No description available.

Corn (Zea mays) is frequently infected by a soil fungal pathogen Aspergillus flavus. The fungus produces aflatoxins, which cause liver cancer. Maize inbred lines that are resistant to infection by A. flavus have been developed, and these inbred lines provide excellent models for studying molecular mechanisms of maize resistance to the fungus. MicroRNA-like RNAs (milRNAs) recently identified in A. flavus had been found to be correlated with aflatoxin production conditions, suggesting that the milRNAs might play a role in the regulation of aflatoxin production. In this research, small RNAs were isolated from kernels of maize (resistant Mp719 and susceptible Va35) inoculated with A. flavus NRRL 3357 (aflatoxigenic) and NRRL 21882 (nonaflatoxigenic) and then subjected to RNA sequencing. Sequencing had identified 69 A. flavus milRNAs and 691 Z. mays miRNAs. The differential expression of some maize miRNAs revealed their potential role in response to inoculation, A. flavus growth, and aflatoxin production.

No description available.

Growth Hormone (GH), also known as somatotropin, is a protein hormone secreted from the anterior pituitary gland. GH action results in longitudinal bone growth in children and adolescents and continues later to control a variety of metabolic reactions in adults. DNA polymorphisms leading to a decrease in GH expression result in persons of short stature, whereas those leading to overexpression of GH produce either acromegaly or gigantism. In order to diagnose and treat patients with altered GH production, understanding the control of GH expression is thus clinically relevant. In order to understand how transcription of GH is determined by selective regulatory modulators, two rat somatotroph cell lines were studied that differed in their relative differentiation: MtT/S cells are considered as fully differentiated somatotrophs that express GH exclusively; MtT/Se cells are related somatotrophs with GH expression that is responsive to estrogen treatment. These cell lines were obtained from the Riken Cell Bank (Japan) and then grown and cultured in accordance with established protocols. After harvesting of cells from each line, total RNA was extracted using a rapid affinity method (Qiagen). After validation of the RNA integrity index of each RNA isolate (Affymetrix), they were sent to an off-site laboratory (NovoGene) for RNA-Seq analysis. Samples were examined in triplicate for comparison using standard procedures for adapter ligation, library construction, amplification and sequencing. In our previous work with single gene quantitative RT-PCR, we measured expression of 9 separate targets, primarily transcriptional controllers, in these cell lines. Now, the use of RNA-Seq quantifies the levels of all mRNAs in each sample without the need for specific primers or probes. Therefore, it is possible to find nearly all of the mRNAs that demonstrate changing abundance without requiring prior evidence of their role in the terminal differentiation of the MtT/S from MtT/Se cells. After extensive QC for validation, initial analysis of the data shows more than 36 million validated sequenced reads from each replicate sample with >96.39% mapping to the reference rat genome sequence. Basic analysis shows that 13,012 expressed genes were 87% similar, with 1,175 (9.0%) unique to MtT/Se cells and 484 (3.7%) genes selectively present in MtT/S cells. Of these, 329 genes were upregulated in expression while only 57 were reduced. For comparison with our previous study, we are now confirming differences in GH transcripts and the transcription factors/regulators previously measured, particularly the GH regulator Zn16, a protein with 16 zinc fingers known to bind to the GH promoter DNA. Further analysis of the RNA-Seq profile is focused on the unique and unidentified changes in expression that differentiate these cell lines. Analysis of the differentiation of MtT/S and MtT/Se cells will further understanding of GH regulation in the body.

No description available.

