RNA sequencing (RNA-seq) technologies facilitate the characterisation of genes and transcripts in different cell types as well as their expression analysis across various conditions. Due to its ability to provide in-depth insights into transcription and post-transcription mechanisms, RNA-seq has been extensively used in functional genetics and transcriptomics, system biology and developmental biology in animals, plants, diseases, etc. The aim of this project is to use mathematical and computational models to integrate big genomic and transcriptomic data from high-throughput technologies in plant biology and develop new methods to identify which genes or transcripts have significant expression variation across experimental conditions of interest, then to interpret the regulatory causalities of these expression changes by distinguishing the effects from the transcription and alternative splicing. We performed a high resolution ultra-deep RNA-seq time-course experiment to study Arabidopsis in response to cold treatment where plants were grown at 20^oC and then the temperature was reduced to 4^oC. We have developed a high quality <i>Arabidopsis thaliana</i> Reference Transcript Dataset (AtRTD2) transcriptome for accurate transcript and gene quantification. This high quality time-series dataset was used as the benchmark for novel method development and downstream expression analysis. The main outcomes of this project include three parts. i) A pipeline for differential expression (DE) and differential alternative splicing (DAS) analysis at both gene and transcript levels. Firstly, we implemented data pre-processing to reduce the noise/low expression, batch effects and technical biases of read counts. Then we used the limma-voom pipeline to compare the expression at corresponding time-points of 4^oC to the time-points of 20^oC. We identified 8,949 genes with altered expression of which 2,442 showed significant DAS and 1,647 were only regulated by AS. Compared with current publications, 3,039 of these genes were novel cold-responsive genes. In addition, we identified 4,008 differential transcript usage (DTU) transcripts of which the expression changes were significantly different to their cognate DAS genes. ii) A TSIS R package for time-series transcript isoform switch (IS) analysis was developed. IS refers to the time-points when a pair of transcript isoforms from the same gene reverse their relative expression abundances. By using a five metric scheme to evaluate robustly the qualities of each switch point, we identified 892 significant ISs between the high abundance transcripts in the DAS genes and about 57% of these switches occurred very rapidly between 0-6h following transfer to 4^oC. iii) A RLowPC R package for co-expression network construction was generated. The RLowPC method uses a two-step approach to select the high-confidence edges first by reducing the search space by only picking the top ranked genes from an initial partial correlation analysis, and then computes the partial correlations in the confined search space by only removing the linear dependencies from the shared neighbours, largely ignoring the genes showing lower association. In future work, we will construct dynamic transcriptional and AS regulatory networks to interpret the causalities of DE and DAS. We will study the coupling and de-coupling of expression rhythmicity to the Arabidopsis circadian clock in response to cold. We will develop new methods to improve the statistical power of expression comparative analysis, such as by taking into account the missing values of expression and by distinguishing the technical and biological variabilities.

Cancer is the collective term used to describe a diverse group of diseases that share certain hallmarks, which in turn enables the affected cells to sustain an uncontrolled cell growth. Despite the increasing efforts and advances in cancer therapies, cancers are still responsible for approximately 10% of all the deaths worldwide. Furthermore, the increase in the average human lifespan will further contribute to the cancer incidences. This brings the necessity to focus our efforts on early detection and effective diagnosis methods. With the advances in high-throughput genomics technologies, gene expression signatures have gained attention as a novel method in cancer diagnostics. These signatures are identified by simply comparing the expression levels of genes in tumor and control samples. Here, we propose an alternative method based on the probe expression level measurement of 3&rsquo / UTR of candidate genes. We chose breast cancer as a model and performed an in silico analysis on publicly available gene expression datasets of Affymetrix chips to analyse 3&rsquo / UTR shortening during breast cancer situation. Overall, our analysis suggests that shortening of 3&rsquo / UTR is a significant mechanism observed in breast cancer .

This project concerns an exploration of the presence and interactions of long non-coding RNA transcripts in an experimental atherosclerosis mouse model with relevance for human abdominal aortic aneurysm development. 187 long noncoding RNAs, two of them entirely novel, were found to be differentially expressed between angiotensin II treated (developing abdominal aortic aneurysms) and non-treated apolipoprotein E deficient mice (not developing aneurysms) harvested after the same period of time. These transcripts were also studied with regards to co-expression network connections. Eleven previously annotated and two novel long non-coding RNAs were present in two significantly disease correlated co-expression groups that were further profiled with respect to network properties, Gene Ontology terms and MetaCore© connections.

