Toxinology in the proteomics era: a review on arachnid venom proteomics

Abstract The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000s. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms (scorpion venom) AND (proteome) for scorpion venomics, and (spider venom) AND (proteome) for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.(AU)

Avaliação do microbioma cutâneo de gatos com dermatofitose pela técnica de sequenciamento de alto rendimento e caracterização de proteínas imunodominantes do Microsporum canis para o sorodiagnóstico da dermatofitose felina

A dermatofitose e uma doenca fungica comumente vista no atendimento ambulatorial de felinos. A sua importancia clinica e epidemiologica reside no fato de ser uma dermatopatia cosmopolita, infecciosa, contagiosa e zoonotica. Atualmente, o cultivo fungico e a principal ferramenta de diagnostico utilizada, porem a sua morosidade na emissao do resultado indica a necessidade do desenvolvimento de novas estrategias de diagnostico. A aplicacao de tecnologias de imunodiagnostico, bem como tecnologias de sequenciamento da microbiota, tem um grande potencial para contribuir na identificacao e desenvolvimento de novos alvos diagnosticos. O objetivo desse estudo foi identificar e caracterizar as proteinas imunodominantes do Microsporum canis para aplicabilidade no sorodiagnostico da dermatofitose felina. Alem disso, objetivou-se avaliar a composicao e a diversidade das microbiotas fungica e bacteriana de gatos com dermatofitose, gatos assintomaticos (carreadores de M. canis) e gatos higidos. Para os ensaios de caracterizacao das proteinas de M. canis, foram colhidas amostras cutaneas e de sangue de 70 felinos: sintomaticos (n= 20), assintomaticos (n= 20), negativos (n = 20) e negativos heterologos (n= 10). As amostras cutaneas foram submetidas a cultivos fungicos para confirmacao diagnostica e subsequente extracao de M. canis e as amostras de sangue foram avaliadas por Western blot uni e bidimensional. Para a realizacao dos ensaios de sequenciamento da microbiota, foram colhidas amostras cutaneas, pelos metodos carpete e escova de dentes, de 45 felinos: sintomaticos (n= 15), assintomaticos (n= 15) e negativos (n= 15). Essas amostras foram submetidas ao sequenciamento de alto rendimento na plataforma Illumina utilizando analise dos genes 16S rRNA e ITS1. Na avaliacao bidimensional das proteinas de M. canis, foi observado que elas se encontravam majoritariamente na faixa de pH de 7 a 3. Ainda, o WB revelou um amplo perfil antigenico com proteinas variando de 150 a 20 kDa em peso molecular. As proteinas de 30 e 42 kDa demonstraram intensa imunorreatividade em felinos sintomaticos e assintomaticos e baixa imunorreatividade nos negativos. O teste de Western blot conseguiu discriminar bem os gatos positivos dos gatos negativos para M. canis demonstrando elevados indices de sensibilidade (92,5%) e especificidade (90%) diagnosticas. Quanto aos resultados da analise de sequenciamento, foi observado que gatos sintomaticos e assintomaticos apresentaram um padrao semelhante na composicao e distribuicao da microbiota. Adicionalmente, nao foi observado diferenca estatistica na distribuicao relativa do Microsporum entre esses grupos. Os resultados do imunodiagnostico sugerem que as proteinas de 30 e 42 kDa apresentam elevada acuracia diagnostica e podem ser utilizadas em teste de WB, bem como sao potenciais candidatas a purificacao e subsequente utilizacao em ensaios enzimaticos. Adicionalmente, conclui-se que a atividade proteolitica do M. canis possui maior acao em ph acido. Em relacao aos dados do sequenciamento, conclui-se que o desencadeamento da dermatofitose esta mais relacionado a fatores intrinsecos referentes a imunidade do hospedeiro do que com fatores relacionados ao dermatofito ou com a distribuicao/composicao da microbiota comensal. Adicionalmente, conclui-se que embora o M. canis seja o agente causal e fator determinante para o desencadeamento da dermatofitose, sua acao isolada nao e suficiente para desencadear a enfermidade.

