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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_simone_ribeiro_lucho.pdf: 5607081 bytes, checksum: 6b6e595948041f7d37a85b68d28e2313 (MD5) Previous issue date: 2018-04-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Atualmente, existe uma demanda global por fontes adicionais de edulcorantes naturais e compostos antioxidantes e as plantas de Stevia rebaudiana, além de possuirem edulcorantes, chamados de glicosídeos de esteviol (GSs) também apresentam grande quantidade de compostos fenólicos. Diante disso, o presente estudo centrou-se em explorar a ação elicitora de quatro reguladores de crescimento, metil jasmonato (MeJa), espermidina (SPD), ácido salicílico (AS) e paclobutrazol (PBZ) e de diferentes concentrações de NaCl no conteúdo dos GSs (esteviosídeo e rebaudiosídeo A) e compostos fenólicos, bem como, sobre a capacidade antioxidante total e nível de expressão dos genes envolvidos nos três estágios da via de biossíntese dos GSs em S. rebaudiana. Para isso, foram realizados quatro estudos, sendo que o primeiro teve por objetivo avaliar a estabilidade da expressão de sete genes candidatos a normalizadores (18S, Actina, Aquaporina, Calmodulina, Fator de Alongamento-1a, Malato desidrogenase e Ubiquitina) para posteriormente serem usados em estudos de expressão gênica. Inicialmente as plantas de estevia foram cultivadas in vitro e posteriormente passaram para um sistema hidropônico de fluxo contínuo com raízes flutuantes. Os tratamentos foram compostos pelo controle, somente com solução de Hoagland (50%) e os demais contendo a mesma solução adicionados de 100 μM de MeJa, SPD e AS. Após 24 h da aplicação dos tratamentos, as folhas destas plantas foram coletadas em um período de cinco dias. Os resultados indicaram que os genes Ubiquitina e Actina podem ser usados para normalizar os dados de expressão gênica em Stevia rebaudiana, pois ambos os genes apresentaram alta estabilidade. O segundo estudo visou avaliar o efeito de MeJa, SPD, AS e PBZ sobre os níveis de expressão dos genes relacionados à biossíntese dos GSs e também sobre o conteúdo de esteviosídeo e rebaudiosídeo A. As condições experimentais foram as mesmas do primeiro estudo, exceto o tratamento com PBZ, que neste estudo constituiu mais um tratamento. Os resultados forneceram evidências diretas de que os tratamentos com MeJa e SPD resultaram em aumento na transcrição dos genes relacionados à biossíntese dos GSs, enquanto que o PBZ causou uma diminuição na expressão, principalmente naqueles genes que codificam as enzimas caurenoides (GGDPS, CDPS, KS, KO). O tratamento com AS não afetou a transcrição dos genes UGT85C2, UGT74G1 e UGT76G1 e causou uma diminuição nos níveis de esteviosídeo. O terceiro estudo teve por objetivo avaliar se algum dos compostos elicitores (MeJa, SPD, AS ou PBZ) eram capazes de aumentar o conteúdo de compostos fenólicos e a capacidade antioxidante das plantas de estevia. As condições experimentais foram as mesmas do segundo estudo. Nossos resultados mostraram que a exposição ao MeJa propiciou maior produção de compostos fenólicos solúveis totais, carotenoides e adicionalmente uma atividade antioxidante aprimorada. Por outro lado, a adição de SPD, AS e PBZ não mostrou aumento significativo em nenhum dos parâmetros avaliados. O quarto e último experimento foi realizado in vitro e teve como objetivo avaliar o efeito de diferentes concentrações de NaCl nos parâmetros de crescimento, açúcares solúveis totais, GSs e compostos fenólicos, bem como, na capacidade antioxidante e no nível de expressão dos genes envolvidos na biossíntese dos GSs em S. rebaudiana. Para atender este objetivo plantas de estevia foram inicialmente cultivadas in vitro em meio MS (50%) com diferentes concentrações de NaCl. Os resultados mostraram que as maiores concentrações de NaCl diminuíram alguns parâmetros de crescimento (número de raízes, massa seca e fresca) enquanto aumentaram a capacidade antioxidante (ensaio FRAP), ácidos hidroxicinâmicos e teor de açúcares solúveis totais. Além disso, vários genes que codificam importantes enzimas (CMS, CMK, HDR e UGT76G1) da via biossintética dos GSs foram regulados positivamente após o tratamentos com NaCl. Os resultados dos quatro estudos permitiram novos insights sobre os mecanismos de respostas