RNA interference (RNAi) is a post-transcriptional gene silencing process elicited by double-stranded RNA, such as micro-RNA (miRNA) and small interfering RNA (siRNA). They are 18-25 nucleotide long, small non-coding RNAs acting as critical regulators in eukaryotic genome expression. They play an important role in regulating a wide range of biological processes such as cell cycle control, differentiation, aging and apoptosis. However, their role in supporting skeletal development and bone homeostasis is still poorly understood. Osteoporotic fractures constitute a tremendous and growing problem in our ageing populations, with an annual incidence of approximately 60000 osteoporotic fractures in Sweden. Osteoporosis is referred as the “Silent epidemic” because bone loss is gradual and a basically symptomless development until a fracture occurs. Results presented in this thesis provide a novel insight into crucial roles of   miRNAs in regulating bone homeostasis. The initial aim for the thesis was to perform global miRNA expression profiling in human bone cells, and to correlate these levels to global mRNA levels. We identified and functionally characterized several miRNAs that were differentially expressed and acted in important bone signaling pathways such as the Wnt and BMP pathways. These miRNAs included hsa-miR-29b, hsa-miR-30c2 and hsa-miR-125b, which we found targeting genes highly relevant to bone metabolism e.g. COL1A1, SPARC, RUNX2, BGLAP and FRZB. Thereafter, the effect on the microRNAome upon external stimuli (e.g., Dexamethasone and Parathyroid hormone) was assessed by SOLiD sequencing. We observed a substantial difference in the expression of miRNAs between PTH and DEX treated cells. Understanding the changes in miRNAome in human bone cells under different conditions could provide new insight in bone remodeling, specifically differentiation and functional properties of osteoblasts. Based on these studies, we furthermore identified Dlx5 as potential common target of miR-203 and miR-320b and these miRNAs negatively regulate BMP-2-induced osteoblast differentiation. To activate the RNAi pathway, siRNA or miRNA molecules must be conveyed into the cytoplasm of target cells. Since challenges in cellular delivery of these small silencing RNA molecules so far have limited their clinical utility, we developed a new siRNA design that demonstrates a novel carrier-free cellular delivery. This development could potentially have a major impact in RNAi therapeutics. In conclusion, this thesis provides novel insight of miRNAs that play a major role in the regulation of bone remodeling and differentiation and functional properties of osteoblasts. Our findings may have diagnostic and/or therapeutic implications in disorders of bone metabolism.

Made available in DSpace on 2014-06-11T19:26:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-04Bitstream added on 2014-06-13T18:54:07Z : No. of bitstreams: 1 nicolaujunior_n_me_jabo.pdf: 694144 bytes, checksum: a56e5908ca3860a419359d11194f9013 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O acúmulo de um açúcar com propriedades osmoprotetoras, a trealose (a-D-glicopiranosil-[1,1]-a-D-glicopiranose), é muito comum em microorganismos, invertebrados e em algumas plantas superiores. A trealose é sintetizada a partir da UDP-glicose e glicose-6-fosfato num processo de dois passos onde atuam duas enzimas principais, trealose-6-fosfato sintase ou TPS e trealose-6-fosfato fosfatase ou TPP. A TPS e o seu produto a trealose-6-fosfato (T6P) são prováveis sinalizadores para o metabolismo de carboidratos, contribuindo para o aumento da tolerância de plantas ao estresse hídrico.Este trabalho analisou a expressão in si/ico a partir de 110 transcritos de TPS isolados de bibliotecas de tecidos e tratamentos distintos provenientes do projeto SUCEST. Dois destes genes relacionados a TPS, o STPS 1 e o STPS2 apresentaram expressão diferencial em estudos anteriores em cana-de-açúcar sob estresse hídrico. Em plantas as TPSs foram divididas em duas subfamílias (classe I e 11). Em cana-de-açúcar a STPS1 pertence aos genes de classe I, pois, não possuem domínio fosfatase (TPP) ativo, enquanto que a STPS2 possui domínios TPP ativos (classe 11) determinados pela presença de fosfatase boxes. Análises de expressão, baseadas no método de RT-PCR semiquantitativo, foram realizadas com os genes STPS1 e STPS2 em plantas sensíveis e tolerantes ao déficit hídrico nos períodos iniciais de estresse (24h, 72h e 120h). Os resultados mostram que o gene STPS1 é induzido em cultivares tolerantes de cana-de-açúcar sob estresse e reprimido em plantas sensíveis. Já o gene STPS2 não apresenta variações consideráveis nos níveis de expressão para os mesmos tratamentos. / The accumulation of a sugar with osmoprotectant