with "house-keeping" functions. On the other hand, female-biased genes are more related to neural plasticity functions involved in remodeling of synaptic circuits, stress cascades and visual behavior. Additionally, we developed and made available a database - the CajaDB – providing a friendly interface for genomic, expression and alternative splicing data of marmosets together with a series of functionalities that allow the exploration of these data. CajaDB is available at cajadb.neuro.ufrn.br Conclusion Based on knowledge of dimorphic social behavior of male and female common marmosets we discuss that these differences might be linked to particular neuronal circuitry underlying the expression of the adaptive strategies in each sex and related to survival and reproductive behavior traits. Keywords (5-8) Transcriptomics, sexual dimorphism, synaptic plasticity, adaptive strategies, neuropsychiatric primate model, database.

(22) 22. Introduction The use of animal models is necessary to advance the understanding of biomedical, evolutionary and behavioral processes of our own species. Common marmoset (Callithrix jacchus) is a New World monkey that has been extensively studied in the neuroscience field due to similarities to human brain functioning, circuitry and behavior (Carlos et al., 2015; Miller et al., 2016; Hunt et al., 2017). As humans, marmosets form a sophisticated society based on cooperative breeding, which is uncommon among primates (Wobber, Wrangham and Hare, 2010). They also pair-bond (Digby and Barreto, 1993), have rich social signaling systems and cooperatively care for infants (French, 1997; Mota et al. 2006), all features also present in humans. This, together with other marmosets’ characteristics - small size (~300–400 g), easy handling in a lab setting, high reproduction rate and the possibility of gene editing (Miller et al., 2016) - make them a suitable model for molecular and behavioral studies. Social behavior encompasses the actions that individuals performed to survive and to reproduce. The relationship between social behaviors and gene expression has been the subject of intense discussions since this relationship is very complex and involves many spheres of knowledge (Robinson, Fernald and Clayton, 2008; Marler, 2012). Brain functioning, and behavior rely on both genetic and environmental influences, and there is increasing recognition that social information can alter brain behavior and gene expression (Burmeister, Jarvis and Fernald, 2005; Pointer et al., 2013; Whitfield et al., 2014). In the last decades, the study of biological systems at a molecular level has significantly progressed due to the advent of large-scale technologies. Omics sciences have great potential to further the understanding of traits in human diseases and represent an opportunity for new biological insights in common marmosets. RNA-seq, in particular, provides a more precise measurement of transcripts levels and their isoforms than other transcriptomics methods (Wang, Gerstein and Snyder, 2009). The differential transcript levels along male and female subjects of the same species are known as sex-biased gene expression (Grath and Parsch, 2016). This phenomenon may regulate sex differences affecting many biological features including prevalence and/or prognosis of diseases, morphology, neurochemistry, and behavior. Elucidating the molecular basis of such differences is remarkably important for both basic neurobiology and neuro-pathophysiology. Nonetheless, sex differences are still relatively underexplored in neuroscience with a few published studies, most lacking a molecular contextualization (Trabzuni et al., 2013; Gilks, Abbott and Morrow, 2014). To date, the only study including sex differences in expression level identified two female-biased genes (XIST and HSBP1) in the brain of some primates using microarray technology (Reinius et al., 2008). In this study, we analyzed sex-biased gene expression (RNA-Seq technology) and alternative splicing across tissues. We focused on frontal cortex of male and female common marmosets since this brain region is responsible for many higher behavioral functions. To provide the translational value of this species we compared marmosets’ data to humans’ expression data in the frontal cortex. To facilitate the access and analyses of omics data from marmosets, we developed a userfriendly application – the CajaDB - which can be used by the scientific community without solid bioinformatics background. CajaDB is available at http://cajadb.neuro.ufrn.br..

