A fundamental goal in biological research is to gain an understanding of the evolutionary processes and genetic elements that drive speciation. Genes responsible for reproductive isolation in young divergent lineages are particularly poorly known. In this thesis, the speciation genetics of carrion (Corvus (corone) corone) and hooded (C. (corone) cornix) crows were studied. These taxa differ strikingly in colouration and meet in a narrow hybrid zone in Europe, yet appear to be very similar genetically. A major component of reproductive isolation is social selection on colour differences. First, we investigated the genetic basis of plumage divergence between carrion and hooded crows using a candidate gene approach. Nucleotide divergence was confirmed to be low, while there was no evidence for any of the sequenced genes to be associated with colour differences. Second, we performed a simulation study to assess the performance of RNA-seq, a relatively novel approach that we later employed ourselves. We asked how variation in transcriptome complexity and bioinformatic workflow affected the accuracy of gene expression profiling. We generally found reassuring robustness and made a number of specific recommendations. Third, we compared the corticosterone stress response of carrion and hooded crows. In accordance with the hypothesis that the degree of melanization and physiological traits are correlated due to pleiotropy, we found a higher stress response in hooded crows, and detected possibly associated gene expression in pituitary. Fourth, we investigated genomic divergence by assembling a hooded crow reference genome followed by whole-genome resequencing of four European population samples. Northern European carrion crows were more similar to hooded crows than to Spanish carrion crows, pointing towards rampant introgression far beyond the hybrid zone. Nevertheless, several narrow genomic regions harboured high between-taxon divergence and were potentially associated with phenotypic traits. Fifth, we compared whole-transcriptome gene expression profiles between crows, focusing on skin with developing feathers. We used a design that allowed to differentiate between taxon-specific, colour-specific and body patterning effects. Widespread underexpression of genes in the melanogenesis pathway was associated with grey colour, and we detected several genes that may contribute to colour divergence in this system.

Le syndrome de Usher est une maladie génétique associant surdité congénitale et rétinopathie pigmentaire (RP), auxquelles peuvent s'ajouter des troubles vestibulaires. Les différences phénotypiques, distinguées en 3 types cliniques, s'accompagnent d'une hétérogénéité génétique impliquant au moins 10 gènes. Identifier et caractériser les causes moléculaires grâce aux outils d'analyses génétiques disponibles permet d'améliorer la compréhension des mécanismes physiopathologiques à l'origine des symptômes du syndrome de Usher. Dans ce cadre, nous nous sommes inscrits dans une recherche d'exhaustivité des études moléculaires. Dans un premier temps, nous avons ainsi mis en place l'analyse et défini le spectre mutationnel des gènes minoritairement impliqués dans le type II (GPR98 et DFNB31). Nous avons également développé différents outils, notamment pour l'analyse de variants altérant le mécanisme d'épissage ou touchant les régions promotrices des gènes USH2.Ces travaux permettent d'obtenir un taux de détection des altérations conduisant au syndrome de Usher type 2 de 90 %. Ce taux est maintenant similaire à celui observé pour le type 1, qui constituait jusqu'ici la référence.Nous avons, dans un second temps, développé le séquençage nouvelle génération (NGS) appliqué à l'exome Usher. L'objectif de cette analyse était de tester la faisabilité et l'efficacité de cette approche, en vue de son éventuelle utilisation en diagnostic moléculaire. La définition des critères de qualité et la mise en place de la priorisation des variants ont été réalisées sur un groupe contrôle. L'étude a ensuite été étendue sur une cohorte de patients. Les résultats obtenus montrent qu'une utilisation en diagnostic est possible mais restera dépendante de l'amélioration de la technique du séquençage, de son analyse et des outils bioinformatiques pour interpréter le volume de données ainsi généré. / Usher syndrome is a genetic disorder combining sensorineural hearing loss (HL) and retinitis pigmentosa (RP). Some patients will also exhibit vestibular areflexia (VA). Clinical and genetic heterogeneity is recognized as the 3 clinical subgroups, defined mainly on the degree of HL and VA, can be caused by mutations in one of the 10 known genes. It is important to use all accessible genetic tools to identify and characterize molecular origin in order to improve the knowledge of the physiopathological mechanisms causing Usher Syndrome.In this context, we have developed an exhaustive approach. In a first step, we have implemented the analysis and established the mutational spectrum of the 2 minor USH2 genes (GPR98 and DFNB31). In addition, we have developed several tools, in particular to study variants susceptible to alter splicing or lying in the promoter regions of the USH2 genes.Thanks to this work, the USH2 mutation detection rate has now been raised to 90%, similar to that of USH1.We have then designed a targeted exome of the Usher genes to be sequenced using the GS Junior system (Roche 454). The aim of the study was to test the feasibility of this new technics for a possible transfer to diagnostic facilities. Quality criteria and variant priorization were set up on a control cohort (previously studied in one of the USH gene). The study has then been extended on a patient cohort. Our results indicate that NGS Usher-exome can be used in molecular diagnostics but improvement of the reliability of the sequencing technology, bioinformatics tools and dedicated databases is essential.

