Massively parallel whole transcriptome sequencing and its ability to generate full transcriptome data at the single transcript level provides a powerful tool with multiple interrelated applications, including transcriptome reconstruction, gene/isoform expression estimation, also known as transcriptome quantification. As a result, whole transcriptome sequencing has become the technology of choice for performing transcriptome analysis, rapidly replacing array-based technologies. The most commonly used transcriptome sequencing protocol, referred to as RNA-Seq, generates short (single or paired) sequencing tags from the ends of randomly generated cDNA fragments. RNA-Seq protocol reduces the sequencing cost and significantly increases data throughput, but is computationally challenging to reconstruct full-length transcripts and accurately estimate their abundances across all cell types. We focus on two main problems in transcriptome data analysis, namely, transcriptome reconstruction and quantification. Transcriptome reconstruction, also referred to as novel isoform discovery, is the problem of reconstructing the transcript sequences from the sequencing data. Reconstruction can be done de novo or it can be assisted by existing genome and transcriptome annotations. Transcriptome quantification refers to the problem of estimating the expression level of each transcript. We present a genome-guided and annotation-guided transcriptome reconstruction methods as well as methods for transcript and gene expression level estimation. Empirical results on both synthetic and real RNA-seq datasets show that the proposed methods improve transcriptome quantification and reconstruction accuracy compared to previous methods.

No description available.

Next Generation Sequencing (NGS) is a new technology that has revolutionized the way we study living organisms. Where previously only a few genes could be studied at a time through targeted direct probing, NGS offers the possibility to perform measurements for a whole genome at once. The drawback is that the amount of data generated in the process is large and extracting useful information from it requires new methods to process and analyze it. The main contribution of this thesis is the development of a novel experimental method coined tagRNA-seq, combining 5’tagRACE, a previously developed technique, with RNA-sequencing technology. Briefly, tagRNA-seq makes it possible to identify the 5’ ends of RNAs in bacteria and directly probe for their type, primary or processed, by ligating short RNA sequences, the tags, to the beginnings of RNA molecules. We used the method to directly probe for transcription start and processing sites in two bacterial species, Escherichiacoli and Enterococcus faecalis. It was also used to study polyadenylation in E. coli, where the ability to identify processed RNA molecules proved to be useful to separate direct and indirect regulatory effects of this mechanism. We also demonstrate how data from tagRNA-seq experiments can be used to increase confidence on the discovery of anti-sense transcripts in bacteria. Analyses of RNA-seq data obtained in the context of these experiments revealed subtle artifacts in the coverage signal towards gene ends, that we were able to explain and quantify based Kolmogorov’s broken stick model. We also discovered evidences for circularization of a few RNA transcripts, both in our own data sets and publicly available data. Designing the tags used in tagRNA-seq led us to the problem of words absent from a text. We focus on a particular subset of these, the minimal absent words (MAWs), and develop a theory providing a complete description of their size distribution in random text. We also show that MAWs in genomes from viruses and living organisms almost always exhibit a behavior different from random texts in the tail of the distribution, and that MAWs from this tail are closely related to sequences present in the genome that preferentially appear in regions with important regulatory functions. Finally, and independently from tagRNA-seq, we propose a new approach to the problem of bacterial community reconstruction in metagenomic, based on techniques from compressed sensing. We provide a novel algorithm competing with state-of-the-art techniques in the field. / <p>QC 20150930</p>

Il periodo di transizione (TP) delle vacche da latte è caratterizzata da disfunzione del sistema immunitario e dalla comparsa di fenomeni infiammatori. La tesi ha presentato una vasta revisione della letteratura seguita da 3 articoli sperimentali. Nel capitolo II sono stati investigati i cambiamenti delle citochine pro-infiammatorie (PIC) nel TP. I livelli di PIC hanno mostrato una elevata variabilità in tarda gravidanza, ma i livelli più alti hanno mostrato un’associazione con i problemi di salute e le prestazioni dopo il parto. Nel capitolo III, l'attività immunitaria di vacche in transizione è stata valutata utilizzando un test ex vivo di stimolazione del sangue con lipopolisaccaridi (WBA) e un test cutaneo alla carragenina. I risultati hanno rivelato che il sistema immunitario è molto sensibile in prossimità del parto. Entrambi i test descrivono i cambiamenti del sistema immunitario durante il TP. Nel capitolo IV, è stata valutata l’espressione genica dei leucociti durante il TP con la tecnica dell’ RNA-Seq. Confrontando i geni differenzialmente espressi con i risultati del capitolo II e III sono stati resi noti i cambiamenti funzionali dei leucociti. Complessivamente, queste ricerche contribuiscono a definire meglio la fisiologia della fase di transizione della vacche da latte. / The transition period of dairy cows is characterized by immune dysfunction and inflammatory like conditions. The thesis presented a wide review literature followed by 3 research papers. Chapter II investigated the pattern of changes of pro-inflammatory cytokines (PIC) around parturition and discovered an association with periparturient health status. PIC levels showed a high variability in late pregnancy but the highest levels demonstrated a good relationship with health troubles and performance after calving. In Chapter III, immune activity of transition cows were evaluated using: an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test. Results revealed that immune system is very sensitive around calving in respect to both tests, with a significant increase of pro-inflammatory cytokines and a reduction of the skin thickness after carrageenan challenge. Thus, both tests are able to describe the complex changes of the immune system combined to conventional metabolic and immune parameters. In Chapter IV, changes of leukocyte gene expression were evaluated from 20 days before to 7 days after calving using RNA-seq technique. Comparing the differentially expressed genes with the results of Chapter II and III were disclosed fundamental functional changes in leukocytes. Overall, these researches contribute to define better the physiology of the most vulnerable phase of dairy cows.