Dermatophytosis is a superficial fungal disease commonly seen in dermatological practice. This mycosis is an important skin disease of cats because it is contagious, infectious and can be transmitted to people. Currently, several diagnostic techniques are available to confirm clinical suspicion, and the fungal culture remains one of the most employed diagnostic tools. However, dermatophytes are slow-growing fungi and it may take several weeks for results. Therefore, the development of new diagnostic tests as immunodiagnostic, as well as microbiota sequencing Technologies can contribute to the proper identification and development of new diagnostic targets. The aim of this study was to identify the immunodominant proteins of Microsporum canis for the serological diagnosis of feline dermatophytosis. Additionally, this study aimed to evaluate the composition and diversity of skin fungal and bacterial communities of cats with dermatophytosis, asymptomatic cats (carriers of M. canis), and healthy cats. For the M. canis protein study, skin and blood samples were collected from 70 cats: symptomatic (n=20), asymptomatic (n=20), negative (n=20) and heterologous negative (n=10 ). Skin samples were submitted to fungal cultures for diagnostic confirmation and subsequent extraction of M. canis, and blood samples were evaluated by Western blot. For the microbiota study, skin samples were collected by combing the coat using a sterile polyester carpet and toothbrush, from 45 cats: symptomatic (n=15), asymptomatic (n=15) and negative (n=15). Samples were submitted to next-generation sequencing on the Illumina platform using 16S rRNA and ITS1 gene analysis. In the two-dimensional evaluation of M. canis proteins, it was observed that they were distributed in the pH range of 7 to 3. Furthermore, the WB revealed a broad antigenic profile with proteins ranging from 150 to 20 kDa. The 30 and 42 kDa proteins showed strong immunoreactivity in symptomatic and asymptomatic samples and low immunoreactivity in negative samples. The Western blot test was able to distinguish positive and negative cats for M. canis, showing high sensitivity (92.5%) and specificity (90%). The microbiota analysis showed that symptomatic and asymptomatic cats presented a similar pattern in the composition and distribution of skin microbiota. Additionally, no statistical difference was observed in the relative distribution of Microsporum between these groups. Immunodiagnostic results suggest that proteins of 30 and 42 kDa present high diagnostic accuracy and can be applied in WB tests, as well as being potential candidates for purification and subsequent application in serological assays. Additionally, it is concluded that the proteolytic activity of M. canis occurred mainly in acidic ph. Regarding the sequencing data, it is concluded that dermatophytosis is more linked to the host's immunity than to factors associated with the dermatophyte or distribution of the commensal microbiota. Additionally, it is concluded that although M. canis is the causative agent and responsible for triggering dermatophytosis, its isolated activity is not sufficient to trigger the disease.

Desempenho e Microbiota fecal de potros desmamados equinos e muares em sistema de pastejo rotacionado

O crescimento dos potros é marcado por mudanças dietéticas e essas variações proporcionam a maturação na microbiota presente no trato digestório, no entanto o desmame é um momento decisivo, visto que os potros terão que se alimentar de forma eficiente para garantir a mantença e desenvolvimento. Esta pesquisa avaliou se os equídeos, forrageiras e períodos após o desmame refletiram em diferenças no crescimento e ganho de peso de potros desmamados, na microbiota e na diversidade bacteriana fecal. O experimento foi conduzido em delineamento inteiramente casualizado em arranjo fatorial 2x3x2, sendo: 1º fator potros Equus caballus e os potros híbridos muaresEquus asinusx Equus caballus; o 2º fator pastagens com os cultivares de Coast-cross, Florakirk e Tifton-85; e 3º fator períodos I (período imediato pós-desmame) e II (período tardio pós-desmame). Realizou-se mensurações de peso corporal e medidas lineares em 12 potros recém-desmamados, sendo seis potros da raça Mangalarga Machador e seis potros muares resultantes do acasalamento de jumento Pêga e éguas Mangalarga Marchador. Os potros foram mantidos nas pastagens de Cynodondactylon: Coast-cross, Florakirk e Tifton-85, em manejo rotacionado em cada tipo de forrageira, em intervalos de 20 dias, durante quatro meses. Em cada rotação de pastagem, amostras fecais foram coletadas em seis potros (3 equinos e 3 muares), no início e ao final do pastejo, para análises bromatológicas e microbiológicas. As técnicas dependentes de cultivo foram utilizadas para obter contagens de microrganismos com função fibrolíticas e independentes de cultivo para avaliar a composição da população bacteriana. Realizou-se o sequenciamento de nova geração IlluminaMiseq da região V3-V4 do gene que codifica o RNA ribossomal 16S. As variáveis de desempenho animal, contagens em meio de cultivo, abundâncias relativas e índices de riqueza e diversidade foram submetidos a análise de variância utilizando o teste F (p<0,05) e normalidade dos resíduos através do teste Shapiro Wilk (p>0,05). As médias foram comparadas através do Teste Tukey (p<0,05). Foram realizadas as análises multivariadas: Permanova (p<0,05) e NMDS (Ordenação multidimensional não métrica) a partir dos dados das distribuições da OTUs. Comparou-se os grupos de tratamentos através do Lefse (análise discriminante linear do tamanho do efeito) com base nos grupos observados na análise de ordenação. Observou-se o predomínio da microbiota fecal de potros desmamados pelo filo Firmicutes (57%) seguido por Bacteroidetes (22%). Os muares apresentaram maior diversidade de espécies fibrolíticas indicando que eles possuem uma eficiência superior na degradação das fibras. Os fatores espécies, forrageiras e períodos após desmame refletiram em diferenças na comunidade bacteriana fecal nas contagens bacterianas, nas abundâncias relativas, nos índices de diversidade e no pH fecal. Apenas as forrageiras e períodos promoveram diferenças nas variáveis de desempenho corporal e as forrageiras que repercutiram em melhores ganhos foram o Florakirk e o Coast-cross. No período imediato ao desmame houve maior desenvolvimento dos animais e os potros superaram a alturamínima esperada, porém não atingiram o peso vivo estimado de acordo com o padrão racial para equino adulto Mangalarga Marchador.

