Orientador: Wellerson Rodrigo Scarano / Resumo: A fertilidade masculina e a espermatogênese estão diretamente ligadas à capacidade das células de Sertoli em produzir fatores associados ao desenvolvimento das células germinativas. As células de Sertoli expressam receptores para FSH e testosterona e são os principais reguladores da espermatogênese. Aproximadamente 60-70% dos casos de infertilidade masculina são considerados idiopáticos, devido aos mecanismos moleculares envolvidos na espermatogênese ainda serem desconhecidos. Estudos recentes relatam que os microRNAs (miRNAs), são capazes de modular a função testicular durante a espermatogênese e sua expressão alterada pode estar envolvida na infertilidade masculina. miRNAs podem desempenhar papel importante na resposta aos xenobióticos que têm todas as consequências adversas para a saúde. Um grupo importante de compostos orgânicos com potencial tóxico são as dioxinas, como o 2,3,7,8-tetraclorodibenzo-p-dioxina (TCDD). Modelos experimentais de exposição ao TCDD, em camundongos, demonstraram que sua exposição provoca baixa contagem de espermatozóides e atraso na puberdade. Neste estudo, analisamos o efeito do TCDD nas células de Sertoli humanas in vitro após 72h a uma dose de 10nM. Nossos resultados mostraram que as enzimas antioxidantes catalase, superóxido dismutase e glutationa peroxidase diminuíram sua atividade e confirmaram o estresse oxidativo causado pelo TCDD nesse tipo celular. 78 miRNAs apresentaram expressão alterada, com regulação positiva de 73 e regulação negat... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Male fertility and spermatogenesis are directly linked to the ability of Sertoli cells to produce factors associated with the development of germ cells. Sertoli cells express receptors for FSH and testosterone, and are the major regulators of spermatogenesis. Approximately 60-70% of male infertility cases are considered idiopathic, due to the molecular mechanisms involved in spermatogenesis are still unknown. Recent studies report that microRNAs (miRNAs) are capable of modulating spermatogenesis in testicular function and its altered expression may be involved in male infertility. miRNAs may play a role in response to xenobiotics that have all the adverse consequences for health. An important group of organic compounds that are potentially toxic are the dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Experimental models of exposure to TCDD in mice showed that its exposure causes low sperm count and delayed puberty. In this study, we analyzed the effect of TCDD on human Sertoli cells after a exposure of 72h in vitro at a dose of 10nM. Our results showed that the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase decreased their activity and confirmed the oxidative stress caused by TCDD in this cell type. 78 miRNAs showed altered expression with upregulation of 73 miRNAs and downregulation of 5 miRNAs compared to the control group. Regarding the gene expression profile, 51 genes showed deregulated, of which 46 genes with upregulation and d... (Complete abstract click electronic access below) / Doutor