Improving the efficiency by which crops use nutrients is critical for maintaining high crop productivity while reducing fertility management costs and eutrophication related to fertilizer runoff. The native crucifer and halophyte, Yukon Eutrema salsugineum, was used in this study. Yukon E. salsugineum is closely related to important Brassica crops and thrives in its native habitat on soil that is low in available phosphate (Pi) and high in sulfur (S). To determine how Yukon E. salsugineum copes with low Pi, leaf transcriptomes were prepared from four week-old plants grown in controlled environment chambers using soil lacking or supplemented with Pi and/or S. This thesis focused on using bioinformatic approaches to assemble, analyze and compare the transcriptome profiles produced by the Yukon E. salsugineum plants undergoing four nutrient combinations of high and/or low Pi and S. The objective of the study was to identify traits associated with altered S and/or Pi with the prediction based on other species that low Pi, in particular, would pose the greatest stress and hence elicit the greatest transcriptional reprogramming. Transcriptome libraries were generated from four treatment groups with three biological replicates each. Reads in each library were mapped to 23,578 genes in the E. salsugineum transcriptome with an average unique read mapping ratio of 99.52%. Surprisingly, pairwise comparisons of the transcriptomes showed little evidence of Pi-responsive reprogramming whereas treatments differing in soil S content showed a clear S-responsive transcriptome profile. Principal Component Analysis revealed that the low variance quaternary Principal Component distinguished the transcriptomes of plants undergoing low versus high Pi treatments with differential gene expression analysis only finding 11 Pi-responsive genes. This outcome suggests that leaf transcriptomes of Yukon E. salsugineum plants under low Pi are largely undifferentiated from plants provided with Pi and is consistent with Yukon E. salsugineum maintaining Pi homeostasis through fine-tuning the expression of protein-coding and non-coding RNA rather than large-scale transcriptomic reprogramming. Previous research has shown Yukon E. salsugineum to be very efficient in its use of Pi and this work suggests that the altered expression of relatively few genes may be needed to develop Pi-efficient crops to sustain the crop demand of a growing population. / Thesis / Master of Science (MSc)

Glicocorticoides (GCs) modulam a reprodução, interferindo na produção de hormônios gonadais, estradiol (E2) e progesterona (P4). Concomitantemente, E2 e P4 também influenciam a liberação de cortisol. Além disso, em altas concentrações os GCs interferem na via de sinalização da insulina prejudicando o funcionamento dos órgãos, incluindo os do sistema reprodutivo bem como a produção de P4 pelo corpo lúteo (CL). O CL utiliza glicose para manter sua atividade, e o cortisol interfere na via de sinalização da glicose em diversos tecidos, porém ainda não existem estudos quanto à sua atividade no CL canino, tampouco quanto à presença de genes relacionados à via de sinalização dos GCs neste órgão. Os objetivos deste estudo foram caracterizar a expressão dos genes relacionados à via de sinalização dos GCs e da insulina em CL de cadelas cíclicas e identificar expressão diferencial destes genes entre os dias 20 e 60 do diestro, que correspondem à fase final de crescimento do CL e à fase de regressão luteínica, respectivamente. Ainda, avaliar as concentrações basais de cortisol salivar (CS) e metabólitos fecais de glicocorticoides (MGFs) em diferentes dias do diestro (10, 20, 30, 40, 50 e 60) de cadelas cíclicas, comparando com os níveis de E2 e P4 já publicados; correlacionar os valores obtidos nas duas técnicas; e estabelecer um intervalo de valores basais de CS e MFGs para o diestro. Para esta pesquisa foram selecionadas 28 cadelas cíclicas saudáveis; foi realizado o sequenciamento de nova geração (RNA-Seq) dos CLs coletados de 18 animais em dias específicos do diestro (dia 10, 20, 30, 40, 50 e 60 pós-ovulação); das outras 10 cadelas foram coletadas amostras de fezes e saliva, durante todo o diestro, e realizada mensuração da concentração de MFGs e CS. Nos resultados de RNA-Seq foi identificada a expressão dos genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. Os genes NR3C1 (p>0,3; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,6) e SLC2A4 (pCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,02; FDRCortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,1) não apresentaram expressão diferencial no diestro. Os genes HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) mostraram maior expressão no dia 20 do diestro, quando comparado ao dia 60. O gene IRS1 (p=0,0006; FDR= 0,006) estava mais expresso no dia 60 do diestro em relação do dia 20. As dosagens hormonais demonstraram que no dia 10 do diestro (CS=0,0656 ± 0,0237µg/dL e MGFs=110,41 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 46,51pg/ng) as duas mensurações apresentaram concentrações menores que nos demais dias do diestro (CS=0,1027 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 0,0496µg/dL e MFGs=220,22 Cortisol. Corpo lúteo. Insulina. Diestro. RNA-Seq 183,74pg/ng). A concentração média de CS=0,0972µg/dL (0,011-0,246µg/dL) e MFGs=189,875pg/ng (9pg/ng-1067,2pg/ng) foi definida para o diestro canino. Estes resultados sugerem que o CL canino também é influenciado pelo cortisol circulante da mesma forma que outros tecidos já estudados, podendo interferir na via de sinalização da insulina e, consequentemente, prejudicar seu funcionamento e o sucesso reprodutivo. Além disso, em relação aos resultados das dosagens hormonais, os níveis baixos de CS e MFGs no dia 10 do diestro corroboram com os achados em literatura e com a queda de E2 neste mesmo período, o que sugere uma associação entre a produção de ambos os hormônios. / Glucocorticoids (GCs) modulate reproduction by interfering in the gonadal hormones production, estradiol (E2) and progesterone (P4). Likewise, E2 and P4 influence release of cortisol. Furthermore, high concentrations of GCs may harm the functioning of organs, including reproductive organs and progesterone (P4) production by the corpus luteum (CL). CL utilizes glucose to maintain its own activity and cortisol interferes with the glucose signaling pathway in several tissues. Studies characterizing cortisol activity and expression of genes related to GC signaling pathways in canine CL are still scarce. The aims of this study were to characterize the expression of genes related to GCs and insulin signaling pathways in cyclic canine CL and to identify differential expression of these genes between day 20 and day 60 of diestrus which correspond, respectively to luteal final growth and regression phases. Moreover to evaluate the basal concentrations of salivary cortisol (SC) and fecal glucocorticoid metabolites (FGMs) on different days of diestrus (10, 20, 30, 40, 50 e 60) in cyclic bitches, moreover to compare E2 and P4 levels already published. As well as to correlate the values of SC and FGMs, and also to establish an interval of basal concentrations of SC and FGMs for diestrus. For this research 28 healthy cyclic bitches were selected; RNA-Seq of the corpora lutea from 18 animals on specific days of diestrus (day 10, 20, 30, 40, 50, 60) was made. Saliva and fecal samples were collected from the other 10 bitches during diestrus phase. Enzyme immunoassay was made to measure the concentrations of SC and FGMs. The RNA-Seq results identified the expression of the genes NR3C1, HSD11B1, HSD11B2, SLC2A4, INSR e IRS1. No difference on the expression of NR3C1 (p>0,3; FDR>0,6) and SLC2A4 (p>0,02; FDR>0,1) was observed during diestrus. Nevertheless HSD11B1 (p=0,0001; FDR=0,0021), HSD11B2 (p=0,013; FDR=0,06), INSR (p=0,002; FDR=0,01) showed a higher expression on day 20 of diestrus and the gene IRS1 (p=0,0006; FDR= 0,006) presented higher expression on day 60 of diestrus. Results related to hormonal evatuations showed lower SC and FGMs concentrations on day 10th (SC=0,0656 ± 0,0237µg/dL and FGMs=110,41 ± 46,51pg/ng) than on the other days of diestrus (SC=0,1027 ± 0,0496 µg/dL e FGMs=220,22 ± 183,74 pg/ng). Average concentrations of SC=0,0972µg/dL (0,011-0,246µg/dL) and FGMs=189,875pg/ng (9pg/ng-1067,2pg/ng) were defined for canine diestrus. These data suggest that canine CL is influenced by cortisol similarly to other tissues in which cortisol may interfere in the insulin signaling pathway and, consequently, its function. Besides that, lower levels of SC and FGMs on day 10th of the diestrus corroborate with the literature data and with the decrease in E2 production at the same period. This data suggest that the production of both hormones are associated.