The developmentoffoalsissurroundedbydietarychanges, andthesevariationsprovide for maturation in the microbiota present in digestivetracts. However, weaning is a turning point as foals will need to feed efficiently to ensure their maintenance and growth. This research evaluated whether equine species, forage and post weaning periods reflect differences in growth and weight gain of weaned foals, the microbiota and bacterial diversity of fecal samples. The research was carried out in completely randomized design in 2x3x2 factorial arrangement, being: 1st factor - Equus caballus foals and the mule foals Equus asinus x Equus caballus; the 2nd factor - pastures with Coast-cross, Florakirk and Tifton-85 cultivars; and 3rd factor - periods I (immediate post-weaning period) and II (late post-weaning period). Body weight measurements and linear measurements were carried out in 12 newly weaned foals, six foals of the Mangalarga Machador breed and six mule foals resulting from the mating of Pêga donkey and Mangalarga Marchador mares. The foals were kept in Cynodon dactylon pastures: Coast-cross, Florakirk and Tifton-85, in rotational management in each type of forage, at intervals of 20 days, for four months. In each pasture rotation, fecal samples were collected from six foals (3 horses and 3 mules), at the beginning and at the end of grazing, for bromatological and microbiological analyses. Culture-dependent techniques were used to obtain counts of microorganisms with fibrolytic function and culture-independent techniques to evaluate the composition of the bacterial population. Illumina Miseq next-generation sequencing of the V3-V4 region of the gene encoding 16S ribosomal RNA was performed. The variables of animal performance, counts in culture medium, relative abundances and richness and diversity indices were submitted to analysis of variance using the F test (p<0.05) and normality of residuals was tested by the Shapiro Wilk test (p>0.05). The means were compared using the Tukey test (p<0.05). Multivariate analyses were performed: Permanova (p<0.05) and NMDS (Non-metric multidimensional ordination) from the data of OTUs distributions. The treatments were compared using Lefse (linear discriminant analysis of effect size) based on the groups observed in the ordination analysis. The fecal microbiota of weaned foals was observed to be dominated by the phylum Firmicutes (57%) followed by Bacteroidetes (22%). Mules showed a higher diversity of fibrolytic species, indicating that they have a higher efficiency in fiber degradation. The factors species, pastures and periods reflected in differences in the fecal bacterial community in: bacterial counts, relative abundance, diversity indices and fecal pH. Only the pastures and periods promoted differences between the body performance measures, the forages that resulted in better gains were Florakirk and Coast-cross. In the period immediately after weaning, there was greater development of the animals and the foals exceeded the minimum expected height, but did not reach the estimated body weight according to the breed standard for the mature Mangalarga Marchador horse.