Orientador: Célia Regina Nogueira / Resumo: O sistema esquelético é um sistema complexo com intenso metabolismo composto de células, proteínas e minerais. Os osteoblastos, são células fundamentais para o tecido, desempenhando duas funções principais: formação óssea e regulação da reabsorção por meio da modulação da osteoclastogênese. Sendo assim, essas células desempenham funções primordiais para o desenvolvimento e manutenção óssea. Diversas moléculas sistêmicas atuam no tecido, sendo os hormônios tireoidianos um destes. Eles são fundamentais para o metabolismo ósseo já que alterações hormonais culminam em desordens ósseas. Osteoblastos possuem receptores nucleares para T3 e apesar de pouco compreendido, ele afeta diversos aspectos do desenvolvimento da célula, assim como vias modulatórias da remodelação óssea. Diversas linhagens celulares têm sido utilizadas para estudos de osteoblastos, sendo as células-tronco mesenquimais derivadas do tecido adiposo humano (hA-CTMs) um modelo promissor para osteoindução. Desta forma, o objetivo deste trabalho foi verificar a influência do T3 suprafisiológico (T3S) na expressão gênica diferencial em osteoblastos diferenciados a partir de hA-CTMs. As células obtidas de 3 doadores foram submetidas a osteoindução por 16 dias com coquetel de diferenciação (dexametasona, ácido ascórbico e β-glicerofosfato) e caracterizadas pela presença de osteocalcina, fosfatase alcalina e matriz mineralizada. O tratamento com T3 (10-8M) foi realizado por 72h e o RNA foi extraído para preparação das bib... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The skeletal system is complex and have an intense metabolism compound with cells, proteins and minerals. The osteoblasts are fundamental cells to the tissue, executing two mainly functions: bone formation and regulation of reabsorption through osteoclastogenesis modulation. Therefore, these cells perform primordial functions to the bone development and maintenance. Several systemic molecules act in the tissue and the thyroid hormones are one of them. They are fundamental to the bone metabolism, once hormone alterations result in bone disorders. Osteoblasts have T3 nuclear receptors, and although not well elucidated it affects many aspects of the cell development so as pathways that modulates the bone remodeling. Several cells lineages have been used in osteoblasts studies, and human adipose-derived Mesenchymal stem-cells (hA-MSCs) are a promising model to osteoinduction. Thus, the aim of this study was to investigate the supraphysiological T3 (T3S) influence in the gene differential expression in osteoblasts differentiated from hA-MSCs. The cells obtained from three donators were submitted to osteoinduction for 16 days with a differentiation cocktail (dexamethasone, ascorbic acid, β-glycerophosphate) and characterized by the presence of osteocalcin, alkaline phosphate and mineralized matrix. The cells were treated with T3 (10-8M) for 72h and the RNA was extracted to mRNA library prepare and sequenced by Illumina platform. The bioinformatic analysis included the software: Fas... (Complete abstract click electronic access below) / Mestre

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald Reeck / Pea aphids (Acyrthosiphon pisum) are sap-sucking insects that feed on the phloem sap of some plants of the family Fabaceae (legumes). Aphids feed on host plants by inserting their stylets between plant cells to feed from phloem sap in sieve elements. Their feeding is of major agronomical importance, as aphids cause hundreds of millions of dollars in crop damage worldwide, annually. Salivary gland transcripts from plant-fed and diet-fed pea aphids were studied by RNASeq to analyze their expression. Most transcripts had higher expression in plant-fed pea aphids, likely due to the need for saliva protein in the aphid/plant interaction. Numerous salivary gland transcripts and saliva proteins have been identified in aphids, including a glutathione peroxidase. Glutathione peroxidases are a group of enzymes with the purpose of protecting organisms from oxidative damage. Here, I present a bioinformatic analysis of pea aphid expressed sequence tag libraries that identified four unique glutathione peroxidases in pea aphids. One glutathione peroxidase, ApGPx1 has high expression in the pea aphid salivary gland. Two glutathione peroxidase genes are present in the current annotation of the pea aphid genome. My work indicates that the two genes need to be revised.

abstract: Circular RNAs (circRNAs) are a class of endogenous, non-coding RNAs that are formed when exons back-splice to each other and represent a new area of transcriptomics research. Numerous RNA sequencing (RNAseq) studies since 2012 have revealed that circRNAs are pervasively expressed in eukaryotes, especially in the mammalian brain. While their functional role and impact remains to be clarified, circRNAs have been found to regulate micro-RNAs (miRNAs) as well as parental gene transcription and may thus have key roles in transcriptional regulation. Although circRNAs have continued to gain attention, our understanding of their expression in a cell-, tissue- , and brain region-specific context remains limited. Further, computational algorithms produce varied results in terms of what circRNAs are detected. This thesis aims to advance current knowledge of circRNA expression in a region specific context focusing on the human brain, as well as address computational challenges. The overarching goal of my research unfolds over three aims: (i) evaluating circRNAs and their predicted impact on transcriptional regulatory networks in cell-specific RNAseq data; (ii) developing a novel solution for de novo detection of full length circRNAs as well as in silico validation of selected circRNA junctions using assembly; and (iii) application of these assembly based detection and validation workflows, and integrating existing tools, to systematically identify and characterize circRNAs in functionally distinct human brain regions. To this end, I have developed novel bioinformatics workflows that are applicable to non-polyA selected RNAseq datasets and can be used to characterize circRNA expression across various sample types and diseases. Further, I establish a reference dataset of circRNA expression profiles and regulatory networks in a brain region-specific manner. This resource along with existing databases such as circBase will be invaluable in advancing circRNA research as well as improving our understanding of their role in transcriptional regulation and various neurological conditions. / Dissertation/Thesis / Appendix file containing list of enriched pathways and functions identified in Chapter 4 / Doctoral Dissertation Biomedical Informatics 2018