fisiológicas, bioquímicas e moleculares das plantas de estevia após a aplicação de diferentes elicitores e abriu novos caminhos a serem explorados que assegurem o bom uso dessas fontes naturais de compostos, bem como a sobrevivência e sustentabilidade desta espécie. / Currently, there is a global demand for additional sources of natural sweeteners and antioxidant compounds. Stevia rebaudiana plants, besides having steviol glycosides (SGs) sweeteners, also present a large amount of phenolic compounds. Therefore, the present study is focused on the elicitor action of four growth regulators, methyl jasmonate (MeJa), spermidine (SPD), salicylic acid (AS) and paclobutrazol (PBZ) and different concentrations of NaCl in the content of SGs (stevioside and rebaudioside A) and phenolic compounds, as well as on the total antioxidant capacity and level of expression of the genes involved in the three stages of the SGs biosynthesis pathway in S. rebaudiana. For this, four studies were carried out, the first of which was aimed at evaluating the stability of the expression of seven candidate genes for normalizers (18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1-α, Malate dehydrogenase, and Ubiquitin) in gene expression studies for the later use. Initially the stevia plants were cultivated in vitro and later passed to a hydroponic continuous flow system with floating roots. The treatments were composed by the control, only with Hoagland solution (50%) and the others containing the same solution added with 100 μM MeJa, SPD and SA. After 24 h of application of the treatments, the leaves of these plants were collected in a period of five days. The results indicated that the Ubiquitin and Actin genes can be used to normalize the gene expression data in Stevia rebaudiana, since both genes presented high stability. The second study aimed at evaluating the effect of MeJa, SPD, SA and PBZ on the expression levels of genes related to SGs biosynthesis and also on the content of stevioside and rebaudioside A. The experimental conditions were the same as in the first study except the treatment with PBZ, which in this study constituted another treatment. The results provided direct evidence that MeJa and SPD treatments resulted in increased transcription of genes related to SGs biosynthesis, whereas PBZ caused a decrease in expression, especially in those genes encoding caurenoid enzymes (GGDPS, CDPS, KS , KO). Treatment with SA did not affect the transcription of UGT85C2, UGT74G1 and UGT76G1 genes and caused a decrease in stevioside levels. The third study aimed to evaluate if any of the elicitor compounds (MeJa, SPD, SA or PBZ) were able to increase the content of phenolic compounds and the antioxidant capacity of stevia plants. The experimental conditions were the same as in the second study. Our results showed that exposure to MeJa provided higher production of total soluble phenolic compounds, carotenoids and in addition an improved antioxidant activity. On the other hand, the addition of SPD, SA and PBZ showed no significant increase in any of the evaluated parameters. The fourth and last experiment was carried out in vitro and had the objective of evaluating the effect of different concentrations of NaCl on growth parameters, total soluble sugars, SGs and phenolic compounds, as well as on the antioxidant capacity and level of expression of the genes involved in biosynthesis of SGs in S. rebaudiana. To meet this objective, stevia plants were initially cultured in vitro in MS medium (50%) with different concentrations of NaCl. The results showed that the higher concentrations of NaCl decreased some growth parameters (number of roots, dry and fresh mass) while there was an increase in the antioxidant capacity (FRAP test), hydroxycinnamic acids and total soluble sugars. In addition, several genes encoding important enzymes (CMS, CMK, HDR and UGT76G1) from the SGs biosynthetic pathway were positively regulated after NaCl treatments. The results of the four studies allowed new insights into the mechanisms of physiological, biochemical and molecular responses of stevia plants after the application of different elicitors and opened new avenues to be explored that ensure the good use of these natural sources of compounds as well as the survival and sustainability of this species.