APPLYING NEXT GENERATION SEQUENCE DATA IN ANIMAL BREEDING

No Melhoramento Animal existe um histórico de utilizar dados fenotípicos dos melhores animais de uma geração para produzir a próxima geração. Entretanto, é sabido que a herdabilidade das características de interesse depende de uma base genética e podem ser explicadas através do DNA. Na genética quantitativa é usado o modelo básico que diz que o fenótipo é a soma do genótipo (composição genética do indivíduo) e do ambiente, entretanto, genótipos diferentes podem desempenhar resultados superiores ou inferiores dependendo do ambiente em que estão inseridos, com isso, houve a necessidade de incluir o fator interação entre o genótipo e o ambiente. Com o desenvolvimento tecnológico, o sequenciamento de nova geração (NGS) tem trazido avanços no entendimento de características complexas no melhoramento animal, com o sequenciamento completo do genoma dos animais domésticos foi possível melhor entender como variantes causais e estruturais tem influenciado o fenótipo. Com o uso do NGS é possível quantificar o mRNA que está sendo expresso de um determinado gene que influencia determinada característica (transcriptoma RNA-Seq), descobrir alterações epigenéticas (ChIP-Seq) e, então, conhecer melhor os mecanismos moleculares que modulam a característica e tecido em questão; aumentar a densidade de marcadores de nucleotídeos simples (SNP) com o sequenciamento do genoma completo (WGS) e então, descobrir associações genômicas mais significativas e mais precisas. Com o advento do NGS veio a necessidade de o desenvolvimento poder computacional e novas ferramentas capazes de analisar e armazenar a quantidade de dados gerada. Em função disso, o desenvolvimento de softwares capazes de realizar o controle de qualidade, mapeamento das sequencias contra o genoma de referencia e, quantificar os transcritos (no caso de RNASeq) tem se tornado necessário e, lidar com esse tipo de análise pode ser difícil para aqueles que não tem familiaridade com sistemas computacionais. Visto isso foi desenvolvido um pipeline de uso amigável para analisar dados de RNA-Seq, realizando controle de qualidade, mapeamento e contagem de sequencias. O BAQCOM (Bioinformatics Analysis of Quality Control and Mapping) tem se mostrado eficiente e rápido. Utilizando dados de RNA-Seq objetivou-se conhecer o perfil global de expressão gênica envolvido em Condronecrose Bacteriana com Osteomielite em frangos (BCO), que é desenvolvida nas placas de crescimento ósseo do fêmur e tíbia e colonizada por bactérias oportunistas. Utilizando amostras de tíbia de seis frangos de linhagem comercial (três afetados por BCO e três sadios), foram encontrados 192 genes diferencialmente expressos (FDR < 0.05) divididos em 63 upregulados e 129 downregulados. 26 genes e sete fatores de transcrição foram encontrados downregulados explicando BCO em tíbia, podendo concluir que a BCO em frangos pode ser causada pela baixa expressão de genes relacionados ao crescimento ósseo e, que, a proliferação bacteriana parece ser um processo secundário. Outro cenário para o uso de NGS é o mapeamento fino de região de QTL (quantitative Traits Loci). Uma região de QTL no cromossomo 5 associada com espessura de toucinho foi encontrada em quatro linhagens comercial de suínos (sintético baseado em large-white, Pietrain, Landrace e Large-White). Espessura de toucinho é importante para eficiência alimentar e qualidade de carne, além de ser reservatório energético. Embora Genome Wide Association Study (GWAS) tem sido amplamente usada associada à fenótipos, ainda há a necessidade de reduzir a região de associação se o objetivo é achar mutações causais. Por essa razão objetivou-se reduzir esta região de QTL usando dados de WGS, RNA-Seq e ChIP-Seq. A partir do SNP mais significativo do GWAS (leadSNP SSC5:66103958), os haplótipos foram postos em fase e selecionados haplótipos de tamanho 41 SNPs (20 < leadSNP > 20). Foi selecionado o haplótipo mais frequente entre as linhagens utilizadas, então foi possível identificar uma região de 5 SNPs (2 < leadSNP > 2) presente em todas as linhagens. A partir desta região foi realizado o mapeamento fino, primeiro foi utilizado WGS e foi identificado três variantes candidatas (SSC5:66097445, SSC5:66099282 e SSC5c66103958). Nesta região entre SSC5:66097445-66103958 foram encontradas marcas epigenéticas H3K27me3 e H3K4me3 utilizado dados de ChIP-Seq de macrófagos alveolar de suínos, indicando regulação de expressão gênica durante período de desenvolvimento pre-natal. Em função disso foram utilizados RNA-Seq de embriões e fetos, onde foi possível encontrar alta expressão do gene CCND2, que está relacionado ao desenvolvimento e diferenciação de tecido adiposo, sendo um forte candidato a gene causal para espessura de toucinho. É possível, também, concluir que nesta região pode ter elementos regulatórios envolvidos na expressão do gene CCND2 no desenvolvimento embrionário e que, o impacto epigenético durante a vida embrionária pode impactar a produção de características na vida adulta. Nessa tese foi usado dados de sequenciamento, genotipagem, fenótipo, transcriptoma e ChIP-Seq, integrando todos para estreitar regiões de interesse no genoma, assim, resultando e propondo ferramentas para melhorar o melhoramento animal no futuro.