The recent advent of High-Throughput Sequencing (HTS) methods has triggered a revolution in gene regulation studies. Demand has never been higher to process the immense amount of emerging data to gain insight into the regulatory mechanisms of the cell. We address this issue by describing methods to analyze, integrate and interpret HTS data from different sources. In particular, we developed and benchmarked Pyicos, a powerful toolkit that offers flexibility, versatility and efficient memory usage. We applied it to data from ChIP-Seq on progesterone receptor in breast cancer cells to gain insight into regulatory mechanisms of hormones. Moreover, we embedded Pyicos into a pipeline to integrate HTS data from different sources. In order to do so, we used data sets from ENCODE to systematically calculate signal changes between two cell lines. We thus created a model that accurately predicts the regulatory outcome of gene expression, based on epigenetic changes in a gene locus. Finally, we provide the processed data in a Biomart database to the scientific community. / La llegada reciente de nuevos métodos de High-Throughput Sequencing (HTS) ha provocado una revolución en el estudio de la regulación génica. La necesidad de procesar la inmensa cantidad de datos generados, con el objectivo de estudiar los mecanismos regulatorios en la celula, nunca ha sido mayor. En esta tesis abordamos este tema presentando métodos para analizar, integrar e interpretar datos HTS de diferentes fuentes. En particular, hemos desarollado Pyicos, un potente conjunto de herramientas que ofrece flexibilidad, versatilidad y un uso eficiente de la memoria. Lo hemos aplicado a datos de ChIP-Seq del receptor de progesterona en células de cáncer de mama con el fin de investigar los mecanismos de la regulación por hormonas. Además, hemos incorporado Pyicos en una pipeline para integrar los datos HTS de diferentes fuentes. Hemos usado los conjuntos de datos de ENCODE para calcular de forma sistemática los cambios de señal entre dos líneas celulares. De esta manera hemos logrado crear un modelo que predice con bastante precisión los cambios de la expresión génica, basándose en los cambios epigenéticos en el locus de un gen. Por último, hemos puesto los datos procesados a disposición de la comunidad científica en una base de datos Biomart.