Connaitre et appréhender les mécanismes de la diversification des espèces est important pour la gestion des écosystèmes, la prévision des impacts des changements climatiques et la compréhension de la biodiversité actuelle et passée. Le but de cette thèse est de comprendre et de déceler les mécanismes génétiques à l’origine de la diversification des espèces en présence de flux de gènes. Cette thèse se focalise sur le modèle biologique Symphonia globulifera, qui compte deux écotypes : le S.globulifera spécialiste de la terra firme et le S.sp1, spécialiste des bas-fonds. Ces deux écotypes montrent une faible différenciation génétique, malgré la présence de deux phénotypes différents.Une première étape a été de mettre en évidence la présence d’une adaptation locale au sein de cette espèce, par le biais d’une expérimentation de jardins de transplantation réciproques, permettant d’expliquer la répartition des écotypes dans leur habitat naturel. Ensuite, dans le but d’identifier les mécanismes sous-jacent à cette adaptation locale, j’ai testé l’hypothèse que la méthylation des gènes pourrait être une marque de l’épigénétique contribuant à la divergence des écotypes, par l’utilisation d’enzyme sensibles à la méthylation dans un protocole de génotypage AFLP. Enfin, un séquençage haut-débit du transcriptome des écotypes en jardins de transplantation réciproques m’a permis de mettre en évidence une expression différentielle des gènes entre les écotypes, qui pourrait expliquer les différences phénotypiques observées entre les écotypes malgré une faible différentiation génétique. Ces travaux de thèse s’appuie, ainsi, sur des données de traits phénotypiques, de génotypage AFLP et de séquençage à haut débit du transcriptome pour montrer la valeur importante de la régulation des gènes dans la divergence des écotypes adaptés localement, et un faible rôle de la méthylation de l’ADN dans l’établissement cette adaptation locale. / Understanding of the mechanisms driving diversification of species is a significant way to improve the management of ecosystems, predict the impacts of climate change and understand the actual and past biodiversity level. The aim of this thesis is to understand and comprehend genetic mechanisms behind the diversification of species in the presence of gene flow. This thesis is focused on the biological model Symphonia globulifera, which presents two ecotypes: the S.globulifera, specialist of seasonally flooded lowlands and S.sp1, specialist of terra firme. These two ecotypes show low genetic differentiation, despite the presence of two apparent phenotypes. A first part of this thesis was to test the presence of local adaptation of this using reciprocal transplant experiment gardens, allowing the understanding of the ecotypes distributions in their natural habitats. Then, this local adaptation in the presence of gene flow, directed me to the regulation of gene methylation in order to see the role this brand of epigenetics can have in the divergence of the ecotypes. In a third part of the thesis, new generation sequencing of the transcriptome ecotypes in reciprocal gardens transplantations allowed me to show the evidence of gene regulation to differentiate the ecotypes. This work thesis is based on phenotype records data, AFLP genotyping and high-throughput sequencing of the transcriptome, in order to show the important value of gene regulation in the divergence of the locally adapted ecotypes, and a weak role of DNA methylation in the establishment of local adaptation.

