Ácaros Brevipalpus yothersi Baker (1949) são vetores do citrus leprosis virus C (CiLV-C), o mais comum causador da leprose dos citros. Esta é considerada, atualmente, a doença viral de maior importância na cultura dos citros no Brasil e ocorre também em países da América do Sul e Central, além do México, na América do Norte. A doença prejudica a vida útil da planta, devido à queda de folhas e frutos, e seca de ramos, podendo levá-la à morte quando o ataque é demasiadamente severo. O controle e manejo da leprose são basicamente atribuídos ao uso de acaricidas, demandando dos produtores um acréscimo significativo no orçamento por defensivos agrícolas e potencial risco ao ambiente. Nos últimos anos, análises de expressão gênica (transcriptoma), envolvendo ácaros praga da agricultura, resultaram em informações importantes como o envolvimento de genes específicos no processo de resistência a acaricidas e desenvolvimento biológico. No entanto, ainda pouco se sabe sobre a interação do ácaro B. yothersi com o CiLV-C, tornando o estudo de transcriptoma muito significativo para a obtenção de informações aprofundadas sobre o atípico padrão vírus-vetor do patossistema leprose. Assim, através do sequenciamento de RNA (RNAseq), foi investigado o perfil de expressão diferencial entre ácaros B. yothersi virulíferos e avirulíferos. O RNAseq foi realizado em sistema Illumina HiSeq 2500, e os dados analisados com base em linguagem R e ferramentas do software Bioconductor. Os reads sequenciados foram mapeados no genoma de B. yothersi e foi feita análise de expressão utilizando DESeq2. Foram observados 5.690 genes diferencialmente expressos (GDE), sendo 2.736 transcritos induzidos em ácaros virulíferos. Os GDE foram analisados em banco de dados do NCBI, buscando por proteínas similares em artrópodes. Dentre os transcritos induzidos em B. yothersi, potencialmente em resposta ao CiLV-C, foram encontrados genes relacionados ao processo de detoxificação de xenobióticos, metabolismo primário e imunidade, com possível envolvimento na interação vírus-vetor. A expressão de 23 genes foi verificada por RT-PCR quantitativo em tempo real (RT-qPCR). As análises indicaram que os genes envolvidos em detoxificação são induzidos durante a interação entre o ácaro da leprose e o CiLV-C. / The false spider mite Brevipalpus yothersi Baker (1949) is recognized as vector of citrus leprosis virus C (CiLV-C), the most common causal agent of citrus leprosis disease. Currently, it is considered the viral disease of major importance in citrus in Brazil and occurs in countries of South and Central America, as well as in Mexico, North America. It reduces the lifespam of the plants due to leaf and fruit drop, dried branches, and can lead to death when the attack is severe. The management of leprosis is based primarily on the chemical control of the mite vector, increasing significantly the cost of production and harming the environment. On the last years, gene expression (transcriptome) analyzes involving phytophagous mites resulted in important data, such as the involvement of specific genes in the process of resistance to acaricides and biological development. However, there is little information about B. yothersi-CiLV-C interaction, making the transcriptome a very interesting tool to obtaining data regarding the atypical virus-vector relationship of the leprosis pathosystem. RNA sequencing (RNAseq) was used to investigate the differential expression profiles between viruliferous and aviruliferous B. yothersi mites. The RNAseq was performed using the Illumina HiSeq 2500 system, the data were analyzed using the R language and Bioconductor software packages. The sequenced reads were mapped on the genome of B. yothersi and expression analysis was performed in DESeq2. We identified 5.690 differentially expressed genes (DEGs), of which 2.736 transcripts were induced in viruliferous mites. The DEGs were analyzed in NCBI database, searching for similar proteins in arthropods. Among the transcripts induced in response to CiLV-C, there are genes related to the detoxification of xenobiotics, primary metabolism, immunity and possible involvement in the virus-vector interaction. Expression of 23 genes was verified by quantitative real-time RT-PCR (RT-qPCR). The analyzes indicate that detoxification genes are induced during the interaction between the false spider mite and CiLV-C.