(23) 23. Materials and methods Tissue samples and data source Common marmoset reference genome (Worley et al., 2014) and transcriptome were downloaded from the UCSC genome browser (version calJac3). Public RNA sequencing (RNA-seq) reads showing high sequence coverage from Cortez et al., (2014) project was downloaded from the SRA/NCBI. This project included Illumina sequencing data of liver, heart, frontal cortex, cerebellum, kidney and gonads tissues from male and female marmosets. Human (Homo sapiens) genome (version hg19) was downloaded from the UCSC genome browser. RNA-seq reads of human frontal lobe were downloaded from the SRA/NCBI, data of Brawand et al., 2011. More information of the samples analyzed is available at Supplementary table 1. Data processing All RNA-seq reads were mapped using TopHat v2.1. 0 (alignment with Bowtie2 - 2.2.5) (Langmead et al., 2009; Trapnell et al., 2010) to the reference genome assembly. Cufflinks 2.2.1 (Goff, Trapnell and Kelley, 2012) was used for assembling and to estimate the abundance of transcripts in FPKM (fragments per kilobase of transcript per million mapped reads) values for all genes in the genome. Several packages within BioConductor 3.5 (Gentleman et al., 2004) were used for gene expression data analysis. Sex-biased gene expression and alternative splicing, and set enrichment analysis Sex-biased expression in the frontal cortex of marmoset and human tissues was defined as the normalized difference between expression in males and females: Δ = (m–f) / (m+ f), where Δ = –1 means female expression only, Δ = 0 means unbiased expression, and Δ = 1 means male expression only (Cheng and Kirkpatrick, 2016). Female- and male-biased genes were defined as the Δ interval of [-1.0-0.5] and [0.5-1.0], respectively (equivalent to z-score 2.0 for genes and 1.5 for isoforms). To identify Gene Ontology (GO) Consortium (2015) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched for particular subsets of sex-biased genes, hypergeometric test for overrepresentation was used (p < 0.01, enrichGO and enrichKEGG in R). To identify and visualize alternative splicing events (Exon skipping, alt. 5′ border, alt. 3′ border and intron retention) we used the Splicing Express (Kroll et al., 2015). Sex-biased alternative splicing events were defined as one splicing variant presenting a relative expression greater than or equal to 75% of the total expression in the given tissue of male and female. Statistical analysis All statistical analyses were performed using R (R Development Core Team, 2009) 3.3 (http:// www.R-project.org). P-values were adjusted by false-discovery rate (FDR) (Benjamini et al., 1995). Enrichment procedures of sex-biased genes were tested by Monte Carlo simulations (1,000 random sampling sets). During each simulation, a random set was generated with the same size of the investigated set (for male- and female-biased genes). Significance (p-mcarlo) was defined as the number of genes in a given category by the number of random sampling (1000). Application for interactive visualization of data The CajaDB, a database available in http://cajadb.neuro.ufrn.br, provides a friendly interactive visualization tool for genomic, expression and alternative splicing data, including tools for.

(24) 24. enrichment analysis and protein-protein-network. More detailed information of this application will be discussed elsewhere. Results Sex-biased gene expression To identify sex biased gene expression in the marmoset tissues, we analyzed public data of RNA-seq from frontal cortex, cerebellum, kidney, heart, liver, and gonads from Cortez et al. (2014). Sex-biased expression was defined as the normalized difference between expression in males and females: Δ = (m–f) / (m+ f). A value of Δ = –1 means expression only in females, Δ = 0 means expression was unbiased, and Δ = 1 means the corresponding gene was expressed only in males (Cheng and Kirkpatrick, 2016). Tissue-based studies (Mayne et al., 2016) commonly show that sex-biased expression tends to be higher in the gonads compared to other tissues, as also found in this study (Figure 1). By comparing sex-biased expression across tissues, we observed that 17,1% of sex-biased genes show an expression bias in more than one tissue. There has been a growing need to understand higher aspects of common marmoset’s cognition since this species is being increasingly used as a model for the study of human social circuits. Although rodents are widely used in behavioral neuroscience, the largest part of the primate prefrontal cortex – part of which is implicated in neuropsychiatric disturbs - has no homolog in other mammals (Wise, 2008). We focused on frontal cortex since this specific brain region is responsible for many higher behavioral functions and of major importance for marmosets as an experimental model.. Figure 1 - The total number of sex-biased genes across tissues: kidney, cerebellum, frontal cortex, heart, liver and gonads. From all the tissues analyzed, gonads presented more sex-biased genes (ovary – 429, testis – 849). Followed by liver (male – 150, female – 441), heart (male – 214,.