Após o seqüenciamento completo do genoma humano, a busca e caracterização do conjunto completo de genes humanos constitui-se no principal desafio nesta área de investigação, sendo o passo limitante para o progresso na exploração dos dados contidos no seqüenciamento deste genoma. O projeto de transcriptoma denominado \"Transcript Finishing Initiative\" (TFI) surgiu neste contexto, com o objetivo principal de gerar fragmentos parciais de transcritos humanos, que não haviam sido descritos previamente e determinar sua seqüência, para iniciar a caracterização de novos genes humanos. A estratégia utilizada foi q alinhamento de todas as seqüências ORESTES e ESTs disponíveis com a seqüência pública do genoma humano e o agrupamento j c1usterização destas com base nas coordenadas deste genoma. Algumas das regiões que não eram cobertas por estas seqüências foram, então, completadas, por RT-PCR, utilizando-se primers ancorados nos clusters vizinhos. Cada par de clusters de ESTs selecionado para validação experimental foi designado como uma Unidade do \"Transcript Finishing\" (TFU), tendo sido validadas experimentalmente, pelo grupo TFI, Um total de 211 TFUs foram validadas, sendo que 197 seqüências consenso foram submetidas ao Genbank (CF272536-CF272733). Atualmente, apenas um pequeno número destas seqüências ainda são considerados genes novos, sem que haja um cDNA depositado em banco de dados; contudo para um número considerável destas TFUs não existe qualquer caracterização sobre sua função. Na tentativa de contribuir para melhor caracterização dos genes identificados no projeto TFI, e tendo, como base, a linha de pesquisa do laboratório, que busca genes diferencialmente expressos envolvidos em transformação maligna/tumorigênese, o presente trabalho propôs a utilização das seqüências TFUs para estudar sua possível associação com tumores de glia humanos e outros tipos de tumores (de próstata e de mama). Para tanto, as TFUs foram analisadas \"in silico\" para estabelecer seu grau de ineditismo como um novo gene ou um gene sem função conhecida, e, para análise de sua expressão diferencial entre tecidos normais e tumorais de cérebro, próstata e mama. Para validar estas análises computacionais na bancada, foram gerados macro- e microarranjos de DNA utilizando-se as TFUs disponíveis como clones físicos ou amplicons, para o rastreamento com sondas das linhagens celulares A172 e T98G de glioblastomas. Os resultados das análises destes dados foram confirmados por PCR quantitativo tanto nas linhagens como em amostras clínicas de astrocitomas que apresentam diversos graus de malignidade. Como resultado, foi possível organizar um Banco de Clones Físicos de TFUs, além de identificar e selecionar uma TFU (168), cuja expressão correlaciona diretamente com o grau de malignidade dos tumores de glia. Esta seqüência corresponde a um novo gene, já que não existe a seqüência de cDNA completo nos bancos de dados. Em vista disto, a TFU168 foi selecionada para estudos funcionais posteriores, que já estão em andamento, através da obtenção de suaseqüência completa de cDNA para ensaios de superexpressão e do silenciamento gênico através de RNAi. / Upon complete sequencing of the human genome, identification and characterization of the complete set of human genes constitutes the major challenge in this research field, constituting the limiting step for progress in exploration of the informations contained in the genome sequencing data. The Transcript Finishing Initiative (TFI) transcriptome project arose in this context, aiming at the generation, sequencing and characterization of partial new human transcripts and genes. The strategy adopted was the alignment of the alI the available ORESTES and EST sequences data with the public human genome sequence and their clusterization based on the coordinates of this genome. Thus, some of the regions which were not cover by ESTs and ORESTES (gaps) were then completed by RT-PCR using primers anchored in the neighboring clusters. Each pair of EST clusters selected for experimental validation was named Transcript Finishing Unit (TFU). A large number (211) of TFU s were validated and 197 -consensus sequences were submitted to the Genbank (CF272536-CF272733). At present, only a few of these sequences are considered as new genes without a full-Iength cDNA sequence deposited in the data bank, however, no functional characterization is yet available for a large number of these sequences. In an attempt to contribute to further characterization of these genes identified in the TFI project and keeping in mind the main interest of our laboratory, which is the identification of differentially expressed genes in tumor versus normal tissue, the present work aims at utilizing these TFUs to find differentially expressed genes associated with human glial tumors and with other kinds of tumors. To this end, these sequences were first subjected to in silico analysis in order to establish their degree of ineditism (new sequences and/or sequences with unknown function) and their expression profile between normal and tumoral tissues of brain, mammary gland and prostate. To validate this computational analysis, DNA macro- and microarrays were generated with the TFU sequences and screened with cDNA probes obtained from the A172 and T98G glioblastomas cell lines. The results of these screenings were confirmed by quantitative PCR both in cell lines and in tumor samples of different degrees of malignancy. The results obtained in this work allowed the organization of a TFUs Physical Clones Bank and the identification and selection of one sequence (TFU 168), whose expression is directly re1ated to the degree of tumor malignancy. This sequence constitutes a new gene, since no complete cDNA sequence is available in the data banks. Therefore, TFU168 was selected for further functional studies by obtaining its full-Iength cDNA sequence to be used for over expression by silencing this gene using RNAi.