The main theme of this thesis research is concerned with developing a computational pipeline for processing Next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate tens of millions of short reads for each DNA/RNA sample. The alignment of a large volume of short reads to a reference genome is a key step in NGS data analysis. Although storing alignment information in the Sequence Alignment/Map (SAM) or Binary SAM (BAM) format is now standard, biomedical researchers still have difficulty accessing useful information. In order to assist biomedical researchers to conveniently access essential information from NGS data files in SAM/BAM format, we have developed a Graphical User Interface (GUI) software tool named SAMMate to pipeline human transcriptome quantification. SAMMate allows researchers to easily process NGS data files in SAM/BAM format and is compatible with both single-end and paired-end sequencing technologies. It also allows researchers to accurately calculate gene expression abundance scores.

As neuropatias periféricas hereditárias são um grupo heterogêneo de doenças relacionadas que afetam o sistema nervoso periférico. Elas podem ser classificadas de acordo com a velocidade de condução motora nos membros superiores (tipo 1 - CMT1, tipo 2 - CMT2 ou intermediário - iCMT), de acordo com o padrão de herança (autossômicas dominantes, autossômicas recessivas ou ligadas ao X) e quanto ao fenótipo de apresentação (neuropatias hereditária sensitivo e motora - CMT, neuropatia hereditária sensitiva - HSN ou neuropatia motora hereditária distal - dHMN). O uso das tecnologias de sequenciamento de nova geração (NGS) para diagnóstico de pacientes com neuropatia hereditária é particularmente eficiente uma vez que representa uma doença Mendeliana com mais de 90 genes diferentes relacionados. Foram avaliados 30 pacientes com diferentes subtipos de neuropatia hereditária (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN e 3 HSN). Foram identificadas 6 mutações (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) e 7 variantes provavelmente patogênicas (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1). Com uma taxa de positividade de 43.3%. As variantes provavelmente patogênicas foram consideradas como a causa da apresentação fenotípica apresentada pelos pacientes baseado na frequência de variantes nos bancos de população normal, no efeito bioquímico das variantes sobre a estrutura proteica e pela análise in silico. No entanto, essas variantes necessitam de evidências adicionais que confirmem sua patogenicidade. Foram identificadas variantes novas nos genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 e FLVCR1 ampliando a variabilidade genotípica desses genes. A associação das mutações identificadas nos genes VRK1, KIF1A, IGHMBP2 e FLVRC1 permitiu a expansão dos fenótipos relacionados a esses genes. Mutações no gene VRK1 podem causar uma dHMN com sinais de liberação piramidal e envolviemento preferencial do compartimento posterior da perna. Transtorno do espectro autista pode ser observado em associação a mutações no gene KIF1A e mutações no gene FLVRC1 podem causar um fenótipo grave caracterizado por insensibilidade congénita a dor e acromutilações. Mutações no gene IGHMBP2 podem causar uma sobreposição entre os fenótipos SMARD1/CMT2S com disautonomia restrita ao trato gastro intestinal. Esse estudo demonstra que o uso de WES para o diagnóstico molecular de doenças geneticamente heterogêneas como as neuropatias hereditárias é uma ferramenta útil. / The hereditary peripheral neuropathies are a heterogeneous group of genetic disorders in which peripheral nervous system degeneration leads to weakness, atrophy and loss of sensation. It can be classified according motor conduction velocities in the upper limbs (type 1 - CMT1, type 2 - CMT2 or intermediate - iCMT), according to inheritance pattern (autosomal dominant, autosomal recessive or X linked) and according to the mainly group of fibres clinically involved (hereditary sensory and motor neuropathy - CMT, hereditary sensory neuropathy - HSN or distal hereditary motor neuropathy - dHMN). The use of next generation sequencing technologies (NGS) for the diagnosis of patients with genetic diseases is well established, as CMT is a Mendelian disease with more than 