properties, the trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside), is very common in microorganisms, invertebrates and in some higher plants. Trehalose is synthesized from the UDP-glucose and glucose-6-phosphate in a process of two steps where two main enzymes act, trehalose-6-phosphate synthase or TPS and trehalose-6¬phosphate phosphatase or TPP. The TPS and its product the trehalose-6-phosphate (T6P) are probable signaling molecules in the carbohydrate metabolism, contributing to enhance the plants tolerance to water stress. This work analyzed the in silico expression from 110 transcripts of TPS acquired from different libraries of tissues and treatments proceeding from the SUCEST project. The result showed that the TPS gene is present in ali tissues of the plant. Two of these TPS genes, the STPS1 and the STPS2 showed differential expression in previous studies in sugarcane under water stress. In plants the TPSs are divided in two subfamilies (class I and 11). In sugarcane the STPS 1 belongs to the class I genes, therefore, it does not have an active phosphatase (TPP) domain, whereas, the STPS2 has an active TPP domain (classroom 11) determined by the presence of phosphatase boxes. Expression analyses, based in the semi-quantitative method of the reverse transcription¬polymerase chain reaction (RT-PCR), were performed with the STPS1 and STPS2 genes in water-stress susceptible and tolerant sugarcane cultivars in the initial periods of stress (24h, 72h and 120h). The results show that the STPS1 gene is up¬regulated in the tolerant cultivar under stress and down-regulated in susceptible plants.The STPS2 gene does not show considerable variations in the expression levels under the same treatments.

Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-09-20T18:39:28Z No. of bitstreams: 1 DissAMN.pdf: 2525250 bytes, checksum: d1ef9cb6cbb03453941fe9349c5da36a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-21T12:59:57Z (GMT) No. of bitstreams: 1 DissAMN.pdf: 2525250 bytes, checksum: d1ef9cb6cbb03453941fe9349c5da36a (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-21T13:00:05Z (GMT) No. of bitstreams: 1 DissAMN.pdf: 2525250 bytes, checksum: d1ef9cb6cbb03453941fe9349c5da36a (MD5) / Made available in DSpace on 2016-09-21T13:00:12Z (GMT). No. of bitstreams: 1 DissAMN.pdf: 2525250 bytes, checksum: d1ef9cb6cbb03453941fe9349c5da36a (MD5) Previous issue date: 2014-10-31 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The majority of species in the genus Anastrepha is endemic to the Neotropics and many are of great economic importance because they inflict great damage to several different fruit crops. Brazil has low insertion in international markets because of the demand for quality products with no pesticide residues, which force the improvement of control techniques. Thus, an alternative to insect pests control is the Sterile Insect Technique (SIT), which reduces the pest population by mass release of sterile insects. However, sterilization by radiation has many side effects, reducing the competitiveness of released males relative to wild males. For this reason, different strategies have been used, such as the production of transgenic individuals not only in order to sterility but also increasing the vitality presented by these males compared to wild males. To accomplish that, an important approach could be the study of genes involved in the reproductive process. In this work, we selected nine candidate genes for expression studies of our transcriptome data, which have the potential of being involved in the reproductive process in Anastrepha because they belong to gene families that have already been associated to the reproductive process, and showed differential expression between the contrast of virgin and post-mating of transcriptome data in Anastrepha flies. Since no qPCR study has been done to date in Anastrepha, we tested several reference genes for normalization of expression data. The genes Rpl18, Rps17 and Ef1a were deemed suitable and used here to standardize studies of expression between life stages of A. obliqua. The qPCR gene expression analysis revealed interesting gene expression patterns for AttA, Obp56a and Obp99c, which increases the potential of these genes being involved in the reproductive process. Obp56a showed a higher expression in virgin females in contrast to postmating, whereas AttA and Obp99c showed higher expression in male in contrast to females, which may be interesting for genetic control techniques. We applied the RNAi silencing technique with AttA and Obp99c, aiming to generate information about the participation of these genes in reproduction. Thus, our results pointed for three candidate genes that could be interesting for population control techniques, with potential