O corpo lúteo (CL) canino é responsável pela síntese de E2 durante o diestro. O E2 atua de forma autócrina e/ou parácrina sobre essa glândula. O mecanismo de atuação do E2 depende da razão entre receptor alfa (ERα) e receptor beta (ERβ). A ligação ao ERα tem efeito proliferativo e ao ERβ antiproliferativo. O objetivo deste trabalho foi entender a sinalização mediada pelo ERα e ERβ no processo de formação e regressão do CL. CLs foram obtidos de cadelas não prenhes (n=30) nos dias 10, 20, 30, 40, 50 e 60 (n=5/grupo) após a ovulação (po). No dia da ovariossalpingohisterectomia foi colhido sangue para mensuração das concentrações de P4 e E2. Dezoito CLs (n=3/grupo) foram submetidos ao sequenciamento de RNA (RNAseq). Os genes diferencialmente expressos (DE) identificados pelo RNAseq foram submetidos ao software oPOSSUM3 para identificação das regiões de ligação mais representadas nos fatores de transcrição relacionados aos genes do ERα (ESR1) e ERβ (ESR2). A análise de expressão temporal revelou a presença de 5.116 diferencialmente expressos (DE) em pelo menos uma comparação e 1.106 não foram anotados ainda no genoma canino, destes genes DE 295 apresentavam regiões de ligação de fatores de transcrição em comum com ESR1 e ESR2. Dez genes DE foram selecionados para validação dos resultados de RNAseq através do qPCR; usando o GAPDH como gene de referência. Desses genes, quatro apresentaram regioes em comum com ESR2 (LEF-1; PAPPA; NDGR2 ATP1A1) e um com ESR1 (CAV1) e os demais controlam proliferação celular (CTNNB1; CCND1; IGFBP 3, 4 e 5). A expressão proteíca de PAPPA e IGFBP 3, 4 e 5 (componentes do sistema IGF) foi avaliada também por imunohistoquímica. Durante a primeira metade do diestro, nossos resultados indicam que a sinalização mediada pelo E2 ocorre via interação do ERα com CAV-1 (sinalização não genômica), ativando as vias de sinalização IGF e WNT/βcatenina, regulando o processo de proliferação celular. Enquanto na segunda metade, o ERβ regularia a expressão gênica de NDGR2 e ATP1A1, contribuindo para a regressão do CL. Assim nos resultados sugerem que o E2 atue tanto como um fator luteotrófico e quanto regulador da regressão do CL canino. / The canine corpus luteum (CL) is responsible for E2 synthesis during diestrus, which acts in an autocrine and / or paracrine manner in this gland. The E2 mechanism of action depends on the ratio between alpha (ERα) and beta (ERβ) receptor The binding to ERα has a proliferative effect whereas to ERβ an antiproliferative. The objective of this study was to understand the signaling mediated by ERα and ERβ in the formation and regression of the CL. CLs were obtained from non-pregnant bitches (n = 30) on days 10, 20, 30, 40, 50 and 60 (n = 5 / group) post-ovulation (po). On the day of ovariohysterectomy blood was collected for measurement of P4 and E2 concentrations. Eighteen CLs (n = 3 / group) were subjected to RNA sequencing (RNA-Seq). Genes differentially expressed (DE) identified by RNA-Seq were submitted to oPOSSUM3 software for detection of over-represented transcription factors binding sites (TFBS) related to the ERα (ESR1) and ERβ (ESR2) genes. The temporal expression analysis revealed the presence of 5116 differentially expressed (DE) genes in at least one comparison, and 1106 genes, which have not been recorded to the canine genome yet From these, 295 genes showed TFBS in common with ESR1 and ESR2. Ten DE genes were selected to validate the results of RNA-Seq by qPCR; using GAPDH as reference gene. Of these genes, four had TFBS in common with ESR2 (LEF-1; PAPPA; NDGR2; ATP1A1) and one with ESR1 (CAV1) and others genes were chosen because they control cell proliferation (CTNNB1; CCND1, IGFBP 3, 4 and 5). Protein expression of the IGF related genes (PAPPA and IGFBP 3, 4 and 5) was also evaluated by immunohistochemistry. During the first half of diestrus, it appears that E2 mediated signaling occurs via interaction of ERα with CAV-1 (non-genomic signaling), activating signaling pathways of IGF and WNT / βcatenin, then regulating the cell proliferation process. Whereas in the second half, ERβ appears to regulate NDGR2 and ATP1A1 gene expression, contributing to the regression of the CL. Thus our results suggest that E2 may act as a luteotrophic and also a luteolytic factor in the canine CL.