Le but de notre étude était de mettre en place une méthode qui puisse nous permettre d’identifier, en aveugle, des perturbateurs de voies biologiques, et qui soit d’une part généralisable pour toute espèce de poissons et d’autre part applicable quel que soit le cours d’eau considéré. Nous nous sommes intéressés aux gènes différentiellement exprimés dans le foie, en utilisant la technologie du séquençage Illumina de banques ADNc. Nous avons étudié trois espèces de cyprinidae (C. nasus, P. toxostoma, S. cephalus) dans le bassin de la Durance, servant à l’alimentation en eau de plusieurs millions de personnes. Nous avons mis en place une suite logicielle pour inférer un transcriptome pour chacune des trois espèces étudiées, et effectué un travail en bioinformatique pour l’identification des spécimens hybrides. Cette méthode nous permet d’assigner les 596 millions de séquences générées (293 spécimens) à l’une des trois espèces et à 16606 gènes identifiés. Les résultats biologiques montrent des variations de l’expression de gènes touchant des voies biologiques associées à des réponses aux xénobiotiques le long de l’axe amont-aval de la rivière. Ils montrent aussi que les spécimens échantillonnés dans le canal EDF présentent des réponses atténuées aux xénobiotiques par rapport aux individus en rivière. Ce résultat peut s’expliquer par l’effet de dilution des polluants dans une masse d’eau plus importante. Cette étude met en évidence les capacités adaptatives des populations de poissons à court terme, via des modifications de l’expression des gènes à un ensemble de perturbateurs environnementaux. Ce travail permet d’envisager la mise en place d’un outil de gestion incontournable. / The aim of our study was to establish a method that allows the identification (in blind) of biologicalpathway disrupters, for all species of fish and applicable regardless of the watercourse considered. Wefocused on differentially expressed genes (and the biological pathways in which they act) in the liver,using the Illumina sequencing technology of cDNA libraries. We studied three species of cyprinidae (C.nasus, P. toxostoma, S. cephalus) in the Durance basin that constitutes water resource for several millionpeople. We have implemented a pipeline to infer the transcriptome for each of the three species studied,and developed a bioinformatics framework for the identification of hybrid specimens. This methodallows us to assign the 596 million sequences generated (representing 293 specimens) to one of the threespecies and to 16,606 identified genes. The biological results display variations in the expression of genesaffecting biological pathways associated with xenobiotic responses (estrogens, Hap, heavy metals) alongthe upstream-downstream axis of the river. They also yield that the specimens sampled in the EDFchannel displayed an attenuated responses to xenobiotics, in comparison to individuals that inhabitethe river, possibly a benefit of the dilution effect of pollutants in a larger body of water. This studyhighlights short-term adaptive capacities (acclimation) of fish populations to a set of environmentaldisrupters via changes in gene expression levels. It will open a way to an essential tool for managementpolicies.

Inúmeros aspectos da reprodução em Apis mellifera já foram extensamente divulgados, no entanto, os mecanismos reguladores da manutenção do estado estéril das operárias, bem como aqueles que permitem a ativação de seus ovários, ainda estão para serem descobertos. Por exemplo, a organização dos folículos ovarianos em crescimento e a arquitetura e papel das células foliculares neste processo. Além disso, para compreender o processo de ativação dos ovários em um contexto mais amplo, também é necessária uma investigação da síntese e maturação de diferentes classes de RNAs as quais modelam redes de interações gênicas extremamente complexas. Portanto, neste doutorado, tivemos como objetivo realizar 1- uma análise morfológica dos ovários ativos de operárias de A. mellifera obtidos em condições orfandade, com ênfase nas células foliculares e 2- um estudo aprofundado da regulação da expressão gênica (genes estruturais e reguladores) que é de fundamental importância para ligar os genótipos aos fenótipos. A análise morfológica dos ovários de operárias de A. mellifera