90 different related genes already reported. We evaluated 30 patients with all subtypes of hereditary neuropathy (3 CMT1, 12 CMT2, 8 iCMT, 4 dHMN and 3 HSN). Six mutations (SH3TC2, GDAP1, MME, IGHMBP2, 2 AARS) and 7 likely pathogenic variants (KIF1A, DRP2, MME, MPZ, VRK1, SIGMAR1, FLVCR1) were detected, leading to a positive rate of 43.3%. Likely pathogenic variants were considered based on their frequency in normal population, in silico analysis and segregation with phenotype. Despite they have strong evidences to support their causative status further evidence of their pathogenicity is required. New variants were identified in the genes MPZ, KIF1A, DRP2, IGHMBP2, VRK1, SIGMAR1 and FLVCR1 amplifying their genotypic variability. The mutations identified in VRK1, KIF1A, IGHMBP2 and FLVRC1 expanded their phenotype spectrum. Mutations in the VRK1 gene may cause dHMN with upper motor neuron signs. Autistic spectrum disorder may be observed in association with mutations in the KIF1A gene and mutations in the FLVRC1 gene may cause a severe phenotype characterized by congenital insensitivity to pain and acromutilations. Mutations in the IGHMBP2 gene may cause an overlap between SMARD1 and CMT2S phenotypes with organ specific dysautonomia. This study demonstrates that WES is a powerful tool for molecular diagnosis of hereditary neuropathies. Additionally, this study provides new information on the mutations in the VRK1, KIF1A and FLVRC1 genes by adding new mutations and increasing the phenotypic variability of the neuropathies associated with these genes.This study demonstrates WES is a powerful tool for molecular diagnosis of hereditary neuropathies.

Les milliers d’espèces de microorganismes présentes dans les océans sont essentiellement connus pour être planctoniques ou benthiques. Moins décrits, de nombreux micro-organismes colonisent également la surface et le tube digestif des macro-organismes marins, formant des communautés appelées microbiomes. Ces microbiomes ont des conséquences cruciales sur la fitness de leur hôte. Les récents progrès en biologie moléculaire ont ouvert la voie à une caractérisation des différentes facettes de sa biodiversité, à la fois taxonomique, phylogénétique, et fonctionnelle. L’objectif de cette thèse est donc de caractériser la biodiversité des microbiomes cutanés des organismes marins, d’identifier ses échelles de variabilité, ses déterminants, et son importance à l’échelle de l’écosystème. Dans un premier temps j’ai mesuré l’efficacité d’indices de biodiversité à détecter des signaux écologiques dans le cas spécifique de communautés microbiennes. Puis, j’ai décrit le microbiome cutané des principaux grands clades d’animaux marins (poissons téléostéens, cétacés et invertébrés de plusieurs classes). J’ai démontré que le microbiome cutané était très différent des communautés présentes dans l’eau environnante. J’ai aussi montré qu’il était variable, à la fois entre individus et entre espèces, mais ne présentait pas de patron de phylosymbiose. Enfin, j’ai évalué la contribution de la diversité des microbiomes cutanés à la diversité de la communauté microbienne globale d’un écosystème corallien. J’ai ainsi démontré que les animaux marins hébergent collectivement une richesse microbienne presque vingt fois supérieure à celle de l’eau les environnant, et 75% de la richesse phylogénétique à l’échelle de l’écosystème. Dans un contexte d’érosion massive de la diversité des macro-organismes marins, ces résultats soulignent la nécessité d’évaluer plus exhaustivement la biodiversité microbienne marine et sa vulnérabilité face aux pressions anthropiques. / Oceans contain thousands of microbial species playing crucial roles for the functioning of the marine ecosystem. These microorganisms are present everywhere in the water column. Some microorganisms also colonize the surface and the digestive tract of marine macro-organisms, forming communities called microbiomes. These microbiomes have positive effects for their host’s fitness. The diversity of these marine animal surface microbiome is still largely understudied, despite recent progress in molecular biology that now permits to fully assess its different facets of biodiversity, i.e. taxonomic, phylogenetic and functional. The goal of this thesis is therefore to