of being involved in the reproductive process, which stimulates further researches in Anastrepha obliqua. / A maioria das espécies do gênero Anastrepha é endêmica à região neotropical e diversas têm importância econômica por causar grandes prejuízos às culturas de frutos. O Brasil tem baixa inserção no mercado internacional de frutas frescas devido às exigências por produtos de qualidade e sem resíduos de agrotóxicos, o que força o aprimoramento das técnicas de controle de insetospraga. Uma das alternativas é a Técnica do Inseto Estéril (SIT), que visa à redução da população da praga pela liberação em massa de insetos estéreis. Porém, a esterilização por radiação traz muitos efeitos colaterais, diminuindo a competitividade dos machos liberados em relação aos machos selvagens. Por esse motivo, diferentes estratégias têm sido utilizadas como a produção de indivíduos transgênicos visando não só a esterilidade como também o aumento do vigor apresentado por esses machos em relação aos selvagens. Para isso, uma abordagem importante seria o estudo de genes envolvidos no processo reprodutivo. Neste trabalho selecionamos nove genes para estudos de expressão por qPCR candidatos de nossos dados de transcriptomas com potencial de estarem envolvidos no processo reprodutivo em Anastrepha por pertencerem a famílias já relacionadas ao processo reprodutivo, além de apresentarem expressão diferencial entre os transcriptomas de machos virgens e machos pós-cópula. Como nenhum estudo de qPCR havia sido feito ainda em Anastrepha, testamos diversos genes de referência para a normalização dos dados de expressão. Os genes rpl18, rps17 e ef1a foram considerados adequados e padronizados para estudos de expressão entre fases de vida de A. obliqua. As análises de expressão por qPCR revelaram que os genes AttA, Obp56a e Obp99c apresentam padrões de expressão interessantes para investigação e aumentam o potencial desses genes estarem de fato participando do processo reprodutivo. Obp56a apresentou maior expressão em fêmeas virgens em relação às pós-cópula. Já AttA e Obp99c apresentaram maior expressão em machos em relação às fêmeas, fato que pode ser interessante para técnicas de controle genético. Aplicamos a técnica de silenciamento por interferência por RNA nesses genes, com o objetivo de trazer informações sobre a participação desses genes na reprodução. Dessa forma nossos resultados apontam para três genes candidatos que podem ser interessantes para técnicas de controle de populações, com potencial de estar participando do processo reprodutivo o que estimula futuras investigações destes em Anastrepha obliqua.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-27T20:41:15Z No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T18:45:59Z (GMT) No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-04T18:46:07Z (GMT) No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) / Made available in DSpace on 2016-10-04T18:46:15Z (GMT). No. of bitstreams: 1 DissWJSD.pdf: 2131217 bytes, checksum: 65c3deee90f5da124b4a13c80563af29 (MD5) Previous issue date: 2015-08-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Iron (Fe) is an essential micronutrient for cellular homeostasis. Structural component of proteins or enzyme cofactor, Fe has participation in important metabolic pathways that include oxidative metabolism, oxygen transport, cell proliferation and immune system function. Despite of its essentiality, Fe has a toxic potential to cells when in excess. So, a sophisticated system is needed to coordinate the process of absorption, recycling, use and storage. Mutations in genes related to homeostasis of this mineral may potentially alter the cellular distribution and storage. Furthermore, the Fe levels affect biological pathways such as carbohydrate and lipid metabolism. Iron content in cattle muscle has been associated with many sensory and technological parameters of meat quality. However, to date, studies that evaluate how the iron levels in the muscle can alter gene expression and the consequences for the metabolism in cattle are still absent. Therefore, this study aims to identify differentially expressed genes, metabolic pathways, gene interactions and potential regulatory biological mechanisms of physiological processes related to meat quality parameters. Longissimus dorsi (LD) muscle were collected at slaughter for total RNA extraction and determination of CFe by optical emission spectrometry (ICP OES). Eight Nelore steers, who are representatives of extreme value for Genetic Genomic Estimate (GEBV) for iron content (CFe), were selected from a reference population of 373 animals. The sequencing of the total mRNA of extreme animals was carried out from the next generation Illumina technology, which resulted in average l9.13 million of