Orientador: João Pessoa Araújo Junior / Resumo: A análise de transcriptoma é essencial para determinar a relação entre as informações codificadas num genoma, a sua expressão e variação genotípica. O transcriptoma é o conjunto completo de transcritos e a quantidade gerada, relacionado a um estágio de desenvolvimento específico ou condição fisiológica da célula. Estes transcritos podem ser mapeados utilizando genomas como referência, para investigação de expressão do genes. Para isso, exige procedimentos de mineração de grandes volumes de dados de RNA-Seq para extrair conhecimentos biológicos. As ferramentas de bioinformática foram desenvolvidas para facilitar e agilizar essas análises, transformando dados em informações. Assim, alguns desses recursos foram empregados na análise de dados gerados pelo RNAseq, a partir de RNAm de cultura celular MDCK infectada por herpesvírus canino tipo 1 (1CaHV-1 - Canid alphaherpesvirus type 1) em diferentes momentos pós infecção. Dessa forma, foi realizado um dual RNAseq, em que foi avaliado tanto a expressão gênica celular do hospedeiro como do patógeno viral, no curso da infecção. Para isso, foram analisados o transcriptoma das atividades celulares e os processos envolvidos no ciclo de infecção viral, até o momento 32h-pi. Assim, foram identificados as atividades de respostas celulares à infecção viral, mecanismos regulatórios induzidos pelo vírus, transcrição de genes virais imediatos, iniciais e tardios. Dentre eles, foi verificado a elevação da expressão do gene COX-2 induzido pela... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

>Magister Scientiae - MSc / Lung cancer remains the leading cause of cancer deaths worldwide, with the majority of cases attributed to non-small cell lung carcinomas. At the time of diagnosis, a large percentage of patients present with advanced stage of disease, ultimately resulting in a poor prognosis. The identification circulatory markers, overexpressed by the tumour tissue, could facilitate the discovery of an early, specific, non-invasive diagnostic tool as well as improving prognosis and treatment protocols. The aim was to analyse gene expression data from both microarray and RNA sequencing platforms, using bioinformatics and statistical analysis tools. Enrichment analysis sought to identify genes, which were differentially expressed (p < 0.05, FC > 2) and had the potential to be secreted into the extracellular circulation, by using Gene Ontology terms of the Cellular Component. Results identified 1 657 statically significant genes between normal and early lung cancer tissue, with only 1 gene differentially expressed (DE) between the early and late stage disease. Following statistical analysis, 171 DE genes selected as potential early stage biomarkers. The overall sensitivity of RNAseq, in comparison to arrays enabled the identification of 57 potential serum markers. These genes of interest were all downregulated in the tumour tissue, and while they did not facilitate the discovery of an ideal diagnostic marker based on the set criteria in this study, their roles in disease initiation and progression require further analysis.