In Animal Breeding there is a history of using phenotypic data of the best animals from one generation to produce the next one. However, it is known that the heritability of interest traits depends on a genetic basis and can be explained by the DNA. In quantitative genetics, the basic model says that the phenotype is the sum of the genotype (genetic composition of the individual) and the environment, however, different genotypes can perform superior or inferior results depending on the environment in which they are inserted, with that, there was the need to include an interaction factor at the genotype and the environment. The development of Next Generation Sequencing (NGS) has brought advances in the understanding of complex traits in animal breeding, with the Whole Genome Sequencing (WGS) of the domestic animals it was possible to better understand how causal and structural variants have influenced the phenotype. With the use of NGS it is possible to quantify the mRNA that is being expressed of a certain gene that influences a certain trait (transcriptome - RNA-Seq), discover epigenetic changes (ChIP-Seq) and, then, better understand the molecular mechanisms that modulate the trait and tissue in question; increase the density of Simple Nucleotide Polymorphism Markers (SNP) with WGS and then discover more significant and more accurate genomic associations. With the advent of NGS came the need for the development of computational power and new tools capable of analyzing and storing the amount of data generated. As a result, the development of software capable of performing quality control, mapping sequences against the reference genome and quantifying the transcripts (in the case of RNA-Seq) has become necessary and, dealing with this type of analysis can be difficult for those unfamiliar with computer systems. For this reason, a userfriendly pipeline was developed to analyze RNA-Seq data, carrying out quality control, mapping and sequence counting. BAQCOM (Bioinformatics Analysis of Quality Control and Mapping) has been shown to be efficient and fast. Using RNA-Seq data, it was aimed to know the global gene expression profile involved in Bacterial Chondronecrosis with Osteomyelitis (BCO) in chicken, which is developed in the bone growth plates of the femur and tibia followed by opportunistic bacteria. Using tibia samples from six commercial broilers (three affected by BCO and three healthy), 192 differentially expressed genes (FDR <0.05) were found (63 upregulated and 129 downregulated). 26 genes and seven transcription factors were found downregulated, explaining BCO in tibia, concluding that BCO in chickens may be caused by the low expression of genes related to bone growth and that bacterial proliferation seems to be a secondary process. Another scenario for the use of NGS is the fine mapping of QTL (quantitative Traits Loci) regions. A QTL region on chromosome 5 associated with backfat thickness was found in four commercial pig lines (synthetic based on large-white, Pietrain, Landrace and Large-White). Backfat is important for feed efficiency and meat quality, as well as being a storage of energy. Although the Genome Wide Association Study (GWAS) has been widely used in association with phenotypes, there is still a need to reduce the associated region if the goal is to find causal mutations. For this reason, it was aimed to reduce this QTL region using data from WGS, RNA-Seq and ChIPSeq. From the most significant SNP of GWAS (leadSNP - SSC5:66103958), haplotypes were phased and haplotypes of size 41 SNPs (20 20) were selected. The most frequent haplotype was selected among the four breeds, so it was possible to identify a region of 5 SNPs (2 2) cross breed. Fine mapping was carried out using this region, first WGS was used and three candidate variants were identified (SSC5:66097445, SSC5:66099282 and SSC5:66103958). In this region between SSC5:66097445-66103958 epigenetic marks H3K27me3 and H3K4me3 were found using ChIP-Seq data from pig alveolar macrophages, indicating regulation of gene expression during the prenatal development period. From this result, RNA-Seq from embryos and fetuses were used, where it was possible to find high expression of the CCND2 gene, which is related to the development and differentiation of adipose tissue, being a strong candidate for a causal gene for backfat. It is also possible to conclude that in this region there may be regulatory elements involved in the expression of the CCND2 gene in embryonic development and that the epigenetic impact during embryonic life can impact the production of traits in adult life. In this thesis, sequencing, genotyping, phenotype, transcriptome and ChIP-Seq data were used, integrating them all to narrow down regions of interest in the genome, thus resulting and proposing tools to improve animal breeding in the future.