reads per sample after quality control and trimming. Data analysis carried out by Tuxedo Suite pipeline identified 49 annotated and differentially expressed genes (DE) (FDR <0.05) between groups of extremes for GEBV value for CFe. From the DE genes, 18 genes were up-regulated and 31 down-regulated for animals of low GEBV for CFe. Candidate genes for meat quality traits were identified in this study and they are related to transport and lipid metabolism. Other pathways identified through functional enrichment analysis include cell growth and development, function of the hematological system, among others. Canonical signaling pathways (interferon signaling, thyroid receptor activation (TR/RXR) and complement system) and canonical metabolic pathways (biosynthesis of stearate, fatty acid biosynthesis and palmitate biosynthesis) were also identified. Although this study did not identify genes with direct role in the regulation of Fe content, our results suggest biological pathways influenced by this mineral and contribute with information to the understanding of their participation in processes affecting quality of meat. This information will be useful in developing strategies that contribute to the production of better quality meat, healthy and nutritionally rich. In addition, this information may help in understanding of metabolic disorders in other species, including humans. / O ferro (Fe) é um micronutriente essencial à homeostase celular. Necessário como componente estrutural de proteínas ou cofator enzimático, o Fe participa de vias metabólicas importantes que incluem metabolismo oxidativo, transporte de oxigênio, proliferação celular e funcionamento do sistema imune. Apesar de essencial, apresenta um potencial tóxico às células quando em excesso. Por isso, é necessário um sofisticado sistema que coordene os processos de absorção, reciclagem, uso e armazenamento. Mutações em genes relacionados à homeostase desse mineral podem potencialmente alterar a sua distribuição e armazenamento celular. Ademais, os níveis de Fe afetam vias biológicas, tais como metabolismo de carboidratos e lipídeos. O conteúdo de Fe no músculo em bovinos tem sido associado a diversos parâmetros sensoriais e tecnológicos de qualidade de carne. Entretanto, até a presente data, são escassos estudos que avaliem como os níveis de ferro no músculo podem alterar a expressão gênica e quais as consequências para o metabolismo em bovinos. Portanto, o presente estudo tem como objetivo principal identificar genes diferencialmente expressos, vias metabólicas, interações gênicas e potenciais mecanismos biológicos que participam de processos fisiológicos relacionados à regulação do ferro e de parâmetros de qualidade da carne. Amostras do músculo Longissimus dorsi (LD) foram coletadas no momento do abate para extração de RNA total e determinação do conteúdo de ferro (CFe) por espectrometria de emissão óptica (ICP OES). Oito machos castrados da raça Nelore, representantes dos extremos para Valor Genético Genômico Estimado (GEBV) para CFe foram selecionados a partir de uma população referência de 373 animais. O equenciamento do mRNA total dos animais extremos foi realizado a partir da tecnologia de nova geração Illumina, o qual resultou em média 9,13 milhões de reads por amostra após o controle de qualidade. Por meio da análise de dados realizada pelo Tuxedo Suíte pipeline foram identificados 49 genes anotados diferencialmente expressos (DE) (FDR <0,05) entre os grupos de extremos para o valor de GEBV para CFe. Dentre os genes DE, 18 genes apresentaram-se up-regulated e 31 down-regulated para os animais do grupo de baixo GEBV para CFe. Genes candidatos para características de qualidade de carne foram identificados no presente estudo e estão relacionados ao transporte e metabolismo de lipídeos. Outras vias identificadas por meio das análises de enriquecimento funcional incluem crescimento e desenvolvimento celular, função do sistema hematológico, entre outras. Vias canônicas de sinalização (sinalização do interferon, ativação do receptor da tireóide (TR/RXR) e sistema complemento) e metabólicas (biossíntese do estearato, biossíntese de ácidos graxos e biossíntese do palmitato) foram também identificadas. Embora o presente estudo não tenha identificado genes com papel direto na regulação do conteúdo de Fe, nossos resultados apontam rotas biológicas influenciadas por esse mineral e contribui com informações para o entendimento da sua participação em vias que afetem a qualidade da carne. Essas informações serão úteis no desenvolvimento de estratégias que contribuam para a produção de carne de qualidade, saudável e nutricionalmente rica. Além disso, essas informações poderão auxiliar no entendimento de distúrbios metabólicos em outras espécies, inclusive a humana.

