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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) analise_transcriptoma_arroz_estresse_frio.pdf: 1983607 bytes, checksum: 89744effdc48619cef6643fb7d1e353c (MD5) Previous issue date: 2014-03-06 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O estresse por frio pode trazer prejuízos econômicos significativos em diversas fases do ciclo da cultura do arroz. Em arroz (Oryza sativa L.), há grande variabilidade genética para tolerância ao frio sendo a subespécie japonica considerada tolerante e a subespécie indica considerada sensível. Foi realizado sequenciamento de RNA (RNA-Seq) visando identificar genes diferencialmente expressos entre os genótipos BRS Atalanta (indica) (A), Nipponbare (japonica) (N) e bulks formados por RILs (Linhas endogâmicas recombinantes) provenientes do cruzamento entre elas. Bulks (B) com as 15 RILs mais próximas a cada genitor (BA e BN) foram formados com base em dados de genotipagem. As plântulas, tanto da condição tratamento quanto da condição controle, permaneceram por 7d a 28°C. Para análise do transcritoma, as plântulas da condição tratamento (T) foram expostas ao frio numa temperatura de 13°C por 24h e as plantas da condição controle (C) permaneceram mais 24h a 28°C. Após este período foi realizada a coleta das plântulas, extração de RNA total, síntese de cDNA e preparo das bibliotecas para sequenciamento (RNA-Seq). Este foi realizado em sequenciador HiSeq 2000, sendo as reads de 100b single-end. Diversas combinações foram analisadas para obter o número de genes diferencialmente expressos envolvendo as condições BRS Atalanta (controle e tratamento), Nipponbare (controle e tratamento), bulk de RILs mais próximos a cultivar BRS Atalanta (controle e tratamento) e bulk de RILs mais próximos ao genótipo Nipponbare (controle e tratamento). Um número significativo de genes mostra-se diferencialmente expressos em cada condição, sendo de modo geral mais genes estão subexpressos do que superexpressos, exceto para as condições AC x BAC, AT x BAT e NT x BNT. Um dendrograma usando as distâncias de Jensen-Shannon mostra a formação de dois ramos sendo que num deles está a cultivar BRS Atalanta e os bulks formados por sua semelhança genotípica com esta cultivar. No outro ramo estão o genótipo Nipponbare e os bulks formados pela semelhança com o genitor. Genes pertencentes a diversas famílias de fatores de transcrição estão diferencialmente expressos nas diferentes combinações, sendo que para grande parte destas famílias já foi demonstrada sua participação na resposta ao estresse por frio. As conclusões deste trabalho indicam que o número de genes diferencialmente expressos (log2-fold-change ≥ |1.5|) varia grandemente entre as combinações, apresentando como limite superior 1481 genes subexpressos na combinação BNC x BAC e 1017 genes superexpressos em NT x AT, e seis genes subexpressos na combinação NT x BNT e cinco genes superexpressos na combinação NC x BNC. As combinações analisadas mostram a expressão diferencial de um grande número de famílias de fatores de transcrição, muitas delas já descritas como responsivas ao estresse por frio, que apresentam um padrão difuso de expressão entre as subespécies indica e japonica. / Chilling stress can cause significant economic losses in different phases of the cycle of rice. There are a wide of genetic variability for cold tolerance in rice (Oryza sativa L.), japonica subspecies is considered tolerant and subspecies indica is considered sensitive. RNA sequencing (RNA-Seq) was performed to identify differentially expressed genes among the cultivar BRS Atalanta (indica) (A), Nipponbare (japonica) (N) and bulks composed by RILs from crosses between them. Bulks (B) with the 15 closest RILs to each parent (BA and BN) RILs were composed based on SNPs-genotyping data. To analyze the transcriptome, plants of the treatment condition (T), 7d after imbibition were exposed to a chilling temperature of 13°C per 24h and plants of the control condition (C) remained 24h more at 28°C. After this time was performed the collection of seedlings, total RNA extraction, cDNA synthesis and preparation of libraries for sequencing (RNA-Seq) in a HiSeq 2000 sequencer, with 100bp single-end reads. Several combinations were analyzed to obtain the number of differentially expressed genes involving BRS Atalanta conditions (control and treatment), Nipponbare (control and treatment), bulk of RILs closest to BRS Atalanta – BA (control and treatment) and bulks of RILs closest to the cultivar Nipponbare - BN (control and treatment). A significant number of differentially expressed genes are shown in each condition, being generally founded more downregulated than up-regulated genes, except for the combinations AC x AT x NT x BAT and BAC x BNT. A dendrogram using the Jensen-Shannon distance shows the formation of two branches of which one is composed by BRS Atlanta cultivar and the bulks composed by the genotypic similarity with this cultivar. In the other branch is the cultivar Nipponbare and the bulks composed by similarity with the parent. Several differentially expressed genes from transcription factors families are present in different combinations, and for many of these families has been demonstrated its involvement in the response to chilling stress. The conclusions of this study indicate that the number of differentially expressed genes (log2 fold-change ≥ |1.5|) greatly changed between combinations, with an upper limit of 1481 down-regulated genes in the combination BAC x BNC and 1017 up-regulated genes in the combination NT x AT, six genes are up-regulate in the combination NT x BNT and five genes are upregulated in NC x BNC combination. The combinations analyzed showed differential expression of a large number of families of transcription factors, many of which have been described as responsive to cold stress, showing a diffuse pattern of expression between indica and japonica groups.