Les efflorescences de la micro-algue toxique Alexandrium minutum sont en constante augmentation au niveau mondial, accentuées par les apports anthropiques et le réchauffement global, et posent des problèmes écologiques, économiques et sanitaires. Lors d’une exposition à A. minutum, l’accumulation des phycotoxines paralysantes (PSP) dans les tissus semble être différente entre les huîtres diploïdes et triploïdes. Par ailleurs, de nombreuses fonctions physiologiques de l’huître C. gigas sont impactées. L’ensemble des fonctions physiologiques d’un organisme est régulé par des rythmes biologiques. Propriété fondamentale de la vie, les rythmes biologiques permettent aux organismes de se synchroniser et d’anticiper les variations cycliques de l’environnement. Dans mes travaux, je me suis intéressée aux rythmes biologiques des huîtres diploïdes et triploïdes C. gigas et à leurs interactions avec la contamination par les PSP. Une analyse in situ sur un an a mis en évidence une relation jusque-là inconnue entre le comportement valvaire de C. gigas et le cycle d’éclairement de la lune, ainsi qu’une relation fine et subtile des cycles comportementaux nycthéméraux et tidaux avec l’évolution annuelle de la photopériode. Cette relation est modulée par la ploïdie. Par ailleurs, la mise au point d’une approche non-invasive d’interférence par ARN a permis de mettre en évidence l’implication de l’horloge circadienne dans les processus de bioaccumulation des PSP chez C. gigas. Enfin, l’analyse du transcriptome cyclique dans les branchies de C. gigas a mis en évidence qu’au moins 42 % du transcriptome peut être exprimé de façon oscillante. Contre intuitivement, en condition d’alternance jour / nuit, une majorité de transcrits sont ultradiens, trois fois plus nombreux que les transcrits circadiens. Exposées à A. minutum, les huîtres ont montré un remodelage profond de leur transcriptome cyclique, pouvant entraîner la perte de synchronisation de l’huître avec son environnement. / Harmful algal blooms of Alexandrium minutum are constantly increasing at the global level, accentuated by anthropogenic contributions and global warming, causing ecological, economical and sanitary problems. During exposition to A. minutum, paralytic phycotoxins (PSP) accumulation differs between diploid and triploid oysters. Moreover, many physiological functions of the oyster C. gigas are impacted. All physiological functions of an organism are regulated by biological rhythms. As a fundamental property of life, biological rhythms allow organisms to synchronize and anticipate cyclic variations of the environment. In my work, I was interested in the biological rhythms of diploid and triploid oysters C. gigas, and their interactions with PSP contamination. A one-year in situ analysis revealed a previously unknown relationship between valve behavior of C. gigas and the lunar illumination cycle, as well as a fine and subtle relationship of the nycthemeral and tidal behavioral cycles with the annual evolution of the photoperiod. This relationship was modulated by the ploidy. Moreover, the development of a non-invasive approach of RNA interference revealed the involvement of the circadian clock in bioaccumulation processes of PSPs in C. gigas. Finally, analysis of the cyclic transcriptome in the gills of C. gigas showed that at least 42 % of the transcriptome can oscillate. Surprisingly, in day / night entrainment, most of transcripts were ultradians, three times more abundant than circadian transcripts. Exposed to A. minutum, results showed a profound remodeling of the cyclic transcriptome of C. gigas, which could lead to loss of synchronization of the oyster with its environment.