Orientadora: Sonia Marli Zingaretti / Banca: Janete Apparecida Desidério Sena / Banca: Miriam Vergínia Lourenço / Resumo: O acúmulo de um açúcar com propriedades osmoprotetoras, a trealose (a-D-glicopiranosil-[1,1]-a-D-glicopiranose), é muito comum em microorganismos, invertebrados e em algumas plantas superiores. A trealose é sintetizada a partir da UDP-glicose e glicose-6-fosfato num processo de dois passos onde atuam duas enzimas principais, trealose-6-fosfato sintase ou TPS e trealose-6-fosfato fosfatase ou TPP. A TPS e o seu produto a trealose-6-fosfato (T6P) são prováveis sinalizadores para o metabolismo de carboidratos, contribuindo para o aumento da tolerância de plantas ao estresse hídrico.Este trabalho analisou a expressão in si/ico a partir de 110 transcritos de TPS isolados de bibliotecas de tecidos e tratamentos distintos provenientes do projeto SUCEST. Dois destes genes relacionados a TPS, o STPS 1 e o STPS2 apresentaram expressão diferencial em estudos anteriores em cana-de-açúcar sob estresse hídrico. Em plantas as TPSs foram divididas em duas subfamílias (classe I e 11). Em cana-de-açúcar a STPS1 pertence aos genes de classe I, pois, não possuem domínio fosfatase (TPP) ativo, enquanto que a STPS2 possui domínios TPP ativos (classe 11) determinados pela presença de fosfatase "boxes". Análises de expressão, baseadas no método de RT-PCR semiquantitativo, foram realizadas com os genes STPS1 e STPS2 em plantas sensíveis e tolerantes ao déficit hídrico nos períodos iniciais de estresse (24h, 72h e 120h). Os resultados mostram que o gene STPS1 é induzido em cultivares tolerantes de cana-de-açúcar sob estresse e reprimido em plantas sensíveis. Já o gene STPS2 não apresenta variações consideráveis nos níveis de expressão para os mesmos tratamentos. / Abstract: The accumulation of a sugar with osmoprotectant properties, the trehalose (a-D-glucopyranosyl-[1,1]-a-D-glucopyranoside), is very common in microorganisms, invertebrates and in some higher plants. Trehalose is synthesized from the UDP-glucose and glucose-6-phosphate in a process of two steps where two main enzymes act, trehalose-6-phosphate synthase or TPS and trehalose-6¬phosphate phosphatase or TPP. The TPS and its product the trehalose-6-phosphate (T6P) are probable signaling molecules in the carbohydrate metabolism, contributing to enhance the plants tolerance to water stress. This work analyzed the in silico expression from 110 transcripts of TPS acquired from different libraries of tissues and treatments proceeding from the SUCEST project. The result showed that the TPS gene is present in ali tissues of the plant. Two of these TPS genes, the STPS1 and the STPS2 showed differential expression in previous studies in sugarcane under water stress. In plants the TPSs are divided in two subfamilies (class I and 11). In sugarcane the STPS 1 belongs to the class I genes, therefore, it does not have an active phosphatase (TPP) domain, whereas, the STPS2 has an active TPP domain (classroom 11) determined by the presence of phosphatase boxes. Expression analyses, based in the semi-quantitative method of the reverse transcription¬polymerase chain reaction (RT-PCR), were performed with the STPS1 and STPS2 genes in water-stress susceptible and tolerant sugarcane cultivars in the initial periods of stress (24h, 72h and 120h). The results show that the STPS1 gene is up¬regulated in the tolerant cultivar under stress and down-regulated in susceptible plants.The STPS2 gene does not show considerable variations in the expression levels under the same treatments. / Mestre

Gramíneas cultivadas, como trigo, cevada e milho são produtos agrícolas de fundamental importância no Brasil. Entre os fatores causadores de perdas na produção de grãos dessas espécies estão os estresses causados por fitopatógenos como Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), agente causador da fusariose e de difícil controle químico, biológico ou mesmo genético. Uma estratégia que tem se mostrado eficiente no controle de doenças é a utilização de microrganismos antagonistas a diferentes fitopatógenos, dentre os quais destaca-se a bactéria P. agglomerans. O presente trabalho teve como objetivo identificar genes diferencialmente expressos em interações fungo fitopatogênico-microrganismo antagonista, considerando