Philosophiae Doctor - PhD / Drought is the most complex phenomenon that remained to be a potential and historic challenge to human welfare. It affects plant productivity by eliciting perturbations related to a pathway that controls a normal, functionally intact biological process of the plant. Sorghum (Sorghum bicolor (L.) Moench), a drought adapted model cereal grass is a potential target in the modem agricultural research towards understanding the molecular and cellular basis of drought tolerance. This study reports on the genomic and proteomic findings of drought tolerance in sorghum combining the results from in silica and experimental analysis. Pipeline that includes mapping expression data from 92 normalized cDNAs to genomic loci were used to identify drought tolerant genes. Integrative analysis was carried out using sequence similarity search, metabolic pathway, gene expression profiling and orthology relation to investigate genes of interest. Gene structure prediction was conducted using combination of ab initio and extrinsic evidence-driven information employing multi-criteria sources to improve accuracy. Gene ontology was used to cross-validate and to functionally assign and enrich genes. An integrated approach that subtly combines functional ontology based semantic data with expression profiling and biological networks was employed to analyse gene association with plant phenotypes and to identify and genetically dissect complex drought tolerance in sorghum. The gramene database was used to identify genes with direct or indirect association to drought related ontology terms in sorghum. Where direct association for sorghum genes were not available, genes were captured using Ensemble Biomart by transitive association based on the putative functions of sorghum orthologs in closely related species. Ontology mapping represented a direct or transitive association of genes to multiple drought related ontology terms based on sorghum specific genes or orthologs in related species. Correlation of genes to enriched gene ontology (GO)-terms (p-value < 0.05) related to the whole-plant structure was used to determine the extent of gene-phynotype association across-species and environmental stresses.

En Amérique Latine, les punaises hématophages Triatominae transmettent à l’homme le parasite Trypanosoma cruzi, responsable de la maladie de Chagas touchant actuellement 5 millions de personnes. Même si les programmes d’éradication chimique des vecteurs sont efficaces, la maladie persiste du fait de la recolonisation des habitations humaines par des vecteurs provenant d’habitats naturels. Ainsi, certaines espèces présentent une capacité d’adaptation aux anthroposystèmes (processus de domiciliation), alors que d’autres espèces apparentées ne l’ont pas. Comprendre cette capacité d’adaptation est crucial d’un point de vue épidémiologique afin de cibler les espèces présentant un risque pour l’homme. La capacité à s’adapter à un nouvel habitat pourrait être liée à l’évolution du répertoire de gènes du système chimiosensoriel, important pour la perception du milieu. Cette étude a porté sur le système chimiosensoriel des Triatominae dans le but de documenter le processus d’adaptation et donc de domiciliation des vecteurs. Des données transcriptomiques obtenues en séquençage à haut débit ont été utilisées pour annoter et répertorier les gènes chimiosensoriels ainsi que pour comparer leur expression au sein de punaises hématophages d’habitats différents. L’existence d’une relation entre les variations de ces gènes chez différentes espèces de Triatominae et leur capacité d’adaptation à un habitat a par la suite été évaluée. L’espèce T. brasiliensis en voie de domiciliation au Brésil et présentant à la fois des populations sylvatiques, péri-domiciliaires et domiciliaires, et différentes espèces du genre Rhodnius d’habitats variés, ont été étudiées, notamment les deux espèces sœurs, R. robustus, sylvatique en Amazonie et R. prolixus majoritairement domiciliée dans toute son aire de répartition. En l’absence de génomes de références suffisamment proches de T. brasiliensis et des 10 espèces de Rhodnius étudiées, leurs transcriptomes ont été assemblés de novo. Les transcriptomes des deux espèces R. prolixus et R. robustus ont été assemblés par alignement sur le génome de R. prolixus. Chez ces différentes espèces de Triatominae étudiées, l’analyse du répertoire des gènes chimiosensoriels codant les OBPs et CSPs (familles multigéniques) comparé à celui d’autres Paranéoptères a montré des expansions géniques pouvant refléter des processus adaptatifs. Par ailleurs, chez les différentes espèces du genre Rhodnius, il existe une corrélation positive entre le nombre de gènes codant les OBPs et la capacité de domiciliation, suggérant l’implication de cette famille de gènes dans l’adaptation au milieu anthropique. Les analyses d’expression différentielle