Les ARN sont des molécules aux propriétés diverses interagissant les uns avec les autres dans le but de médier des fonctions spécifiques. L’évaluation de l’abondance relative des ARN est une étape cruciale à la compréhension de la stoechiométrie nécessaire à ces fonctions. Cependant, plusieurs biais et limitations s’imposent avec l’utilisation de différentes techniques d’évaluation, dont le séquençage d’ARN (RNA-Seq), qui sont problématiques dans l’estimation de l’abondance de différents types d’ARN. De récentes études ont exposé les avantages d’un protocole novateur de RNA-Seq qui utilise une rétrotranscriptase thermostable d’intron de groupe II (TGIRT) d’origine bactérienne afin de réduire ces biais. Ce mémoire fait la comparaison entre différentes techniques de RNA-Seq afin d’élucider si l’utilisation de TGIRT en RNA-Seq offre une représentation plus juste du transcriptome. Les comparaisons avec les valeurs d’abondance de différents types d’ARN décrites dans la littérature ainsi qu’obtenues expérimentalement par notre groupe pointent au fait que TGIRT donne une meilleure estimation de l’abondance relative des ARN, et plus particulièrement des ARN hautement structurés. Cette meilleure estimation de l’ensemble de la composition du transcriptome permet de faire des observations sur les rapports d’abondance entre des ARN codants et non-codants fonctionnellement apparentés. Notamment, des ratios d’expression constants entre les ARN non-codants associés à des RNP et les ARNm codants pour leur facteurs protéiques ont été observés. Ceci suggère la présence d’une régulation transcriptionnelle commune nécessaire à la stoechiométrie de ces complexes. Une forte disparité dans l’expression des snoRNA et de leurs gènes hôtes, dépendant du type de snoRNA et de gène hôte a par ailleurs été constatée, corroborant une régulation distincte de la stabilité de ces transcrits. Dans l’ensemble, nos données suggèrent que la méthode TGIRT-Seq est la plus appropriée dans l’évaluation du transcriptome entier et ouvre donc la voie à des analyses plus holistiques par RNA-Seq en donnant une estimation plus juste de l’abondance relative des transcrits d’ARN. / Abstract : RNA are molecules with a wide range of properties that can interact with one another to mediate specific function. The evaluation of RNA abundance is a crucial step in understanding the stoichiometry needed for these functions. However, many limitations and biases come with the use of different techniques, including RNA sequencing (RNA-Seq), which affects the estimation of the abundance of different RNA types. Recent studies have exposed the advantages of a new RNA-Seq protocol using a thermostable group II intron reverse transcriptase (TGIRT) of bacterial origin to reduce these biases. This thesis makes the comparison between different RNA-Seq techniques to elucidate if the use of TGIRT in RNA-Seq offers a more representative depiction of the transcriptome. The comparisons with the abundance values given in literature and obtained experimentally by our group agree with the fact that TGIRT gives a better estimation of the relative abundance of RNA, especially highly structured RNA. This better estimation of the transcriptomic landscape allows many observations on the abundance relations between coding and non-coding RNA that are functionally related. Namely, constant expression ratios between RNP associated noncoding RNA and the mRNA that codes for their associated proteins have been observed. This suggests the presence of a common transcriptional regulation which is necessary for the stoichiometry of these complexes. A strong disparity in the expression of snoRNA and their host genes depending on snoRNA and host gene types has also been observed and corroborate a distinct regulation of these transcripts’ stability. In summary, our data suggest that the TGIRT-Seq method is the most appropriate to evaluate the transcriptome and thus opens the way to more holistic RNA-Seq analyses by giving a better estimation of RNA transcripts relative abundance.