como modelo o sistema F. graminearum-P. agglomerans. A construção de uma biblioteca de cDNA de F. graminearum cultivado in vitro proporcionou a geração de 1.983 seqüências válidas, resultando em 1.283 unigenes. As categorias de maior representatividade desta biblioteca foram aquelas constituídas por proteínas envolvidas em vias da informação genética - DNA-RNA-proteína (26 %); proteínas hipotéticas (24 %) e proteínas do metabolismo (16 %). Tanto a categoria de proteínas envolvidas nos processos de desenvolvimento como as envolvidas na percepção a estímulos externos constituíram 10 % dos unigenes. Dentre os genes presumivelmente anotados, foram identificados aqueles codificadores de enzimas de importantes rotas metabólicas como gliceraldeído-3-fosfato-desidrogenase, fosfoglicerato quinases e fosfoenolpiruvato carboxilases, como também componentes produzidos pelo metabolismo secundário como micotoxinas e outras proteínas associadas a estresse e patogenicidade de fungos. Neste trabalho também foi verificado o potencial de antagonismo in vitro da bactéria P. agglomerans frente a três fitopatógenos de trigo: Drechslera tritici-repentis (Died.) Shoem e Bipolaris sorokiniana (Sacc. in Sorok.) e F. graminearum. Foi verificado que a inibição do crescimento destes fungos está associada à liberação de compostos solúveis e voláteis pela bactéria, que foram responsáveis por cerca de 50 % e 40 % de inibição, respectivamente. O perfil da expressão gênica de F. graminearum na interação com a bactéria P. agglomerans foi avaliado via macroarranjo. Dos 1.014 genes avaliados, 29 genes de F. graminearum foram diferencialmente expressos (p < 0,05) durante a interação com a bactéria antagonista, sendo 19 genes induzidos e 10 genes reprimidos. Entre os transcritos induzidos foram identificadas proteínas envolvidas nos processos de defesa e/ou virulência de fungos, cuja expressão foi induzida em resposta a estresses tanto abióticos como bióticos. Dos genes que foram reprimidos, destacaram-se: um transcrito com similaridade a uma proteína com um domínio do tipo dedo de zinco ?zinc finger? que é um fator de transcrição importante no processo de divisão celular, bem como proteínas envolvidas na cadeia respiratória, na modulação protéica e sinalização celular. Os dados do macroarranjo foram validados via transcrição reversa seguida de PCR quantitativo em tempo real (RT-PCRq), metodologia que se mostrou adequada para complementar a análise transcricional obtida por macroarranjo. As informações geradas na análise de antagonismo in vitro, bem como a análise e seqüenciamento dos transcritos, juntamente com a quantificação do nível de expressão na interação, foram fundamentais para compreender o padrão de resposta do fungo F. graminearum na interação com a bactéria P. agglomerans. / Cultivated grasses such as wheat, barley and maize are agricultural products of fundamental economic and social importance in Brazil. Among causing factors of important grain production losses in these species are diseases caused by phytopathogenic fungi such as Fusarium graminearum Schwabe (teleomorfo Gibberella zeae Schw.), the causal agent of fusariosis, a disease of difficult chemical, biological or even genetic control. An efficient and promising strategy to be adopted in order to protect cultivated plants against such diseases is the selection of antagonist microorganisms, amongst them the bacteria Pantoea agglomerans. This microbiota might have an important impact in scab control, isolated or in an integrated management program with chemical treatment. The present work aimed at identifying differentially expressed sequences in pathogenic fungi-antagonistic microorganisms interactions, considering the F. graminearum ? P. agglomerans model. The construction of a cDNA library for F. graminearum grown in PDA medium generated 1,983 valid sequences and provided 1,283 unigenes. The most representative categories in this library were proteins involved in genetic information pathways, DNA-RNA-protein (26 %); hypothetical proteins (24 %); and proteins involved in metabolism (16 %). The protein category involved in developmental processes as well as those related to external stimuli perception comprised 10 % of the obtained unigenes. Among putatively annotated genes, some coding