L’exploitation conjointe des données transcriptomiques et protéomiques permet l’étude détaillée des mécanismes moléculaires induits lors de perturbations environnementales. L’assemblage de données issues du séquençage des ARNs d’organismes dit « non-modèle » permet de produire la base de données pour l’interprétation des spectres générés en protéomique shotgun. Dans ce contexte, les travaux de thèse avaient pour objectif d’optimiser l’interprétation et l’analyse des données protéomiques par le développement de concepts innovants pour la construction de bases de données protéiques et l’exploration de la biodiversité. La première étape s’est concentrée sur la mise au point d’une méthode de pré-traitement des données de séquençage basée sur les résultats d’attribution protéomique. La deuxième étape a consisté à travailler sur la réduction de la taille des bases de données en optimisant les paramètres de la recherche automatisée des régions codantes. La méthode optimisée a permis l’analyse de 7 groupes taxonomiques de Gammaridés représentatifs de la diversité retrouvée in natura. Les bases de données protéomiques ainsi produites ont permis l’analyse inter-population de 40 protéomes individuels de Gammarus pulex répartis sur deux sites de prélèvement (pollué vs référence). L’analyse statistique basée sur une approche « individu-centré » a montré une hétérogénéité de la réponse biologique au sein d’une population d’organismes suite à une perturbation environnementale. Différents sous-groupes de mécanismes moléculaires induits ont été identifiés. Enfin, l’étude de la transversalité de biomarqueurs peptidiques identifiés chez Gammarus fossarum a permis de définir les peptides communs à l’aide de l’ensemble des données protéomiques et transcriptomiques. Pour cela, un logiciel d’exploration des séquences peptidiques a été développé permettant de proposer de potentiels biomarqueurs substituts dans le cas où les peptides définis ne sont pas applicables à certaines espèces de gammare. Tous ces concepts s’intègrent dans une démarche pour améliorer et approfondir l’interprétation des données par protéogénomique. Ces travaux entrouvrent la porte à l’analyse multi-omique d’individus prélevés in natura en considérant la biodiversité inter-espèce et intra-population. / The exploitation of omics data combining transcriptomic and proteomic enables the detailed study of the molecular mechanisms of non-model organisms exposed to an environmental stress. The assembly of data from the RNA-seq of non-model organism enables to produce the protein database for the interpretation of spectra generated in shotgun proteomics. In this context, the aim of the PhD work was to optimize the interpretation and analysis of proteomic data through the development of innovative concepts for the construction of protein databases and the exploration of biodiversity. The first step focused on the development of a pretreatment method for RNA-seq data based on proteomic attribution results. The second step was to work on reducing the size of the databases by optimizing the parameters of the automated coding region search. The optimized method enabled the analysis of 7 taxonomic groups of Gammarids representative of the diversity found in natura. The proteomic databases thus produced enabled the inter-population analysis of 40 individual Gammarus pulex proteomes from two sampling sites (polluted vs reference). Statistical analysis based on an "individual" approach has shown an heterogeneity of the biological response within a population of organisms induced by an environmental stress. Different subclusters of molecular mechanisms response have been identified. Finally, the study of the transversality of the biomarkers peptides identified with Gammarus fossarum revealed which are the common ones using both proteomic and transcriptomic data. For this purpose, a software for the exploration of peptide sequences has been developed suggesting potential substitute biomarkers when the defined peptides are not available for some species of gammarids. All these concepts aim to improve the interpretation of data by proteogenomics. This work opens the door to the multi-omic analysis of individuals collected in natura by considering inter-species and intra-population biodiversity.