Background: Transcriptome analytic tools are commonly used across patient cohorts to develop drugs and predict clinical outcomes. However, as precision medicine pursues more accurate and individualized treatment decisions, these methods are not designed to address single-patient transcriptome analyses. We previously developed and validated the N-of-1-pathways framework using two methods, Wilcoxon and Mahalanobis Distance (MD), for personal transcriptome analysis derived from a pair of samples of a single patient. Although, both methods uncover concordantly dysregulated pathways, they are not designed to detect dysregulated pathways with up- and down-regulated genes (bidirectional dysregulation) that are ubiquitous in biological systems. Results: We developed N-of-1-pathways MixEnrich, a mixture model followed by a gene set enrichment test, to uncover bidirectional and concordantly dysregulated pathways one patient at a time. We assess its accuracy in a comprehensive simulation study and in a RNA-Seq data analysis of head and neck squamous cell carcinomas (HNSCCs). In presence of bidirectionally dysregulated genes in the pathway or in presence of high background noise, MixEnrich substantially outperforms previous single-subject transcriptome analysis methods, both in the simulation study and the HNSCCs data analysis (ROC Curves; higher true positive rates; lower false positive rates). Bidirectional and concordant dysregulated pathways uncovered by MixEnrich in each patient largely overlapped with the quasi-gold standard compared to other single-subject and cohort-based transcriptome analyses. Conclusion: The greater performance of MixEnrich presents an advantage over previous methods to meet the promise of providing accurate personal transcriptome analysis to support precision medicine at point of care.

Observations of floral development indicate that floral organ initiation in pentapetalous flowers more commonly results in a medially positioned abaxial petal (MAB) than in a medially positioned adaxial petal (MAD), where the medial plane is defined by the stem and the bract during early floral development. It was proposed that the dominant MAB petal initiation might impose a developmental constraint that leads to the evolution of limited patterns of floral zygomorphy in Asteridae, a family in which the floral zygomorphy develops along the medial plane and results in a central ventral (CV) petal in mature flowers. Here, I investigate whether the pattern of floral organ initiation may limit patterns of floral zygomorphy to evolve in pentapetalous angiosperms. I analyzed floral diagrams representing 405 species in 330 genera of pentapetalous angiosperms to reconstruct the evolution of floral organ initiation and the evolution of developmental processes that give rise to floral zygomorphy on a phylogenetic framework. Results indicate that MAB petal initiation is the most common; it occupies 86.2% of diversity and represents the ancestral state of floral organ initiation in pentapetalous angiosperms. The MAD petal initiation evolved 28 times independently from the ancestral MAB petal initiation. Among the 34 independent originations of floral zygomorphy, 76.5% of these clades represent MAB petal initiation, among which only 47% of the clades result a CV petal in mature flowers. The discrepancy is explained by the existence of developmental processes that result in floral zygomorphy along oblique planes of floral symmetry in addition to along the medial plane. Findings suggest that although the early floral organ initiation plays a constraining role to the evolution of patterns of floral zygomorphy, the constraint diverges along phylogenetically distantly related groups that allow the independent originations of floral zygomorphy through distinct development processes in pentapetalous angiosperms. In additional study, the butterfly-like flowers of Schizanthus are adapted to pollination by bees, hummingbirds, and moths. I investigated the genetic basis of the zygomorphic corolla, for which development is key to the explosive pollen release mechanism found in the species of Schizanthus adapted to bee pollinators. I examined differential gene expression profiles across the zygomorphic corolla of Schizanthus pinnatus, a bee-pollinated species, by analyzing RNA transcriptome sequencing (RNA- seq). Data indicated that CYC2 is not expressed in the zygomorphic corolla of Sc. pinnatus, suggesting CYC2 is not involved in the development of floral zygomorphy in Schizanthus (Solanaceae). The data also indicated that a number of genes are differentially expressed across the corolla.

Philosophiae Doctor - PhD / The growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development.