for enzymes of important metabolic routes were identified, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and phophoenolpyruvate carboxylase. Also secondary metabolism compounds, specially micotoxins and proteins related to fungi stresses and pathogenicity were identified. In the present work, the control of three wheat phytopathogens, Drechslera tritici-repentis (Died.) Shoem, Bipolaris sorokiniana (Sacc.in Sorok.) and F. graminearum, using specific isolates of P. agglomerans was demonstrated. It was observed that the 50 % and 40 % growth inhibition of these fungi is associated to the bacteria release of soluble and volatile compounds, respectively. The gene expression profile of F. graminearum during interaction with the bacteria P. agglomerans was evaluated via macroarray. Among the 1,014 analysed genes, 29 F. graminearum genes were differentially expressed (p < 0,05) during its interaction with the antagonist bacteria: 19 genes were induced while 10 genes were repressed. Among the induced transcripts, proteins involved in fungi defense and/or virulence processes were identified, whose expression was induced in reponse to abiotic or biotic stresses. Among the identified repressed genes, a transcript similar to a protein containing a zinc finger-type domain, a transcription factor relevant in cell division, deserves special attention, as well as proteins involved in respiratory chain, in protein modulation and in cell signaling. Additionally, the macroarray data were validated by reverse transcription followed by real-time quantitative PCR (RT-PCRq), a suitable method for complementing transcriptional analysis through macroarray. Finally, the information generated in in vitro pathogenic fungi-antagonistic microorganisms interactions analysis, as well as in the analysis and sequencing of the obtained transcripts, together with the determination of the level of expression during the evaluated interactions were essential for better understanding the response pattern of the fungus F. graminearum in interaction with the bacteria P. agglomerans

We study the tasks of transcript expression quantification and differential expression analysis based on data from high-throughput sequencing of the transcriptome (RNA-seq). In an RNA-seq experiment subsequences of nucleotides are sampled from a transcriptome specimen, producing millions of short reads. The reads can be mapped to a reference to determine the set of transcripts from which they were sequenced. We can measure the expression of transcripts in the specimen by determining the amount of reads that were sequenced from individual transcripts. In this thesis we propose a new probabilistic method for inferring the expression of transcripts from RNA-seq data. We use a generative model of the data that can account for read errors, fragment length distribution and non-uniform distribution of reads along transcripts. We apply the Bayesian inference approach, using the Gibbs sampling algorithm to sample from the posterior distribution of transcript expression. Producing the full distribution enables assessment of the uncertainty of the estimated expression levels. We also investigate the use of alternative inference techniques for the transcript expression quantification. We apply a collapsed Variational Bayes algorithm which can provide accurate estimates of mean expression faster than the Gibbs sampling algorithm. Building on the results from transcript expression quantification, we present a new method for the differential expression analysis. Our approach utilizes the full posterior distribution of expression from multiple replicates in order to detect significant changes in abundance between different conditions. The method can be applied to differential expression analysis of both genes and transcripts. We use the newly proposed methods to analyse real RNA-seq data and provide evaluation of their accuracy using synthetic datasets. We demonstrate the advantages of our approach in comparisons with existing alternative approaches for expression quantification and differential expression analysis. The methods are implemented in the BitSeq package, which is freely distributed under an open-source license. Our methods can be accessed and used by other researchers for RNA-seq data analysis.