(25) 25. female – 264), frontal cortex (male - 117, female – 326), cerebellum (male – 80, female – 20) and kidney (male – 36, female – 61). Expressions at gene-level and isoform-level of male and female in the frontal cortex were compared in common marmosets (C. jacchus) as well as in humans (H. sapiens) (Figure 2). Distribution of male and female bias showed the same tendency in these two species, with a slightly higher number of female-biased genes. As expected, when compared to genes, a higher number of isoforms was observed for both marmosets and humans. Female- and male-biased genes were defined as Δ values lower than -0.5 and Δ values higher than 0.5, respectively. When sex-biased expression of humans and marmosets was analyzed, 45,01% were present in both species at isoform level (791 out of 1757) but the same pattern was not found at gene level analysis. To comprehend the biological processes linked to these sex biased genes, ontology enrichment analyses were performed. Enrichment analysis identifies categories of genes that occur over-represented in a set of genes, when compared to all genes, using statistical approaches and ranking the significantly enriched categories (Subramanian et al., 2005). In general, male and female-biased genes were enriched for different functional categories: male-biased genes for more conserved and broadly expressing "house-keeping" functions whereas female-biased genes were more related to cognitive functions (Figure 3). A list of all genes and isoforms differentially expressed in females and males are available at Supplementary Table 2..

(26) 26. Figure 2 – Expressions in the frontal cortex of marmosets and humans. Correlation of all genes by expression in log2 FPKM is presented in marmoset (A) and human (B), where yellow dots are the sex-biased genes. Distribution of sex-biased expression, defined as the normalized difference between expression in males and females, by gene and isoform levels are shown in C (marmoset) and D (human). We have calculated two p values, the first (p-adjust) related the enrichment step and the second to exhibit the robustness of sampling significance over a random set (p-mcarlo). Among male-biased expression, significant enrichment of genes involved in ATP metabolic process (padjust = 4e-3, p-mcarlo = 1e-4) was found. Among female-biased expression, 13 isoforms characterized an enrichment for visual behavior (p-adjust = 5e-4, p-mcarlo = 3e-4) while 33 female-biased isoforms characterized an enrichment for regulation of synaptic plasticity (p-adjust = 6e-9, p-mcarlo = 6e-4). In humans, 41 female-biased isoforms characterized an enrichment (p-adjust = 1e-4, p-mcarlo = 7e-4) for regulation of synaptic plasticity with 10 genes (out of the 41) being orthologs of marmosets. Genes into these two categories for marmosets were broadly explored through literature review.. Figure 3 – Enrichment analyses for sex-biased at both isoform and gene-level. A) Enrichment analysis (Gene Ontology) for common marmosets. For males, no significant enrichment was.