Philosophiae Doctor - PhD / In this dissertation three computational approaches are presented that enable optimization of reference-free transcriptome reconstruction. The first addresses the selection of bona fide reconstructed transcribed fragments (transfrags) from de novo transcriptome assemblies and annotation with a multiple domain co-occurrence framework. We showed that selected transfrags are functionally relevant and represented over 94% of the information derived from annotation by transference. The second approach relates to quality score based RNA-seq sub-sampling and the description of a novel sequence similarity-derived metric for quality assessment of de novo transcriptome assemblies. A detail systematic analysis of the side effects induced by quality score based trimming and or filtering on artefact removal and transcriptome quality is describe. Aggressive trimming produced incomplete reconstructed and missing transfrags. This approach was applied in generating an optimal transcriptome assembly for a South African isolate of V. inaequalis. The third approach deals with the computational partitioning of transfrags assembled from RNA-Seq of mixed host and pathogen reads. We used this strategy to correct a publicly available transcriptome assembly for V. inaequalis (Indian isolate). We binned 50% of the latter to Apple transfrags and identified putative immunity transcript models. Comparative transcriptomic analysis between fungi transfrags from the Indian and South African isolates reveal effectors or transcripts that may be expressed in planta upon morphogenic differentiation. These studies have successfully identified V. inaequalis specific transfrags that can facilitate gene discovery. The unique access to an in-house draft genome assembly allowed us to provide preliminary description of genes that are implicated in pathogenesis. Gene prediction with bona fide transfrags produced 11,692 protein-coding genes. We identified two hydrophobin-like genes and six accessory genes of the melanin biosynthetic pathway that are implicated in the invasive action of the appressorium. The cazyome reveals an impressive repertoire of carbohydrate degrading enzymes and carbohydrate-binding modules amongst which are six polysaccharide lyases, and the largest number of carbohydrate esterases (twenty-eight) known in any fungus sequenced to date

Infectious diseases are the top leading cause of death worldwide. Identifying essential genes, genes indispensable for survival, has been proven indispensable in defining new therapeutic targets against pathogens, major elements of the minimal set genome to be harnessed in synthetic biology, and determinants of evolutionary relationships of phylogenetically distant species. Thus, essentiality studies promise valuable revenues that can decipher much of biological complexities. Taking advantage of the available microbial sequences and the essentiality studies conducted in various microbial models, we proposed a framework for the prediction of essential genes based on our experimentally verified knowledge of the pathways involved in three essential xiv functions: genetic information processing, cell wall biosynthesis, and energy metabolism. We investigated physiological pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) database and developed a bioinformatics approach to predict essential genes in 13 different microbial species. Our in silico findings matched to a high degree the experimental data derived from essentiality studies conducted on the same microbial models, providing insights about the microbial lifestyles, including energy resources, cell wall structure, and ecological preferences, but not virulence tools and mechanisms. Furthermore, we believe that essential genes have survived the evolutionary purifying selection due to their evolved capacity to re-wire genetic and protein networks in response to any emerging stress. In this sense, an environmental specificity (stress) provides a dominant determinant of an essential gene set. The new challenge was understanding the contribution of the essential genome in S. sanguinis to the coping mechanisms to different clinically relevant stress factors, namely temperature elevation (43oC) and sub-inhibitory concentration of ampicillin, an abundantly prescribed antibiotic for prophylaxis and treatment against S. sanguinis. The current project investigated the transcriptomic and proteomic profiles of essential genes and proteins, using RNA-seq and mass spectrometry respectively, under the impact of the two stressors separately, to elucidate the essential genome and proteome dynamics on a temporal basis and define “pathogenesis signatures” as potential therapeutic targets. We believe that the current findings will help characterize a bacterial model for studying the dynamics of essential genes and assist in designing evidence-based guidelines for drug prescription in clinical practice.