DETECÇÃO DE VARIANTES GERMINATIVAS NO PLASMA DE CADELAS COM NEOPLASIAS MAMÁRIAS POR SEQUENCIAMENTO DE NOVA GERAÇÃO

O câncer de mama é uma das neoplasias malignas mais incidentes em mulheres e cadelas. Em mulheres, sabe-se que mutações patogênicas herdadas nos genes de alta penetrância podem estar associadas ao risco elevado da doença, à resistência a terapias e ao prognóstico reservado ou ruim. Em cadelas, existem muitos dados divergentes sobre o envolvimento dos genes de alta penetrância (BRCA1 e BRCA2) com a carcinogênese mamária e sobre a verdadeira patogenicidade das variantes detectadas. Dessa forma, o objetivo principal deste estudo foi detectar variantes germinativas nos genes de alta e moderada penetrância associados ao câncer de mama em mulheres no plasma de cadelas por meio do sequenciamento de nova geração (NGS). Para tanto, foram coletadas vinte amostras de sangue provenientes de dez cadelas recém diagnosticadas com neoplasias de glândula mamária (RD), cinco cadelas mastectomizadas (MAST) e cinco cadelas sem neoplasia (CTR). Posteriormente, foram realizadas as etapas de extração do DNA livre circulante (plasmático), construção da biblioteca utilizando um painel de genes específicos associados ao câncer de mama em mulheres, incluindo os genes BRCA1, BRCA2, CDH1, STK11, TP53, PALB2 e ATM, e, por fim, análise bioinformática para detecção de variantes: polimorfismos de nucleotídeo único (SNPs), deleções e inserções (indels). Foram detectadas 218 variantes no total, sendo 32,56% (n=71) em regiões codificantes e 13,76% (n=30) classificadas como de moderado impacto, a maioria do tipo missense. Dentre os genes de alta penetrância, o gene BRCA2 foi o que mais apresentou variantes, sendo identificados onze SNPs e dois indels, dessas, destacaram-se p.Thr1425Pro e p.Leu2307del, que foram classificadas como possivelmente patogênicas pelos programas de predição in silico. Em relação aos genes de moderada penetrância, foi identificado um quantitativo menor de variantes, quando comparados aos genes de alta penetrância e, dentre os genes avaliados, o PALB2 merece evidência, pois foram detectadas duas novas variantes possivelmente patogênicas: p.Ala949Val e p.Pro590_Val595delinsLeu. Assim, o sequenciamento de nova geração permitiu identificar grande quantidade de variantes, ainda de significado incerto, nos genes de predisposição ao câncer de mama, e essa condição parece ser semelhante em mulheres. São necessários estudos adicionais para validar as variantes identificadas em cadelas com câncer de mama para que, futuramente, seja possível proporcionar tratamento personalizado às pacientes.

Breast cancer is one of the most common malignant tumors in women and dogs. In women, pathogenic mutations inherited in high penetration genes are associated with increased risk of the disease, resistance to therapies and poorer prognosis. In dogs, there are many divergent data involving high penetration genes (BRCA1 and BRCA2) role in breast carcinogenesis and on true pathogenicity of the detected variants. Therefore, the main objective of this study was to detect germline variants in the genes of high and moderate penetrance associated with breast cancer in women in female dogs plasma using next generation sequencing (NGS). Blood from 20 female dogs were sampled: 10 newly diagnosed with mammary gland neoplasms (ND), 5 mastectomized (MAST) and 5 without neoplasm (CTR). Subsequently, the steps for extracting free circulating (plasmatic) DNA were performed, building the library using a panel of specific genes associated with breast cancer in women, including the genes BRCA1, BRCA2,CDH1, STK11, TP53, PALB2 and ATM, and, at last, bioinformatics analysis for variant detection: single nucleotide polymorphisms (SNPs), and deletions and insertions (indels). 218 variants were detected, 32.56% (n=71) in coding regions, and 13.76% (n = 30) classified as moderate impact, mostly the missense type. Among the high penetration genes, the BRCA2 gene was the one with the most variants 11 SNPs and 2 indels being identified and the variants p.Thr1425Pro and p. Leu2307del stand out, being classified as possibly pathogenic by in silico prediction programs. Regarding the genes of moderate penetrance, a smaller number of variants were identified when compared to the genes of high penetrance, and, among the evaluated genes, PALB2 deserves attention, as 2 new possibly pathogenic variants were detected: p.Ala949Val and p.Pro590_Val595delinsLeu. Thus, new generation sequencing allowed the identification of a large number of variants, still of uncertain significance, in the breast cancer predisposition genes and this condition appears to be similar in women. Additional studies are needed to validate the identified variants in female dogs patients with breast cancer in order to provide them with personalized treatment in the future.