La majorité des Légumineuses sont capables d’établir une symbiose avec des bactéries du sol nommées Rhizobia. Lors de cette interaction symbiotique, un nouvel organe est formé, la nodosité. Dans cet organe, les bactéries fixent l’azote atmosphérique au profit de la plante hôte. Pendant la symbiose Rhizobia-Légumineuse, deux grands changements transcriptômiques ont été observés par différentes technologies, comme le RNASeq (Maunoury et al., 2010) ou les expériences de microarrays (Benedito et al., 2008). Ces grands changements interviennent aux différentes étapes de développements des nodosités et sont médiés par différents régulateurs de l’expression génique comme certains FTs clés et des petits ARN. Ces petits ARN régulateurs sont produits après le clivage de précurseurs de long ARN double brin ou d’ARN en épingle à cheveux par des enzymes particulières de la famille des ribonucléases de type III (RNase III), nommées DICER-LIKE (DCL). De plus, des gènes codant des RNases III additionnelles sont présents dans le génome de plantes et leurs rôles restent encore à être déterminés.Dans cette étude, nous avons caractérisés la famille des RNases III chez Medicago truncatula mais aussi chez d’autres espèces de légumineuse. Nous avons également recherchés l’implication de MtRTL1b, une RNase III, lors du développement des nodosités.Cette RNase III est un orthologue spécifique des nodosités d’AtRTL1, un répresseur de silencing chez Arabidopsis thaliana. Tout d'abord, nous avons montré que l’expression de ce gène est activée juste avant la différenciation et est principalement restreinte à l’interzone, là où les bactéroïdes deviennent totalement différenciés dans les cellules hôtes, et dans la zone de fixation de la nodosité. La répression de l’expression de MtRTL1b, par ARN interférence dans des racines transgéniques, affecte le développement de la nodosité, la fixation de l’azote et la viabilité des bactéroïdes. Un phénotype opposé est observé lorsque MtRTL1b est exprimé de façon ectopique dans la racine. Les analyses des données de séquençage nous ont permis de mettre en évidence que le RNAi conduit à la sous-expression de 1038 gènes, incluant plus de 109 gènes codant des NCRs qui sont des peptides intervenant dans le développement des bactéroïdes et/ou pour leur viabilité dans les nodosités indéterminées. De plus,des gènes impliqués dans les voies métaboliques et la régulation de l’état d'oxydo-réduction mais aussi dans le processus symbiotique, comme la leghémoglobine, sont également sous-exprimés. Des données de séquençage de petits ARN et d’ARN double brins sont en cours d’analyse afin de caractériser les changements dans les populations de petit ARN et identifier les substrats ARN double brin de cette RNase III lors du développement des nodosités. / Almost all Legumes are able to establish symbiosis with soil bacteria called Rhizobia. During this interaction, a new organ is formed, the nodule. In this organ, bacteria fix the atmospheric nitrogen for the host plant. During Rhizobia-Legumes symbiosis twotranscriptomic changes were observed by different technologies like RNAseq (Maunoury et al., 2010) or microarrays experiment (Benedito et al., 2008). These dramatic changes occur at the different steps of nodule development and are mediated by various gene expression regulators including several keys transcription factors and small RNAs. These small regulatory RNAs are produced after cleavage of long double-stranded or hairpin RNA precursors by particular enzymes of the ribonuclease III (RNase III) family, called DICERLIKEproteins (DCL). However, additional RNase III encoding genes are present in plant genomes, whose roles remain to be fully determined.In this work, we characterized the RNAse III family in the model M. truncatula, as well as other legumes species. We also investigated the involvement in nodule development of MtRTL1b, one RNAse III, a nodule-specific orthologue of AtRTL1, a putative silencing repressor in Arabidopsis thaliana. First, we showed that the expression of this gene is activated just before differentiation and is mainly restricted in the interzone, where bacteroid become fully differentiated into the host cells and in the nitrogen fixation zone of the nodule. Repression of MtRTL1b expression, by RNA interference in transgenic roots, affected nodule development, nitrogen fixation and bacteroid viability while an opposite phenotype was observed in roots with ectopic expression of this gene. Then, RNASeq analyses showed that the RNAileads to the down-regulation of 1038 genes, including more than 109 NCRs, encoding peptides involved in bacteroid development and/or viability in indeterminate nodules. Moreover, genes involved in metabolic pathways and redox regulations as well as other genes involved in symbiosis, like leghemoglobins, are also down-regulated. RNAseq of small RNAs and double strand RNAs are under analysis to characterize changes in sRNA populations and identify dsRNA substrates of this RNAse III during nodule development.