Le colza (Brassica napus L.) est une plante de grande culture nécessitant de forts intrants azotés et présentant une faible Efficience d’Utilisation de l’Azote (EUA) principalement due à une faible efficience de remobilisation cet élément durant la sénescence foliaire. Actuellement, une amélioration du bilan agroenvironnemental de la culture du colza, nécessite la mise au point de nouvelles pratiques culturales permettant de maintenir (voire d’améliorer) son rendement tout en limitant les doses d’intrants azotés. Dans ce contexte, l’utilisation d’éléments bénéfiques tels que le silicium (Si) dont les effets ont déjà été démontrés dans la tolérance à certains stress nutritionnels (phosphore et potassium) constitue une alternative intéressante. Les objectifs de cette étude visent à étudier l’effet du Si (i) sur la progression de la sénescence foliaire, l’absorption et la remobilisation de l’azote et les performances agronomiques du colza cultivé en hydroponie ou en conditions de plein champ et soumis ou non à une limitation ou une privation en azote. Nos travaux montrent, qu’en dépit de son caractère faiblement accumulateur en Si, le colza absorbe le Si puis le stocke essentiellement au niveau racinaire. Par ailleurs, un apport de Si de courte durée (7 jours ; 1,7 mM) à des jeunes plantes privées en azote provoque un retard de sénescence des feuilles matures associé un maintien de leur teneur en chlorophylles et de leur activité photosynthétique. De plus, au champ, un apport de Si (12 kg ha-1) à des plantes cultivées avec 160 kg N ha-1 s’accompagne d’une augmentation du rendement grainier (+4,2 quintaux ha-1). L'utilisation de l’indice agronomique «Agronomic Nitrogen Recovery (ANR)» combinée à l’étude de l’expression de gènes codant des transporteurs d’azote (BnaNRT1.1, BnaNRT2.1, BnaAMT1.1) montrent que le Si agit notamment en stimulant l’absorption de l’azote chez le colza. Enfin, une analyse différentielle par RNAseq des transcriptomes racinaires de jeunes plantes traitées ou non avec du Si pendant une courte durée (7 jours ; 1,7 mM), montre que le Si module l’expression de 1079 gènes, 334 étant induits et 745 réprimés. Ce résultat indique qu’en plus de son rôle mécanique, le Si exerce également un rôle signalétique. / Rapeseed (Brassica napus L.) is a crop that requires high nitrogen inputs and has a low Nitrogen Utilization Efficiency (NUE) mainly due to a low remobilization efficiency during leaf senescence. Currently, an improvement of the agro-environmental balance of rapeseed, requires the development of new cultural practices to maintain (or even improve) its yield in response to low nitrogen inputs. In this context, the use of beneficial element such as silicon (Si) with previously demonstrated effects on tolerance to some nutritional stress (phosphorus and potassium), constitutes an interesting alternative. The objectives of this study are to investigate the effect of Si on the progression of leaf senescence, nitrogen uptake and remobilization but also on agronomic performances of rapeseed grown in hydroponics or in field conditions and submitted or not to a limitation or deprivation of nitrogen inputs. Our work shows that despite being considered as a low Si accumulator, rapeseed takes up and stores Si mainly in its roots. In addition, a Si supply for a short time (7 days; 1.7 mM) to young plants deprived of nitrogen leads to a delay of senescence of mature leaves associated with a maintenance of their chlorophyll content and their photosynthetic activity. In addition, in field conditions, a supply of Si (12 kg ha-1) to plants grown with 160 kg N ha-1 leads to an increase of yield (+4.2 quintals ha-1). Furthermore, calculation of the "Agronomic Nitrogen Recovery (ANR)" combined with the expression of genes encoding nitrogen transporters (BnaNRT1.1, BnaNRT2.1, and BnaAMT1.1) shows that Si increases the nitrogen uptake. Finally, a differential analysis by RNAseq of roots transcriptomes from young plants treated or not with Si for a short time (7 days; 1.7 mM), shows that Si modulates the expression of 1079 genes, 334 being induced and 745 repressed. This result indicates that in addition to its mechanical role, Si acts also a signal, a role very poorly demonstrated in the literature.