(27) 27. found at isoform-level. B) Enrichment analysis (Gene Ontology) for humans. For males, no significant enrichment was found at gene-level. In addition, among female-biased genes, a significant enrichment of genes related to both RNA splicing (p-adjust = 1e-3, p-mcarlo =2.9e-3) and RNA processing (p-adjust = 1.49e-9, p-mcarlo =1e-4) was found. Sex-biased alternative splicing The observation of RNA processing and splicing with sex bias in our analysis and the concepts of (1) alternative splicing is one of the main mechanisms controlling the large variability of mRNA and protein isoforms, and (2) sex-bias in alternative splicing is a relevant biological mechanism underlying sex differences lead us to perform a genome-wide screen for sex-biased alternative splicing events. To identify sex biased alternative splicing in the marmoset tissues, we analyzed the same public data of RNA-seq from frontal cortex, cerebellum, kidney, heart, liver, and gonads from Cortez et al. (2014). Sex-biased alternative splicing events were defined as one splicing variant presenting a relative expression greater than or equal to 75% of the total expression in the given tissue of male and female. Table 1 - Number of genes presenting sex-biased alternative splicing events across tissues. Female Tissues. Male. ES Alt 3' Alt 5' IR Total. Frontal Cortex 496. ES Alt 3' Alt 5'. IR. Total. 281. 192. 125 654. 217. 119. 82. 40. 304. Liver. 366. 192. 149. 68. 471. 145. 73. 58. 36. 199. Kidney. 166. 80. 58. 27. 217. 100. 46. 31. 18. 137. Cerebellum. 46. 24. 16. 9. 60. 56. 39. 31. 18. 89. Heart. 364. 182. 141. 68. 467. 214. 120. 84. 55. 303. Gonads. 959. 567. 451. 273 1287. 976. 505. 417. 201 1224. ES – Exon Skipping, Alt 5’ - alternative 5′ splice site, Alt 3’ - alternative 3′ splice site and IR - Intron retention.. Differences in alternative splicing between male and female can be observed in all the tissues analyzed (Table 1), where a higher number of variants were detected in the female for all tissues, except for cerebellum. The most frequent event type was exon skipping, followed by alternative 3’ splicing border, alternative 5’ splicing border and intron retention. A list of all splicing variants in females and males are available at Supplementary Table 3. To better visualize the sex-bias in alternative splicing events (ASEs) in our genes of interest (present in the regulation of synaptic plasticity and visual behavior categories), we show ASEs on a tissue-based perspective (Figure 4). In the DLG4 gene, it was observed a sex-biased alternative 3' Splice Site event - where the green isoform was expressed in male only, in the frontal cortex. Moreover, in the YWHAG gene, it was observed a sex-biased intron retention event, where the red isoform was expressed in male only, for frontal cortex..

(28) 28. Figure 4 – Alternative splicing events in a tissue-based (frontal cortex, heart, cerebellum, kidney, and liver for male and female, testis and ovary) perspective. Sex-specific alternative splicing of DLG4 (alternative 3' splice site) and YWHAG (intron retention) can be observed in the frontal cortex. Both genes are involved in regulation of synaptic plasticity. Discussion Sex differences in molecular context Sexual chromosomes and hormones account for sexual dimorphism but the exact mechanisms for sexual differences are not fully understood (Arnold, van Nas and Lusis, 2009). In a tissue-based perspective, sex-biased expression tends to be higher in the gonads compared to other tissues (Albritton et al., 2014; Mayne et al., 2016). Nonetheless, sex-biased expression found by us for marmosets has been described for other species in kidney (Kwekel et al., 2013), cerebellum (Ziats et al., 2015), frontal cortex (Xu et al., 2014), heart (Isensee et al., 2008), and liver (Zhang et al., 2011). These biases in expression are important regarding sex differences in disease susceptibility related to these tissues (Perucca et al., 2007; Werling and Geschwind, 2013; Regitz-Zagrosek and Kararigas, 2017). Alternative splicing is one of the main mechanisms controlling the large variability of mRNA and protein isoforms, especially in vertebrates. Alternative splicing events (ASEs) can.