Nicotiana benthamiana is now an established platform for molecular farming, the production of biopharmaceuticals in plants. Infiltration with Agrobacterium tumefaciens (agroinfiltration) is commonly used to transiently express one or multiple transgenes in N. benthamiana leaves. Agroinfiltrated N. benthamiana is a flexible and scalable recombinant protein (RP) production platform, but is impeded by low RP yields. Plant proteases can degrade RPs and thus limit RP accumulation. To inform, design and implement strategies for enhancing RP accumulation, I present four papers about proteases and protease inhibitors in agroinfiltrated N. benthamiana. First, I investigated the transcriptome, extracellular proteome and active secretome to understand the plant response to agroinfiltration and investigate the expressed proteases. I show that an extracellular immune response is mounted at the expense of photosynthesis. Comprehensive annotation and monitoring uncover a large, diverse repertoire of proteases in agroinfiltrated leaves, indicating that broad-range depletion of protease activity may be required to enhance RP accumulation. Second, I reviewed the literature on multifunctional plant protease inhibitors (PIs) and grouped them into three types of multifunctional PIs that evolved independently. Third, I screened candidate PIs and discovered that three new, unrelated PIs enhance RP accumulation. I present universal elements of the RP degradation machinery, uncovering new questions on our understanding of the protease network that degrades RPs. Fourth, I identified targets of SlCYS8, a PI that enhances RP accumulation. The target proteases of SlCYS8 are implicated in RP degradation and the high specificity of SlCYS8 can be used to study their role in other processes. By elucidating the immune response to agroinfiltration, by uncovering the N. benthamiana protease repertoire and by providing new tools to deplete the activity of specific proteases, this thesis makes a relevant contribution to both basic plant research and molecular farming.

Homeostatic plasticity is important in neurobiology for stabilising neuronal networks in the face of Hebbian forms of synaptic plasticity that are thought to mediate memory storage. Impairment of homeostatic plasticity has also been implicated in neurological diseases such as Rett syndrome and fragile X syndrome. Homeostatic plasticity can be achieved through scaling of the strength of synaptic connections between neurones or by changes in intrinsic excitability. While homeostatic plasticity has been studied mainly using in vitro preparations, it is for the most part not known whether changes of neural activity in vivo induce homeostatic changes. The molecular pathway responsible for homeostatic plasticity still remains unclear. In this thesis, I have used stereotaxic surgery to over express Kir2.1, an inwardly rectifying potassium channel, in vivo in the brains of adult mice. I show that the expression of Kir2.1 through adeno-associated virus (AAV) does not cause any adverse effects in the dentate gyrus nor the CA1 of the mouse hippocampus. I go on to use slice patch clamp methods to measure the change in electrical properties of granule cells in the dentate gyrus and pyramidal cells in CA1 caused by expression of Kir2.1. I show that the excitability of neurones expressing Kir2.1 was reduced compared to control neurones. By 2 weeks after virus injection the neurones showed homeostatic plasticity in response to Kir2.1 over expression. Interestingly, the mechanism of adaptation was different in different types of cells; dentate gyrus granule cells adapted through change in their intrinsic excitability, whereas CA1 pyramidal cells adapted by modifying the strength of their synaptic inputs. To establish whether induction of homeostatic plasticity is associated with changes in gene expression I used fluorescent activated cell sorting (FACs) to isolate pure population of neurones infected with viruses. I then sequenced RNA extracted from neurones expressing Kir2.1 and control neurones. Analysis of the RNAseq data revealed molecular candidates involved in homeostatic plasticity. In summary, I show that Kir2.1 over expression causes change in excitability and subsequent homeostatic plasticity in vivo. The mechanism of adaptation differs between cell types. RNAseq results identify novel candidates for future investigation.