foi realizada em microscópio de fluorescência ou confocal (priorizou a contagem das células foliculares) e microscópio eletrônico de transmissão, que permitiu a descrição e caracterização, pela primeira vez, da patência em ovários de operárias A. mellifera. Paralelamente, por meio da técnica de RNAseq, foi possível analisar o transcriptoma (miRNAs e mRNAs) de amostras específicas de ovários, em diferentes estados fisiológicos, em rainhas e operárias. Os mRNAs e miRNAs que se destacaram em nossas análises in silico foram validados experimentalmente por RT-PCR com alto grau de reprodutibilidade e em harmonia com o estado fisiológico dos ovários. Os transcritos altamente expressos nos ovários ativados foram: fpps5, cad, obp7, yellow-g e aqueles representados pelo GB42182 e GB44975. Acreditamos estes genes possam fazer parte da rede que regula o processo de ativação dos ovários em A. mellifera. Os miRNAs que se destacaram em nossas análises foram: A) miR-306 e miR-317 - altamente expressos nas amostras de ovários funcionais e B) miR-71 pelo fato de, nas análises in silico, ser o mais forte candidato a alvejar a vitelogenina, e na análise experimental, apresentarem, microRNAs e mRNAs, perfis de expressão antagônicos. A construção de bibliotecas de microRNAs e mRNAs a partir de ovários funcionais e não funcionais de abelhas operárias e rainhas, a análise de expressão, bem como a predição de uma rede de integração nos deu um retrato do sensível equilíbrio reprodutivo que mantém ambas as castas em aparente harmonia dentro da colônia aonde elas assumem, no momento certo, seus papéis nesta sofisticada sociedade empreendendo ou não a reprodução. / Countless aspects of reproduction in Apis mellifera have been widely published, however, the regulatory mechanisms for the maintenance of the sterile state of workers as well as those that allow the activation of their ovaries are still to be discovered, as much as the organization of growing ovarian follicles, the architecture and the role of follicular cells during this process. Furthermore, to understand the activation process of the ovaries in a broader context, it is also necessary to investigate the synthesis and maturation of different classes of RNAs which exemplify networks of gene interactions, extremely complex. Therefore, PhD project, we aimed to approach: 1 - A morphological analysis of active ovaries of A. mellifera workers obtained in queenless conditions, with emphasis on the follicular cells and 2 - A detailed study of the regulation of gene expression (structural and regulatory genes) that is crucial for linking genotypes to phenotypes. Morphologic analysis of workers ovaries of A. mellifera was performed under a fluorescence microscope or confocal (prioritized follicular cell count) and transmission electron microscope, which allowed, for the first time, a description and characterization of the patency of worker ovaries in A. mellifera. Similarly, by RNAseq technique, it was possible to analyze the transcriptome (miRNAs and mRNAs) of specific samples of ovaries at different physiological states, in queens and workers. mRNAs and miRNAs that stood out in our in silico analysis were experimentally validated by RT-PCR with high reproducibility and in harmony with ovaries physiologic state. Transcripts highly expressed in activated ovaries were fpps5, cad, obp7, yellow-g and those represented by GB42182 and GB44975. We believe these genes may be part of the network that regulates ovaries activation process in A. mellifera. miRNAs that stood out in our analysis were: - a) - miR-306 and miR-317 - highly expressed in samples of active ovaries and b) -miR-71 by the fact that the in silico analysis, was the strongest candidate to target vitellogenin, and in experimental analysis, presented antagonistic profile of expression when microRNAs and mRNAs were contrasted. The construction of microRNAs and mRNAs libraries from active and inactive ovaries of worker bees and queens, the analysis expression, as well as the prediction of a integrative network has given us a portrait of the sensitive reproductive balance that keeps both castes of bees in apparent harmony within the colony, where they take each one, at the right time, their roles in this sophisticated society, undertaking or not the reproduction.