No description available.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

O trigo (Triticum aestivum L.) é uma importante cultura no Brasil. Poucas cultivares são recomendadas para produção do tipo sequeiro no Bioma Cerrado onde a escassez de água limita o rendimento de grãos. Aqui reportamos uma análise de transcriptoma do MGS1 Aliança (cultivar de trigo adaptada ao Cerrado) sob estresse de seca. Um grupo de 4.422 transcritos diferencialmente expressos foi encontrado em raízes e folhas. O número de transcritos reprimidos em raiz (1.102) foi menor que os transcritos induzidos (1.706), enquanto o oposto ocorreu em folhas (1,017 induzidos e 647 reprimidos). O número de transcritos comuns entre ambos órgaõs foi 1.249, enquanto 2.124 foram específicos para raíz e 1.049 específicos para folhas. Análises de RT-qPCR de 35 transcritos selecionados ao acaso revelou uma correlação de 0,78 com os dados de transcriptoma. Os transcritos diferencialmente expressos foram distribuídos por todos os cromossomos e componentes do genoma. O número de transcritos no genoma B foi maior do que nos genomas A e D. Ainda, um grande número de transcritos relacionados à seca foi mapeado nos cromossomos 3B, 5B e 2B. Quando consideramos ambos órgãos, 116 diferentes rotas metabólicas foram alteradas. Uma rota em comum, entre as três mais alteradas em ambos órgãos, foi o metabolismo do amido e da sacarose. A comparação de transcritos derivados de raiz e de folha permite a identificação de transcritos importantes relacionados à respota ao estresse de seca em cada um destes órgãos. Os dados obtidos, também, abrem caminho para o desenvolvimento de futuros marcadores e seleção de genes candidatos ligados à característica. Estes resultados são úteis para o entendimento de rotas metabólicas envolvidas na tolerância à seca em trigo. A informação gerada será usada, a mais longo prazo, para propósitos de transgenia. Para isto, a metodologia de duplo-haploides é desejável e uma primeira investigação sobre a eficiência de protocolo se mostrou necessária. Micrósporos são células gaméticas com capacidade de dar origem a uma nova planta via embriogênese in vitro. Plantas duplo-haploides geradas pela cultura de micrósporos isolados são completamente homozigotas e representam uma importante ferramenta para estudos genéticos e melhoramento de plantas O processo androgenético é desencadeado por diferentes pré-tratamentos de estresse, os quais são empregados para mudar os micrósporos da rota gametofítica para a rota esporofítica. Embora a cultura de micrósporos isolados tenha inúmeras vantagens, importantes limitações tem impedido sua apliação em larga escala. Diferenças genotípicas na resposta androgenética e na formação de plantas albinas ainda constituem desafios. Embora o albinismo seja principalmente uma característica genética, pré-tratamentos e meios de cultura apropriados podem evitar este fenômeno até certo ponto. A resposta androgenética de cinco genótipos de trigo brasileiro foi avaliada no presente estudo. Dois pré-tratamentos foram testados: frio (4°C) e ácido 2-hidroxinicotinico (100 mg/L). O frio foi melhor que o pré-tratamento químico, produzindo mais plantas verdes em quatro de cinco genótipos. Somente dois genótipos brasileiros tratados com ácido 2-hidroxinicotinico produziram plantas, e um deles apenas uma única planta albina. Nossos reultados mostram, também, que o meio semilíquido (contendo 10% de Ficoll) promoveu uma maior resposta androgenética que o meio líquido, aumentando o número de embriões e plantas regeneradas. / Wheat (Triticum aestivum L.) is an important crop cultivated in Brazil. Few cultivars are recommended for rainfed production in the Cerrado Biome where water scarcity limits grain yield. Here we report a transcriptome analysis of MGS1 Aliança (a wheat cultivar adapted to the Cerrado) under drought stress. A set of 4,422 differentially expressed transcripts was found in roots and leaves. The number of down-regulated transcripts in roots (1,102) was lower than the up-regulated transcripts (1,706), while the opposite occurred in leaves (1,017 induced and 647 repressed). The number of common transcripts between the two tissues was 1,249, while 2,124 were specific to roots and 1,049 specific to leaves. Quantitative RT-PCR analysis of 35 randomly selected transcripts revealed a 0.78 correlation with the transcriptome data. The differentially expressed transcripts were distributed across all chromosomes and component genomes. The number of transcripts on the B genome was greater than on the A and D genomes. Additionally, a greater number of drought related transcripts was mapped on chromosomes 3B, 5B and 5D. When considering both tissues, 116 different metabolic pathways were changed. One common pathway, among the top three changed pathways in both tissues, was starch and sucrose metabolism. The comparison of root- and leaf-derived transcripts allows the identification of important transcripts related to water stress response in each of these tissues. It also paves the way for future marker development and selection of candidate genes linked to that trait. These results are useful for understanding the metabolic pathways involved in wheat drought response. The information generated will be used for transgenic wheat purposes. For this the doubled-haploid method is desirable and an investigation about the protocol eficiency is needed. Microspores are gametic cells with capacity to give rise to a new plant via in vitro embryogenesis. Doubled haploid plants generated by isolated microspore culture are completely homozygous and represent an important tool for plant genetics and breeding research. This process is triggered by different stress pretreatments, which are employed to switch microspores from gametophytic to a sporophytic pathway. Although isolated microspore culture has innumerous advantages, important limitations have prevented its application on a large scale. Genotypic differences in androgenic response and the formation of albino plants remain great challenges. Although albinism is a major genetic characteristic, appropriated pretreatments and culture medium can avoid this phenomenon to some extent. The androgenic response of five Brazilian wheat genotypes was evaluated in the present study. Two pretreatments were tested: cold (4°C) and 2-hydroxynicotinic acid (100 mg/L). Cold was better than chemical pretreatment, producing more green plants in four out of five genotypes. Only two Brazilian genotypes treated with 2-hydroxynicotinic acid produced plants, and one of them produced a single albino plant. Our results also show that semi-liquid medium (containing 10% Ficoll) promoted a higher androgenic response than did liquid medium, increasing the number of embryos and regenerated plants.