The mitochondria are organelles where energy in the form of ATP (Adenosine Triphosphate) is produced. During low oxygen supply (hypoxia) and mitochondrial uncoupling, ATP synthesis is reduced and AMP (Adenosine Monophosphate) accumulates in the cells. This increase in AMP: ATP ratio stimulates the AMP-Activated Kinase (AMPK) pathway, known to improve healthspan and lifespan by increasing mitochondrial biogenesis (making new mitochondria), decreasing oxidative stress and inflammation, and proteotoxicity (by degrading non-functional organelles and proteins). Here, the life/healthspan extending potential of chronic mild intermittent hypoxia (CMIH) and mitochondrial uncoupling using 2,4 -Dinitrophenol (DNP) was investigated in an emerging model organism, an aquatic crustacean, Daphnia magna. First, the effect of CMIH (4mgO2/L) on longevity in four different genotypes of Daphnia magna was investigated. All individuals were kept in similar conditions with controls in normoxia (8mgO2/L). Hypoxia was created by bubbling compressed nitrogen gas through the water twice daily. Survival was assessing through censuses conducted every 3 days and gene expression changes in response to CMIH were assessed by RNA sequencing using Oxford Nanopore Technology. Briefly, RNA was isolated from genotypes after hypoxic treatments and reverse transcribed to cDNA, libraries were multiplexed and sequenced using Oxford Nanopore MinION for 24-48 hours. Lastly, the effect of prolonged exposure to DNP on longevity was evaluated. Daphnia were chronically exposed to either 0 (control), or 0.1, 1, and 5μM of DNP. Genotypes displayed different tolerance to hypoxia and DNP treatments. Contrary to the expectations, CMIH and DNP reduced longevity, but only in genotypes from permanent ponds, while having no effect on the survival of genotypes from intermittent ponds, arguably better adapted to naturally occurring hypoxic conditions. We uncovered 11 candidate genes that were differentially expressed in these genotypes. In particular, genes involved in mTOR, p53, and sirtuin pathways showed patterns of expression consistent with protection against hypoxia. These pathways are known to regulate autophagy, apoptosis, inflammation, and cell cycle. Because our findings elucidate genotype-specific physiological and transcriptomic responses to respiratory perturbations (CMIH and DNP) we may be able to make a step towards the understanding of a model organism’s response to respiratory stress.

No description available.

Heritable and environmental factors contribute to an individual’s risk of substance abuse and psychosis. Individuals diagnosed with a mental disorder have greater vulnerability for substance abuse. Our laboratory established that neonatal treatment of rats with quinpirole (NQ), a dopamine (DA) D2-like agonist, results in a significant increase of DAD2 receptor sensitivity throughout the animal’s lifetime. An increase of DAD2 receptor sensitivity is relevant to a model of schizophrenia (SZ), although increases of DAD2 receptor activity also occur in a number of clinical disorders, including bipolar disorder, obsessive-compulsive disorder, panic disorder, and major depression. Common amongst these clinical conditions is a dramatic increase in cigarette smoking compared to the general population. We bred NQ-treated male and female rats with their NQ-treated or neonatal saline (NS)-treated counterparts once they reached adulthood to determine whether increases in DAD2 sensitivity were passed to the next generation. Offspring of these animals, regardless of whether one or both founders received NQ-treatment, also demonstrated increases of DAD2 receptor sensitivity both behaviorally and neurobiologically. RNASeq preliminary data revealed an increase in cortisol synthesis and release in F1 generation animals, demonstrating an enhanced response to stress, consistent with a model of drug abuse vulnerability. Consistent with this finding, F1 generation rats demonstrated enhanced nicotine conditioned place preference (CPP) and had an enhanced brain derived neurotrophic factor (BDNF) response to nicotine in the nucleus accumbens (NAcc), a brain area critical to drug reward. The DAD2 receptor forms a triple heteromer with the adenosine A(2A) and metabotropic glutamate type 5 (mGlu5) receptor, such that stimulation of either receptor results in a decrease of DAD2 activity. Therefore, we analyzed whether use of a positive allosteric modulator (PAM) of mGlu5 in the F1 generation would block nicotine CPP and improve sensorimotor gating deficits, which is a hallmark of psychosis. In both experiments, the mGlu5 PAM effectively blocked the enhanced rewarding effects of nicotine and also alleviated sensorimotor gating deficits in this model. In essence, we demonstrate in results reported here that there may be a common therapeutic target for the dual treatment of substance abuse and psychosis.