concernant les différentes populations de T. brasiliensis et les espèces R. prolixus/R. robustus ont montré qu’un certain nombre de transcrits sont différentiellement exprimés selon l’environnement dans lequel ont évolué les punaises notamment des gènes chimiosensoriels (OBPs, CSPs) ainsi que des gènes impliqués dans le rythme circadien et le comportement de recherche alimentaire (Takeout), dans la réponse à des stress environnementaux comme des gènes de détoxification (P450, glutathione S-transférase), dans la résistance à des changements climatiques (Heat-shock protéines) et dans la protection du milieu extérieur (protéines cuticulaires). Ce travail a permis de mettre à la disposition de la communauté scientifique des outils performants pour l’étude du processus de domiciliation des vecteurs de la maladie de Chagas (transcriptome, répertoire de gènes). Il a également permis de révéler des gènes qui pourraient être impliqués dans l’adaptation et/ou la plasticité phénotypique en réponse à un changement d’habitat. La compréhension des bases moléculaires de l’adaptation des vecteurs aux habitations humaines ouvre des potentialités de développer des méthodes alternatives de lutte contre les vecteurs qui pourraient être basées sur une perturbation de la communication chimique. / In Latin America, the bloodsucking bugs (Triatominae, Hemiptera, Reduviidae) are vectors of the parasite Trypanosoma cruzi, which causes Chagas disease. More than five million people are infected. Even if chemical control campaigns are effective against vectors, the disease persists due to the recolonization of human habitations by vectors from natural habitats. Some species have the capacity to adapt to anthroposystems (domiciliation process), while other related species do not. Understanding this capacity to adapt is crucial from an epidemiological perspective to target species at risk to humans. The capacity to adapt to a new habitat could be linked to changes in the repertoire of chemosensory system genes, particularly for odorant binding proteins (OBP) and chemosensory proteins (CSP), which are important proteins to detect various odor stimuli. This study is based on the chemosensory system of Triatominae to document the adaptation process and then the domiciliation of the vectors. Transcriptomic data obtained by high-throughput sequencing were used to annotate and list the chemosensory genes and also to compare their expression in bloodsucking bugs from different habitats. The relationship between changes in these genes in different Triatominae species and their ability to adapt to a new habitat was evaluated. The species T. brasiliensis, which is in the process of domiciliation in Brazil with sylvatic, peridomiciliary and domiciliary populations, and various species of the genus Rhodnius from diverse habitats were studied, especially the two sibling species R. robustus, sylvatic in the Amazonia and R. prolixus mostly domiciliary throughout its geographical range. In the absence of a reference genome for T. brasiliensis, a reference transcriptome via de novo assembly (data 454 and Illumina) was achieved. The reference transcriptomes for 10 Rhodnius species were also established using the de novo assembly method. A genome reference based method on R. prolixus was also used to assemble the transcriptome of the two species R. prolixus and R. robustus. In the different species of the Triatominae studied, the chemosensory gene repertoire showed a high diversity and genic expansions compared to that of others Paraneoptera, which could reflect adaptive process. Furthermore, a positive correlation was shown between the number of OBP genes in Rhodnius species and their domiciliation ability, suggesting that this gene family is involved in the adaptation to anthropogenic environment. The differential expression analyses on the T. brasiliensis populations and the R. prolixus / R. robustus species showed that some transcripts are differentially expressed according to the environment in which the bugs have evolved, especially the chemosensory genes (OBP, CSP) and also genes involved in the circadian rhythm and foraging behavior (Takeout), in the response to environmental stress such as detoxification genes (P450, glutathione S-transferase), in resistance to climatic changes (heat-shock proteins) and in protection from the external environment (cuticular proteins).This work has helped make available to the scientific community powerful tools for studying the process of domiciliation of Chagas disease vectors (transcriptome, gene repertoire). It also revealed genes that could be involved in the adaptation and/or phenotypic plasticity in response to a change in habitat. Understanding the molecular basis of vector adaptation to human dwellings opens the potential to develop new tools to control the disease vectors, for example by disrupting chemical communication.