(29) 29. determine binding properties, intracellular localization, enzymatic activity, protein stability and post-translational modifications, with compatible biological functions associated (Kelemen et al., 2013). Sex-biased ASE is a relevant biological mechanism underlying sex differences (McIntyre et al., 2006; Stolc et al. 2004). Our analysis in common marmosets shows widespread sex-biased ASEs through tissues, which is consistently described in humans and primates (Blekhman et al., 2010; Trabzuni et al., 2013). We focused on sex-biased expression in the frontal cortex of marmosets aiming to clarify some molecular, cellular or systemic mechanisms that might be involved in the physiopathology behind neuropsychiatric disorders and behavior. Genes located on the Y chromosome can be expressed only in males, and this chromosome likely contains 50 to 60 genes that code proteins. In our sex-biased expression analysis, genes mapped to sexual chromosomes were not significant since only 2.6% of the frontal cortex genes of marmosets were located on these chromosomes. Sexual hormones influence on neurogenesis, cell differentiation, apoptosis, axon guidance and synaptogenesis processes (Jazin and Cahill, 2010). In our enrichment analysis for frontal cortex, sex-biased genes fell into categories related to all these processes. From the female-biased genes enriched for regulation of synaptic plasticity category, NTRK2, CREB1, and CRTC1 are possibly associated with CREB1-BDNF-NTRK2 pathway which plays a significant role in brain adaptation to stress (Juhasz et al., 2011). NLGN1, RASGRF1, PRKCZ, SRF, IQSEC2, JPH4, UNC13A, YWHAG, KCNB1, STXBP1, BRAF, and SNAP25 are associated with general functional synaptic plasticity. Furthermore, some genes are specifically related to the glutamatergic system: CPEB3, DLG4, SHISA9, SHISA7, FMR1, ABHD6, and NPTN. Some other genes are linked to morphological (growth or apoptotic) mechanisms: TNR, CNTN4, STAU1, PPP1R9A, CAMK2B, and SNCA. Sex differences in synaptic plasticity pathways have been recently discussed (Bourgeron, 2015; Duman et al., 2016; Dachtler and Fox, 2017). The genes in the visual behavior category (PIAS1, APP, NDRG4, KMT2A, NLGN3, HMGCR, HIF1A, CDK5, ATP1A3, MECP2, and SLC24A2) are possibly associated with different chromaticity in the sexes and visual processing of foraging behavior. Furthermore, male-biased genes enriched for ATP metabolic process. Female and male sexual hormones have been shown to be controlling ATPase activity, and previous observations suggest that ATPase expression levels are sexually dimorphic (Fekete et al., 2008). In the context of neuropsychiatric diseases, schizophrenia prefrontal cortex genes linked to energy metabolism have been found to have modified expression only in males (Qin et al., 2016). Gene expression in social behavior Social behavior encompasses the actions that individuals performed to survive and to reproduce. Correlations between genes and social behaviors involve many biological processes and molecular functions (Robinson, Fernald and Clayton, 2008). Social factors have been demonstrated to influence gene expression as observed in cichlid fish (Astatotilapia burtoni) for ascension to dominance (Burmeister, Jarvis and Fernald, 2005), in adult honey bees (Apis mellifera) during transition from hive work to foraging (Whitfield et al., 2014), in swordtail fish (Xiphophorus nigrensis) in context of sexual selection (Cummings et al., 2008), and in rhesus monkeys (Macaca mulatta) in early parental loss (Sabatini et al., 2007). Differences in expression can emulate sex-biased gene regulatory structures and have been reported having functional importance on behavior (Burmeister, Jarvis and Fernald, 2005; Pointer et al., 2013; Trabzuni et al., 2013). Males and females’ marmosets present different strategies in social behavior. In general, marmosets are considered cooperative breeders which are characterized by flexibility in their reproductive behavior where monogamic, as well as polygynous mating systems, were recorded in.