Thousands of chemical pollutants enter the environment continuously, each with the potential to cause adverse effects in both terrestrial and aquatic organisms. As a result, organisms are often exposed to a mixture of stressors within their habitat. Populations of fish inhabiting most aquatic environments are exposed to time-varying or repeated pulses of exposure, driven by run-off events or spills, or due to their mobility between polluted and clean waters. Therefore, the sustainability of fish populations is critically dependent on their ability to adapt to frequent changes in their local environment. Despite this, legislation to protect the environment from chemical contamination are generally based on toxicological measurements following exposures to single stressors, conducted under optimal laboratory conditions, and that do not take into account the variation in susceptibility of wild populations, or the potential consequences of exposure for the susceptibility of the population during future exposures, including across generations. Increasing evidence is suggesting that a number of chemicals may interact with the epigenome, and that differential responses to pollutants may be modulated, at least in part, via epigenetic mechanisms. However, our understanding of the role of epigenetic mechanisms in normal development in fish models or its susceptibility to exposure to environmental stressors is currently very limited. This thesis aimed to document the mechanisms of genetic and epigenetic responses to industrial pollutants in fish, and to explore the extent to which differential responses can be induced in the lab following exposure during the critical window of embryonic development or in adults. To address these objectives, I performed a series of experiments using both the zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus) as fish models. I first used the zebrafish (Danio rerio) model to investigate the sex-specific transcription and DNA methylation profiles for genes involved in the regulation of reproduction and in epigenetic signalling in the livers and gonads. I provide evidence of the sex-specific transcription of genes involved in reproduction and their regulation by epigenetic signalling in this commonly used vertebrate model and highlight important considerations regarding the use of whole tissues comprised of multiple cell types in epigenetic and transcriptomic studies. I then investigated the potential for exposure to Bisphenol A (BPA) to cause adverse effects on reproduction and to disrupt the expression profiles and promotor DNA methylation of target genes important for reproductive function and epigenetic signalling in the zebrafish. To do this, I exposed breeding zebrafish to a range of BPA concentrations over 15 days and found that BPA disrupted reproductive processes in zebrafish, likely via estrogenic mechanisms, but only at high concentrations. Importantly, exposure to environmentally relevant concentrations of BPA resulted in altered transcription of key enzymes involved in DNA methylation maintenance, and caused changes in promoter DNA methylation. I also conducted a series of repeated exposures to copper in the three-spined stickleback to investigate the extent to which differential susceptibility can be induced in the lab. This work provides evidence that pre-exposure to copper results in differential responses in future exposure scenarios both when the initial exposure occurred in adults and during embryogenesis. For adults, fish appeared to recover completely from the initial exposure following a period of depuration of 30 days, but displayed decreased susceptibility upon re-exposure. In contrast, for fish exposed during the critical windows of embryonic development when epigenetic reprogramming are hypothesised to occur, differential copper accumulation was maintained throughout life. Importantly, the initial exposure caused increased tolerance in the offspring, which was inherited up to the F2 generation. This work provides valuable information regarding potential critical windows of development which may be more susceptible to effects associated with pre-exposure, highlighting that early life exposure to a low concentration of copper can induce differential responses to copper across generations. These data highlight the extent of differential responses to chemical stressors likely to be present in wild populations, and point towards the possibility that effective population management will likely require an in-depth understanding of the exposure history of a given population in order to manage restocking initiatives, and to inform conclusions drawn from toxicity testing studies conducted using individuals originating from wild populations. In addition, these data suggest that it is likely that both epigenetic and genetic changes can contribute to the adaptation of individual populations to their local environment. Finally, other vertebrates including humans have been shown to be exposed to the chemicals tested in this thesis. Therefore, this highlights the potential for these chemicals to also cause toxic effects in humans, potentially via (epi) genetic mechanisms, and advocate the testing of the potential for inheritable phenotypes, such as those described in this thesis, to occur in mammalian models.

Helicobacter pylori infects more than half of the world's population, and is known to be involved in several diseases including gastric cancer. Its close interactions with the stomach and host immune system serves as a good model to study the co-adaptation and co-evolution of the organisms in the stomach micro-environment. In this project, we utilized RNA-seq and data analysis tools to investigate differentially expressed genes by H. pylori in patients at different stages of early gastric cancer development. We also investigated the abundance and diversity of bacterial genera other than H. pylori, and looked for correlations with H. pylori presence and number. For differential gene expression of H. pylori, one gene was differentially expressed between samples of corpus atrophy without metaplasia vs. samples of antrum gastritis, and eight genes were found to be differentially expressed between samples of corpus atrophy with metaplasia vs. samples with pan-gastritis. When samples were clustered into different groups based on the expression data, 52 genes (shared or unique to the specific comparison groups) were found to be differentially expressed, but no apparent patterns were observed that could be explained by medical or sample collection data. For bacterial diversity and abundances, we found several genera colonizing the stomach, of which some have been previously identified. While most of these bacteria colonize regardless of the presence of H. pylori, the abundance of three genera, Wolinella, Campylobacter, and Veillonella, seem to be correlated with the presence of H. pylori.