Differential expression analysis can be a powerful method to investigate expressed differences between closely related species. Our ambition is to highlight differentially expressed nuclear genes to explain the hybrid incompatibilities among the Amazonian, Centro-American and Orinocan semispecies of Drosophila paulistorum. RNA sequencing (RNA-seq) establishes the foundation of the study where we first evaluate the influence of two distinct alignment references. We discover the benefits of concatenating a de novo assembly instead of using the genome reference of a close relative. The bioinformatic pipeline handles the interesting inclusion of D. melanogaster and D. willistoni, where their contribution assists in the search for previously studied speciation genes. Among the down- and upregulated subsets we can see a diverse mix of general biological processes such as regulatory functions and transcriptional factors. In the end we uncover potential indications to why the Amazonian seems to be the least compatible semispecie to produce hybrids. This study provides a competitive working frame for comparative RNA-seq studies between closely related species.

No description available.

A rich diversity of social behaviours exists in the animal kingdom, and these behaviours have evolved to perform a variety of adaptive functions. Social behaviours show variation both among and within species, however the mechanisms that give rise to this variation are not well understood. Using fruit flies (Drosophila melanogaster), my goal was to uncover the genetic and behavioural mechanisms that underpin natural variation in two different social behaviours: sociability and sexual aggression. First, I showed that sociability, which is the tendency of animals to engage in friendly activities together, is influenced by indirect genetic effects (IGEs), and that encounters among individuals drive these effects (Chapter 2). I then showed that sociability and social plasticity have low-moderate heritability (Chapter 3), and sociability is not correlated between the sexes or with activity. I then generated lineages of flies with high and low sociability using artificial selection (Chapter 4). The evolved lineages had significantly diverged sociability which was not associated with fitness measures or nearest-neighbor distances, but was negatively correlated with intrasexual aggression (Chapter 4). Finally, in sexual aggression, which I quantified as male forced copulation rate, I showed that evolved differences and differences due to social plasticity were both associated with the differential expression of many genes, but only a few of these genes were significant in both (Chapter 5). I also showed that these sets of genes are enriched in neuropeptide hormone and serotonin gene ontology categories, and that 4 of 7 chosen genes were validated for their effects on sexual aggression. Overall, this thesis sheds light on the complex mechanisms that underlie variation in these social behaviours, and it paves the way for future research to further elucidate some of these mechanisms, especially on the genetic basis of sociability using the evolved lineages I generated. / Thesis / Doctor of Philosophy (PhD) / Individual animals tend to vary in many traits including social behaviours. Using fruit flies, my goal was to understand what causes individuals to vary in two social behaviours: sociability and sexual aggression. I found that highly sociable flies tended to influence other flies to become more sociable due to a change in how much these flies interacted. I also found that individual differences in sociability are moderately heritable, and the genetic variation contributing to this is different between the sexes. Also, less sociable flies tended to be more aggressive than highly sociable flies. Finally, for sexual aggression, I showed that variation in a male’s success in forcibly mating with a female was associated with changes in the expression of hundreds of genes, but these changes were mostly unique for evolved versus environmentally induced variation. Future work will similarly look to identify genes involved with individual differences in sociability.