(30) 30. free-ranging animals depending on the ecological and social factors (Arruda et al., 2005; Sousa et al., 2005). Animal and human studies suggest that CREB1-BDNF-NTRK2 pathway plays an important role in brain adaptation to stress (Juhasz et al., 2011). The present analysis showed female-biased expression of NTRK2, CREB1 and CRTC1 genes in common marmosets, suggesting a higher demand for this signaling cascade in females. This might be involved with reproductive strategy, where females are based on competition whereas males are based on cooperation (Yamamoto et al., 2010). Synaptic plasticity Sex-differences in cognitive abilities do not simply reflect differences in sex hormones, but also reveal distinctions in synaptic signaling mechanisms (Mizuno and Giese, 2010). Synaptic plasticity (SP) is defined as the process of strengthening or weakening synapses related to development or learning. Some human neuropsychiatric disorders associated with prefrontal cortex dysfunctions show sex differences in prevalence and symptomatology possibly related to synaptic plasticity, such as those demonstrated in major depression disorder (MDD) and autism spectrum disorder (ASD) (Bourgeron, 2015; Duman et al., 2016). One of the factors that are involved with sex biased differences is related to CaMKK and estrogen receptor pathways which present sexual dimorphism with implications for SP in the cerebral cortex (Dachtler and Fox, 2017). Mottron et al., (2015) argued that males have more susceptibility than females to gene disruption involved in SP and evidences from human genetics and transcriptomics, animal models, and studies of cerebral plasticity support the hypothesis of sex-dependent plastic reaction proposed by them. This hypothesis accounts for the high ratio of males in relation to females in autism (males have 4 to 7 higher-fold risk of developing the disorder than females). Gray et al., (2015) showed in humans higher levels of expression of glutamate receptor genes occurring in the dorsolateral prefrontal cortex in MDD and its global prevalence is 2.7% in males and 3.4% in female (Ferrari et al., 2013). The present analysis for common marmoset shows female sex-biased genes related to general regulation of SP, and genes specifically related to the glutamatergic system. Glutamate (LGlu) is the main and most abundant excitatory neurotransmitter in the central nervous system of mammals, playing a crucial role in the mechanisms underlying SP. These mechanisms depend on several glutamate receptors stimulation. Differences in glutamatergic pathways associated with sex biased prevalence of diseases in humans and supported by recent findings reviewed by Dachtler and Fox, 2017 suggest that sexual differences in plasticity functionalities are in accordance with the data currently found for marmosets. Visual processing in the frontal cortex of marmosets The prefrontal cortex (PFC) is essentially an integrative cortex, where sensory and other inputs determine and guide commands and decisions. PFC is implicated in spatial attention through control of eye and head movement and in primates, the prearcuate gyrus plays a role in peripheral aspects of visual attention. This integrative area process visual information to planning and triggers the conjugate movements of saccadic or head–neck movements associated with visual exploration and accuracy of visual stimuli responses (Miller and Cohen, 2001). Marmosets’ color vision is based on two (short-wavelength-sensitive, SWS1 or "blue", and long-wavelength-sensitive, LWS or "red") to three (SWS1, LWS and middle-wavelengthsensitive MWS or "green") cone types in the retina. Each type of cones expresses a different class of visual pigments. All male marmosets are dichromatic (two cone types in the retina) whereas females have either trichromatic vision (three cone types in the retina), like most humans, or.

(31) 31. dichromatic vision like marmoset males (Mitchell et al., 2016). This is due to the New World primates' mechanism of leading to trichromacy, based on single polymorphic X-linked LWS gene with different allelic variants encoding pigments with different spectral peaks. Advantages for trichromatic females over dichromatic ones in detecting orange–colored food items against foliage were described (Caine and Mundy, 2000; Hunt et al., 2017). Thus, differences in visual processing of males and females might account for sexual differences and a higher demand of visual cascades in females. Marmosets as animal model Brain circuitry knowledge that drives social interactions is limited, in part due to the technical limitations of measuring brain activity in humans. Animal models have been and will continue to be demanding to study many aspects of behavior, particularly to decipher the neural basis of human social behavior. Unfortunately, to date, rare existing behavioral paradigms are underlying experimental model. Humans and marmosets share features of cognition and social behavior, where marmosets display social traits such as pair bonding, living in extended family groups and cooperative caring for infants. They markedly exhibit social cognitive skills such as imitation and cooperative breeding (Burkart, Hrdy and Van Schaik, 2009; Miller et al., 2016). Moreover, marmosets fulfill the validation criteria to be a potential experimental model for depression (Galvão-Coelho et al., 2008; Sousa et al. 2015). Changes in the social environment require changes in behavior. At the molecular