A tuberculose ainda é pontuada como uma doença de impacto mundial, sendo considerada desde 1993 um problema de saúde pública global. Uma das grandes preocupações é a contínua prevalência de cepas da Mycobacterium tuberculosis multidroga resistentes, especialmente o genótipo hipervirulento W-Beijing. Acredita-se que este genótipo apresenta alguma vantagem seletiva em relação a outros genótipos da M. tuberculosis, além de estar associado à falha terapêutica, tuberculose extrapulmonar, resistência à vacinação pela BCG e acentuada capacidade de disseminação. Estas cepas apresentam variável capacidade de sobrevivência dentro de macrófagos e do granuloma, modulando vias metabólicas específicas que culminam no escape do sistema imunológico e sucesso na infecção. Buscando entender esta vantagem seletiva e capacidade de persistência na infecção, este estudo teve como objetivo analisar e comparar o perfil transcriptômico de macrófagos infectados com as cepas da M. tuberculosis, W-Beijing 1471 e H37Rv. Os RNAs mensageiros dos macrófagos infectados foram sequenciados em plataforma HiScan Genome Analyzer Illumina. Foram gerados aproximadamente 30 milhões de sequências por amostra, em leituras single reads, com mais de 70% de sequências com valores de score Q de qualidade superior ou igual a 30. Foram mapeados e analisados 35.581 transcritos. Em média, 63% dos genes não apresentaram diferenças nos valores de expressões, 19% tiveram suas expressões reduzidas e 18% dos genes foram classificados como mais expressos, para todas as amostras de macrófagos sequenciadas. Após as análises terciárias e validação por PCR em tempo real, as amostras infectadas com a cepa W-Beijing 1471 apresentaram um aumento nas expressões de IFNs da classe I (p<0,001) e aumento exacerbado de TNF-alfa (p<0,001), comparativamente ao controle e as amostras infectadas com a cepa padrão H37Rv. Aditivamente foi observado um aumento nas expressões de duas quinases, RIPK1 e RIPK3 e de moléculas envolvidas na indução e controle de espécies reativas de oxigênio (ROS), que em infecções por bactérias intracelulares, estão correlacionadas com a morte de macrófagos por necroptose. A cepa hipervirulenta da M. tuberculosis, W-Beijing 1471, apresentou reduzida persistência intramacrofágica e induziu morte precoce dos macrófagos ao quinto dia de infecção. A morte observada nos macrófagos foi associada a ativação de IFNs da classe I/TNF-&#945;/RIPK1/RIPK3 e ROS, indicando necroptose. Ainda, foi observado um aumento na expressão do receptor TLR3 nas amostras infectadas com a cepa W-Beijing, comparativamente as amostras controles e infectadas com a cepa H37Rv. É provável que a ativação inicial dos IFNs da classe I tenha ocorrido via TLR3 através do reconhecimento de dsRNAs da M. tuberculosis. / Since 1993 the tuberculosis is considered as a disease of worldwide impact and a problem of public health. A major concern is the continuing prevalence of multidrug resistant Mycobacterium tuberculosis strains, especially the hypervirulent W - Beijing genotype. It is believed that this genotype has a selective advantage over other genotypes of M. tuberculosis and has being associated with treatment failure, extrapulmonary tuberculosis, resistance to BCG vaccination and marked ability to spread. These strains have varying ability to survive within macrophages and granuloma, modulating specific metabolic pathways that culminate in the escape of the immune system response. To understand this selective advantage and ability to persist in infection, this study aimed to analyze and compare the transcriptomic profile of macrophages infected with strains of M. tuberculosis W - Beijing 1471 and H37Rv. The mRNAs of infected macrophages were sequenced in HiScan Illumina Genome Analyzer platform. Were generated approximately 30 million sequences per sample, in single-reads readings. More than 70 % of sequences had values of Q score superior or equal to 30. Were mapped and analyzed 35,581 transcripts. On average, 63% of the genes showed no differences in the expressions, 19% were downregulated and 18% were upregulated, for all samples sequenced macrophages. After tertiary analysis and validation by real-time PCR, samples infected with the strain W -Beijing in 1471 showed an increase in expression of IFN class I (p <0.001) and exacerbated increase of TNF- alpha (p < 0.001) when compared to the control samples and those infected with standard strain H37Rv. Additively was observed an increase in expressions of the two kinases RIPK1 and RIPK3 and molecules involved in the induction and control of reactive oxygen species (ROS). In infections by intracellular bacteria, activation of RIPK1, RIPK3, ROS and TNF-&#945;, are correlated with death of macrophages by necroptosis. The hypervirulent M. tuberculosis W - Beijing 1471 strain showed reduced persistence inside macrophage and induced early death of macrophages in the fifth day of infection. The death observed in macrophages was associated with activation of IFNs class I/TNF-&#945;/RIPK1/RIPK3 and ROS, indicating necroptosis. Also was observed an increase in the expression of TLR3 receptor in infected samples with W-Beijing 1471 strain compared to controls and those infected with H37Rv strain. Probably the initial activation of IFNs class I occurred by TLR3 through the recognition of M. tuberculosis dsRNAs.