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No. of bitstreams: 1 AnaCarolinaPereiraRocha_DISSERT.pdf: 5861142 bytes, checksum: 39f089afa48682089f4ef1bb8949e060 (MD5) Previous issue date: 2017-09-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A diferencia??o das c?lulas-tronco mesenquimais humanas (CTMh) em osteoblasto segue um programa espec?fico de express?o g?nica, comprometendo-se inicalmente sob a influ?ncia das vias Wnt (Wingless pathway) e das BMPs (bone morphogenetic protein) que direcionam a c?lula a sua transforma??o em osteoblasto. Entretanto, as vias ativadas especificamente durante a fase inicial da diferencia??o s?o ainda pouco exploradas. Assim, o objetivo deste trabalho foi caracterizar o perfil transcricional do in?cio do processo de diferencia??o de CTMh, obtidas da veia do cord?o umbilical, em osteoblasto, a partir da t?cnica de RNA-Seq. A partir dos dados de transcriptoma, foi produzida uma lista ordenada de genes funcionalmente associados, obtida pela me?dia da expressa?o ge?nica tomada sobre genes vizinhos nesta lista, facilitando, assim, a sua interpretac?a?o biolo?gica. Para o estudo de ontologia g?nica e delineamento do perfil de express?o durante a osteog?nese, os processos metab?licos e fun??es moleculares significativamente alterados durante o decorrer do processo de diferencia??o foram analisados em diversas ferramentas (REVIGO, GOrila, PANTHER, LNCipedia e NONCODE) e descritos. Durante a indu??o ? diferencia??o das CTMh em osteoblasto, foi observado o aumento da express?o de genes caracter?sticos do fen?tipo osteobl?stico j? a partir do primeiro dia de diferencia??o. Foram identificados RNAs n?o codificantes, consoante a evolu??o da diferencia??o, bem como genes envolvidos na forma??o de rafts de membrana, j? a partir do terceiro dia de diferencia??o. Durante o terceiro dia de indu??o, genes envolvidos na regula??o da diferencia??o celular e em outros processos biol?gicos que precedem a diferencia??o, como ades?o celular, sinaliza??o e resposta ? fatores qu?micos externos j? apresentaram aumento em sua express?o. O estudo da express?o g?nica durante estes tr?s primeiros dias revelou ainda a diminui??o na express?o de genes envolvidos em processos biol?gicos de manuten??o de metabolismo basal, degrada??o de RNA e organiza??o do citoesqueleto, indicando assim que as mudan?as celulares que levam a c?lula a entrar em diferencia??o podem ter origem durante os tr?s primeiros dias de tratamento osteog?nico. / Human mesenchymal stem cells (hMSC) differentiation into osteoblast follows a specific gene expression program, committing itself initially under Wnt and BMPs pathways influence then differentiating into osteoblasts. However, specifically activated pathways during the first three days of differentiation are still poorly understood. Considering next generation sequencing technologies efficiency, and the lack of characterization in early hMSC differentiation process, in this work we used Illumina RNA-Seq to investigate the changes in these cells transcriptome. We used ex-vivo cultures of two human umbilical cord veins. Data from the complete transcriptome were analyzed in Transcriptogramer for the production of an ordered list of functionally associated genes, obtained by the mean of the gene expression taken on neighboring genes in this list, thus facilitating their biological interpretation. To study gene ontology and gene expression profile during osteogenesis, the metabolic processes and molecular functions significantly altered during the course of the differentiation process were analyzed in several tools (REVIGO, GOrila, PANTHER, LNCipedia and NONCODE) and properly described. During hMSC differentiation, an increase in expression of osteoblastic phenotype genes was observed as soon as the first day of differentiation started. Noncoding RNAs were also identified, depending on the evolution of the differentiation process, as well as genes involved in the formation of membrane rafts, on the third day of differentiation. During the third day of induction, cell differentiation regulation genes and other important genes on biological processes that precede differentiation, such as cell adhesion, signaling and external chemical factors response, have already increased expression. The study of gene expression during these first three days also revealed the decrease in the expression of groups of genes crucial to basal metabolism maintenance, RNA degradation and cytoskeleton organization, thus indicating that the cellular changes that lead the cell into differentiation may originate during the first three days of osteogenic treatment.