level, they rely on the regulation of gene expressions by signaling pathways. This molecular response to perceived social information is known as social plasticity. Decision making and social landscapes interpreting require a neural circuitry adaptable and able to accommodate learning on short and long-term perspectives to understand the situation and consequences of behavior. Gene expression is determining to promote key male and female traits, and it may be conserved during evolution (Reinius et al., 2008; Carlos et al., 2015). In our analysis, 45,01% of sex-biased genes at isoformlevel were present for both species. In the category of regulation of SP, female-biased genes were found in marmosets as well as in humans with 10 genes being orthologs. In this regard, the similarity of these results reinforces this species as a good model to study neuropsychiatric disorders since social behaviors involve homologous neural mechanisms across primate species. CajaDB CajaDB, the most comprehensive molecular database in marmosets, provides an intuitive interface to visualize and explore genomic, transcriptomic and alternative splicing data. Our application not only allows the user to navigate the data but also supports biological analyses such as functional (ontology) enrichment analysis and protein-protein-network. Hopefully, these centralized resources will provide numerous benefits to researchers in addressing scientific questions. More detailed information of this application will be provided elsewhere. Perspectives In order to validate our data, we suggest follow-up experiments (with a higher number of testing individuals) designed to test the sex-differences expressions in visual behavior, synaptic plasticity, and social behavior context here presented. In marmosets, social groups are composed of dominant breeding females and subordinate nonbreeding females. This relation demands positive and harmful interactions into the social group to reach and to maintain the dominance and, consequently, reproductive success. Sex-biased gene expression in the context of dominance was analyzed in turkeys: when subordinate males were compared to the dominants, the overall expression patterns were concordant with their phenotypic status (Pointer et al., 2013). We.

(32) 32. envision that gene expression patterns would be a good strategy to investigate social hierarchy in female marmosets.. Conclusion Findings of the present study reinforce the use of common marmosets as a translational model to study neuropsychiatric disorders. Female-biased genes in frontal cortex were enriched toward cognitive functions while male-biased genes were associated with “housekeeping” functions. This relationship was also present when we analyzed expression data from humans. In concordance with other species, including fish, rodents and humans, sex-biased gene expression and splicing are phenomena present in adult marmoset brain. These findings suggest that the main sexual differences in frontal cortex are largely determined by differential expression of genes from autosomes chromosomes, although genes of sexual chromosomes may also contribute to these sex-biased expressions. The occurrence of a sex-differential expression in marmosets might be involved with a more resourceful stress cascade to lead with social competition present in females. These differences in convergence with plastic events related to remodeling of synaptic circuits are possibly associated with particular mechanisms of the neuronal circuitry underlying the expression of the adaptive strategies in each sex associated with survival and reproduction. References Albritton, S.E., Kranz, A.L., Rao, P., Kramer, M., Dieterich, C., & Ercan, S. (2014). Sex-biased gene expression and evolution of the X chromosome in nematodes. Genetics 197, 865–883. Arnold, A.P., van Nas, A., & Lusis, A.J. (2009). Systems biology asks new questions about sex differences. Trends Endocrinol. Metab. 20, 471–476. Arruda, M.F., Ara??jo, A., Sousa, M.B.C., Albuquerque, F.S., Albuquerque, A.C.S.R., & Yamamoto, M.E. (2005). Two breeding females within free-living groups may not always indicate polygyny: Alternative subordinate female strategies in common marmosets (Callithrix jacchus). Folia Primatol. 76, 10–20. Author, T., Benjamini, Y., Hochberg, Y., & Benjaminit, Y. (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Controlling the False Discovery Rate: a Practical and Powerful Approach to Multiple Testing. J. R. Stat. Soc. 57, 289–300. Blekhman, R., Marioni, J.C., Zumbo, P., Stephens, M., & Gilad, Y. (2010). Sex-specific and lineage-specific alternative splicing in primates. Genome Res. 20, 180–189. Bourgeron, T. (2015). From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat. Rev. Neurosci. 16, 551–563. Brawand, D., Soumillon, M., Necsulea, A., et al. (2011). The evolution of gene expression levels in mammalian organs. [SupMat]. Nature 478, 343–348. Burkart, J.M., Hrdy, S.B., & Van Schaik, C.P. (2009). Cooperative breeding and human cognitive evolution. Evol. Anthropol. 18, 175–186. Burmeister, S.S., Jarvis, E.D., & Fernald, R.D. (2005). Rapid Behavioral and Genomic Responses to Social Opportunity. 3. Caine, N.G., & Mundy, N.I. (2000). Demonstration of a foraging advantage for trichromatic marmosets (Callithrix geoffroyi) dependent on food colour. Proc. Biol. Sci. 267, 439–444. Carlos, J., Belmonte, I., Callaway, E.M., et al. (2015). Perspective Brains , Genes , and Primates. Neuron 86, 617–631..