Beef quality in Brazil is important for both consumers and the food industry due to high demand and competitiveness in the domestic and international markets. Therefore, it is necessary to develop research to improve beef quality of Nellore cattle (Bos indicus), mainly tenderness, one of the main features to add value to meat. New-generation technologies provide accurate, rapid and inexpensive information on the entire genome, showing great advantage over conventional methods for sequencing and gene expression. However, these new technologies generate large database, which require the use of bioinformatics tools for data analyses of sequencing and for a better understanding of biological regulation mechanisms , cellular control, gene interactions, among other applications. In a previous study, samples were collected from the Longissimus dorsi muscle of 790 animals from Nellore cattle and shear force assessments were made 24 hours after slaughter, with seven and 14 days of aging. Aiming to identify differentially expressed (DE) genes, 34 samples from Nellore animals with extreme levels of estimated breeding value (EBV) for shear force (SF) were selected, sequenced by the method of RNA sequencing (RNA-Seq) (Illumina HiScanSQ). This study performed the processing of data generated by RNA-Seq using software QuasiSeq and Cuffdiff. In the QuasiSeq analysis, 22 DE genes were found, while in the Cuffdiff analysis, 113 DE genes were found. To better understand the biological process involved in meat tenderness, integrative analysis identified possible regulators that can explain the activity of transcriptional regulation in this process using partial correlation coefficient with information theory (PCIT), phenotypic impact factor (PIF) and regulatory impact factor (RIF) methods. The genes found in the PCIT analysis USP2, GBR10, ANO1 and TMBIM4; microRNAs found in RIF analysis bta-mir-133a-2 and bta-mir-22, and the genes with high PIF value MB, ENO3, CA3 could be fundamental to unravel the complex molecular mechanisms that control the meat tenderness in Nellore cattle. / A qualidade da carne bovina no Brasil é importante tanto para o consumidor, como para a indústria alimentícia devido à alta competitividade e exigência do mercado nacional e internacional. Portanto, é necessário o desenvolvimento de pesquisas para melhorar a qualidade da carne bovina da raça Nelore (Bos indicus), principalmente a maciez, que é considerada uma das principais responsáveis por agregar valor à carne. Tecnologias de nova geração proporcionam informações precisas, rápidas e baratas de todo genoma, mostrando grande vantagem em relação aos métodos convencionais de sequenciamento e de estudos de expressão gênica. Essas novas tecnologias geram um grande volume de dados, sendo necessário o uso de ferramentas de bioinformática para realizar as análises de sequenciamento e ter uma maior compreensão de mecanismos biológicos de regulação, controle celular, interações gênicas, entre outras aplicações. Em um estudo prévio, foram coletadas amostras do músculo Longissimus dorsi de 790 animais da raça Nelore e foram realizadas avaliações da força de cisalhamento 24 horas após abate, e com sete e 14 dias de maturação. Com o objetivo de identificar genes diferencialmente expressos (DE), foram selecionadas no total 34 amostras de animais da raça Nelore com valores extremos de valor genético estimado (EBV) para força de cisalhamento (SF), e sequenciados pelo método de sequenciamento de RNA (RNA-Seq) (Illumina HiScanSQ). Neste estudo foi realizado o processamento dos dados gerados pelo RNA-Seq através dos softwares QuasiSeq e Cuffdiff. Foram encontrados 22 genes DE para as análises do QuasiSeq e 113 genes DE para as análises do Cuffdiff. Para melhor compreensão dos processos biológicos envolvidos na maciez da carne, análises integrativas identificaram possíveis reguladores que podem explicar a atividade de regulação transcricional neste processo utilizando os métodos do Coeficiente de Correlação Parcial com Teoria da Informação (PCIT), Fator de Impacto Fenotípico (PIF) e Fator de Impacto Regulatório (RIF). Os genes encontrados nas análises análises do PCIT USP2, GBR10, ANO1 e TMBIM4, assim como os microRNAs encontrados nas análises do RIF, bta-mir-133a-2 e bta-mir-22 e os genes de maior valor de PIF MB, ENO3, CA3 podem ser fundamentais para desvendar os complexos mecanismos moleculares que controlam a maciez da carne na raça Nelore.

Agronomic outcomes are the product of a plant's genotype and its environment. Genomic technologies allow farmers and researchers new avenues to explore the genetic component of agriculture. These technologies can also enhance understanding of environmental effects. With a growing world population, a wide variety of tools will be necessary to increase the agronomic productivity. Here I use massively parallel, deep sequencing of RNA (RNA-Seq) to measure changes in cotton gene expression levels in response to a change in the plant's surroundings caused by conservation tillage. Conservation tillage is an environmentally friendly, agricultural practice characterized by little or no inversion of the soil prior to planting. In addition to changes in cotton gene expression and biological pathway activity, I assay the transcriptional activity of microbial symbiotes living in and around the cotton roots. I found a large degree of similarity between cotton individuals in different treatments. However, under conventional disk tillage I did find significantly greater activity of cotton phosphatase and sulfate transport genes, as well as greater abundance of the microbes Candidatus Burkholderia brachynathoides and Arthrobacter species L77. This study also includes the use of high-throughput physical mapping of DNA to examine the genomic structure of a wild cotton species, Gossypium raimondii, which is closely related to the economically significant crop species Gossypium hirsutum. This technology characterizes genomic regions by assembling large input DNA molecules labeled at restriction enzyme recognition sites. I created an efficient algorithm and generated 812 whole genome assemblies from two datasets. The best of these assemblies allowed us to detect 3,806 potential misassemblies in the current release of the G. raimondii genome sequence assembly.

No description available.