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Weed resistance to control measures, particularly herbicides, is a growing problem in agriculture. In the case of herbicides, resistance is sometimes connected to genetic changes that directly affect the target site of the herbicide. Other cases are less straightforward where resistance arises without such a clear-cut mechanism. Understanding the genetic and gene regulatory mechanisms that may lead to the rapid evolution of resistance in weedy species is critical to securing our food supply. To study this phenomenon, we exposed young Arabidopsis plants to sublethal levels of one of four weed management stressors, glyphosate herbicide, trifloxysulfuron herbicide, mechanical clipping, and shading. To evaluate responses to these stressors we collected data on gene expression and regulation via epigenetic modification (methylation) and small RNA (sRNA). For all of the treatments except shade, the stress was limited in duration, and the plants were allowed to recover until flowering, to identify changes that persist to reproduction. At flowering, DNA for methylation bisulfite sequencing, RNA, and sRNA were extracted from newly formed rosette leaf tissue. Analyzing the individual datasets revealed many differential responses when compared to the untreated control for gene expression, methylation, and sRNA expression. All three measures showed increases in differential abundance that were unique to each stressor, with very little overlap between stressors. Herbicide treatments tended to exhibit the largest number of significant differential responses, with glyphosate treatment most often associated with the greatest differences and contributing to overlap. To evaluate how large datasets from methylation, gene expression, and sRNA analyses could be connected and mined to link regulatory information with changes in gene expression, the information from each dataset and for each gene was united in a single large matrix and mined with classification algorithms. Although our models were able to differentiate patterns in a set of simulated data, the raw datasets were too noisy for the models to consistently identify differentially expressed genes. However, by focusing on responses at a local level, we identified several genes with differential expression, differential sRNA, and differential methylation. While further studies will be needed to determine whether these epigenetic changes truly influence gene expression at these sites, the changes detected at the treatment level could prime the plants for future incidents of stress, including herbicides. / Doctor of Philosophy / Growing resistance to herbicides, particularly glyphosate, is one of the many problems facing agriculture. The rapid rise of resistance across herbicide classes has caused some to wonder if there is a mechanism of adaptation that does not involve mutations. Epigenetics is the study of changes in the phenotype that cannot be attributed to changes in the genotype. Typically, studies revolve around two features of the chromosomes: cytosine methylation and histone modifications. The former can influence how proteins interact with DNA, and the latter can influence protein access to DNA. Both can affect each other in self-reinforcing loops. They can affect gene expression, and DNA methylation can be directed by small RNA (sRNA), which can also influence gene expression through other pathways. To study these processes and their role in abiotic stress response, we aimed to analyze sRNA, RNA, and DNA from Arabidopsis thaliana plants under stress. The stresses applied were sublethal doses of the herbicides, glyphosate and trifloxysulfuron, as well as mechanical clipping and shade to represent other weed management stressors. The focus of the project was to analyze these responses individually and together to find epigenetic responses to stresses routinely encountered by weeds. We tested RNA for gene expression changes under our stress conditions and identified many, including some pertaining to DNA methylation regulation. The herbicide treatments were associated with upregulated defense genes and downregulated growth genes. Shade treated plants had many downregulated defense and other stress response genes. We also detected differential methylation and sRNA responses when compared to the control plants. Changes to methylation and sRNA only accounted for about 20% of the variation in gene expression. While attempting to link the epigenetic process of methylation to gene expression, we connected all the data sets and developed computer programs to try to make correlations. While these methods worked on a simulated dataset, we did not detect broad patterns of changes to epigenetic pathways that correlated strongly with gene expression in our experiment's data. There are many factors that can influence gene expression that could create noise that would hinder the algorithms' abilities to detect differentially expressed genes. This does not, however, rule out the possibility of epigenetic influence on gene expression in local contexts. Through scoring the traits of individual genes, we found several that interest us for future studies.