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(36) 36. 5.2 Artigo 2: CajaDB: a molecular database of common marmosets (Callithrix jacchus) (APÊNDICE 2).. A ser submetido a Databases (Oxford) (Fator de impacto 3.290)..

(37) 37. CajaDB: a molecular database of common marmosets (Callithrix jacchus) Viviane Brito Nogueira, Health Sciences Center, Federal University of Rio Grande do Norte, Natal, RN, Brazil. Danilo Oliveira Imparato, Bioinformatics Multidisciplinary Environment, Federal University of Rio Grande do Norte, Natal, RN, Brazil. Sandro José de Souza, Brain Institute, Bioinformatics Multidisciplinary Environment; Federal University of Rio Grande do Norte, Natal, RN, Brazil. Maria Bernardete Cordeiro de Sousa, Brain Institute, Federal University of Rio Grande do Norte, Natal, RN, Brazil. Corresponding Author: Maria Bernardete Cordeiro de Sousa mbcsousa@neuro.ufrn.br Abstract Common marmoset (Callithrix jacchus), a small New World monkey, is a valuable model in different areas of investigation and recently it has been proposed for the study of neurological/neuropsychiatric diseases and cognition. One of the frontiers in neuroscience field requires handling omics large-scale data sets for connecting molecular pathways to nervous system behavior. Hence, to make the omics datasets for this species more accessible for the scientific community without a solid bioinformatics background, we have created CajaDB, a molecular database that provides a friendly interface for genomic, expression and alternative splicing data, including tools for biological analyses. Once lateralization is a crucial aspect of the functional brain architecture for cognitive abilities, we analyzed genes expressed in the marmosets' hemispheres to illustrate the use of CajaDB. We found 24 genes with left hemisphere bias and 25 genes with right hemisphere bias. CajaDB is available at cajadb.neuro.ufrn.br. Keywords (5-8) Transcriptomics, database, neuropsychiatric primate model, laterality, brain asymmetry Introduction Animal models have been crucial in biomedical research once a large part of human experimentation is unfeasible or unethical. Common marmosets (Callithrix jacchus) are widely used as a model in several areas of research. In the neuroscience field, it has progressively gained space for both cognition studies and investigation of neuropsychiatric conditions such as Parkinson's disease (Phillips et al., 2017)⁠, Alzheimer's disease (Philippens et al., 2016)⁠ and depression (Galvão-Coelho et al., 2008; 2017). Besides sharing behavioral characteristics regarding their social organization with humans (Miller et al., 2016)⁠, their use as a model holds advantages of easy handling in a lab setting and reproduction at a rate that makes genetically manipulated models of disorders feasible (Kishi, Sasaki, & Okano, 2014)⁠. Recently, large-scale technologies (the omics, e.g. genomics, transcriptomics, and proteomics) have revolutionized biological studies at a molecular level. The use of data generated by these technologies represents a great opportunity to molecular neuroscience research. One of the greater challenges in the omics is the organization of data to extract new scientific knowledge. Simple levels of data organization do not represent the inherent potential information, and it is.