Life history traits affect the timing and pattern of maturation, reproduction, and survival during an organism’s lifecycle and are the major components influencing Darwinian fitness. Co-evolved patterns of these traits are known as life history strategies (LHS) and variation occurs between individuals, populations, and species. The polymorphisms underlying LHS are important targets of natural selection, yet the underlying genes and physiological mechanisms remain largely unknown. Mapping the genetic basis of a LHS and subsequently unraveling the associated physiological mechanisms is a challenging task, as complex phenotypes are often polygenic. However, in several systems discrete LHS are maintained within the population and are inherited as a single locus with pleiotropic effects. These systems provide a promising starting point for investigation into LHS mechanisms and this thesis focuses on one such strategy - the Alba polymorphism in Colias butterflies. Alba is inherited as a single autosomal locus, expressed only in females, and simultaneously affects development rate, reproductive potential, and wing color. Alba females are white, while the alternative morph is yellow/orange. About 28 of 90 species exhibit polymorphic females, though whether the Alba mechanism and associated tradeoffs are conserved across the genus remains to be determined. In this thesis I primarily focus on the species Colias croceus and integrate results from lipidomics, transcriptomics, microscopy, and genomics to gain insights to the proximate mechanisms underlying Alba and Alba’s evolution within the genus. Lipidomics confirm that, consistent with findings in New World species, C. croceus Alba females have larger abdominal lipid stores than orange, an advantage which is temperature dependent and arises primarily due to mobilized lipids. Gene expression data suggests differences in resource allocation, with Alba females investing in reproduction rather than wing color, consistent with previous findings in other Colias species. Additionally, I identify a morphological basis for Alba’s white wing color. Alba females from C. croceus, an Old World species, and Colias eurytheme, a New World species both exhibit a significant reduction in pigment granules, the structures within the wing scale that contain pigment. This is a trait that seems to be unique to Colias as other white Pierid butterflies have an abundance of pigment granules, similar to orange females. I also map the genetic basis of Alba to a single genomic region containing an Alba specific, Jockey-like transposable element insertion. Interestingly this transposable element​ is located downstream of BarH-1, a gene known to affect pigment granule formation in Drosophila. Finally, I construct a phylogeny using a global distribution of 20 Colias species to facilitate investigations of Alba’s evolution within the genus. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p>

Hypoxia pathways are associated with the pathogenesis of both ischaemic and neoplastic diseases. In response to hypoxia the transcription factor hypoxia‐inducible factor (HIF) induces the expression of hundreds of genes with diverse functions. These enable cells to adapt to low oxygen availability. To date, pan-genomic analyses of these transcriptional responses have focussed on protein-coding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. My thesis aimed at improving understanding of the transcriptional regulation of the non-coding transcriptome in hypoxia. I performed an integrated genomic analysis of both non-coding and coding transcripts by massively parallel sequencing. This was interfaced with pan-genomic analyses of DNAse hypersensitivity and HIF, H3k4me3 and RNApol2 binding in hypoxic cells. These analyses have revealed that hypoxia profoundly regulated all RNA classes. snRNAs and tRNAs are globally downregulated in hypoxia, whilst miRNAs, mRNAs and lncRNAs are both up- and downregulated with an overall trend towards slight upregulation. In addition, a significant number of previously non-annotated (and largely hypoxia upregulated) transcripts were identified, including novel intergenic transcripts and natural antisense transcripts. HIF bound close to genes for mRNAs, miRNAs and lncRNAs that were upregulated by hypoxia, but was excluded from binding at genes for RNA classes that showed global downregulation. This suggests that HIF acts as a transcriptional activator (but not repressor), of lncRNAs as well as mRNAs and miRNAs. Consistent with direct regulation by HIF, many of these hypoxia-inducible, HIF-binding lncRNAs were downregulated following HIF knockdown. Analysis of RNApol2 binding and DNAse HSS signals at HIF transcriptional target genes indicated that HIF-dependent transcriptional activation occurs through release of RNApol2 that is pre-bound to open promoters of lncRNAs as well as mRNAs. In these datasets, NEAT1 was the most hypoxia-upregulated, HIF-targeted lncRNA in MCF-7 cells and, despite binding of both HIF-1 and HIF-2 isoforms at its promoter, was selectively regulated by HIF-2 alone. Furthermore, NEAT1 was induced by hypoxia in a wide range of breast cancer cell lines and in hypoxic xenograft models. Functionally, NEAT1 is required for the assembly of nuclear paraspeckle structures. Increased nuclear paraspeckle formation was observed in hypoxia and was dependent on both NEAT1 and HIF-2. Knockdown of hypoxia-induced NEAT1 significantly reduced cell proliferation and survival and induced apoptosis. Finally, high expression of NEAT1 correlated with poor clinical outcome in a large cohort of breast cancer patients. These findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts and provide new insights into molecular roles of hypoxia-regulated lncRNAs, which may provide the basis for novel therapeutic targets in the future.