describe the diversity of the surface microbiome of marine animals, to assess its variability at different levels, as well as its determinants, and the significance of such diversity at the ecosystem’s scale. Firstly, I have assessed the efficiency of various diversity indices to detect ecological signals in the specific case of microbial communities. Secondly, I have described the surface microbiome of major marine animal clades (teleostean fishes, cetaceans and several classes of invertebrates). I found that these microbiomes are highly distinct from the surrounding planktonic communities. I demonstrated that these microbiomes are variable both between individuals from the same species and between species, but do not show a phylosymbiosis pattern. Last, I assessed the contribution of surface microbiomes to the global microbial community at the scale of a coral reef ecosystem. I demonstrated that marine animal surfaces host almost twenty times more microbial species than the water column, and 75% of the phylogenetic richness present in the ecosystem. In a context of massive erosion of marine macroscopic organisms, it is therefore urgent to exhaustively assess marine microbial biodiversity and its vulnerability facing anthropic pressures.

A Síndrome Ablefaria Macrostomia (SAM) é uma condição rara, onde os pacientes apresentam características clínicas marcantes como o encurtamento ou ausência das pálpebras superiores e inferiores, ausência de sobrancelhas e cílios, macrostomia por defeitos na fusão dos lábios, entre outros. O padrão de herança da síndrome não está elucidado, tendo a herança autossômica dominante com expressividade variável sido sugerida. SAM possui sobreposição fenotípica com a Síndrome de Barber-Say e com a Síndrome de Fraser, porém nenhum gene já descrito apresentou mutação nos pacientes portadores da SAM. A abordagem genômica no estudo de doenças raras tem sido amplamente utilizada, devido principalmente ao surgimento da Nova Geração de Sequenciamento, que possui alto poder de descriminar as seqüencias nucleotídicas com grande cobertura, em um curto período de tempo. No presente estudo o sequenciamento completo do exoma foi realizado, com cinco indivíduos de uma mesma família, três membros afetados e dois não, e permitiu a análise das regiões codificantes nestes indivíduos. A base molecular da Síndrome Ablefaria Macrostomia é aqui sugerida como autossômica dominante, e decorrente da mutação nova não sinônima c.223G>A (p.E75K) no gene TWIST2. Essa mutação patogênica ocasiona a troca de um aminoácido pequeno de carga negativa, o ácido glutâmico, para um aminoácido de cadeia maior carregado positivamente, a lisina. A modelagem in silico da proteína Twist2 mostrou que a estrutura geral tridimensional da proteína não foi alterada, mas a troca do aminoácido ocorre na posição 75 dentro do domínio básico HLH, e pode impedir a formação de dímeros, ou a própria ligação ao DNA. Sugere-se ainda que a heterogeneidade de fenótipos associados a mutações no gene TWIST2, pode ser atribuída às interações que essa proteína é capaz de formar, e a ampla ação regulatória que ela desempenha em diversos genes do desenvolvimento. / Ablepharon-Macrostomia Syndrome (AMS) is a rare condition characterised by absent or hypoplastic eyelids, absent eyebrows and eyelashes, macrostomia caused by fusion defects of the mouth with unfused lateral commissures, as well as other clinical features. The inheritance pattern has not been confirmed and while autosomal dominant inheritance with variable expressivity has been suggested, recessive inheritance has not been ruled out. The phenotype of AMS overlaps that of Barber-Say and Fraser Syndrome, but any reported gene for these syndromes is mutated on AMS patients. The genomic approach for rare disease studies has been widely used mainly due to the emergence of Next Generation Sequencing, which is very effective at determining nucleotide sequences with large coverage in a short period of time. The whole exome sequencing of five family members was undertaken, with three affected and two unaffected, and the coding regions of the individuals were subsequently analysed. The molecular basis of AMS is suggested here as autosomal dominant, and due to a novel non-synonymous mutation c.223G>A (p.E75K), in TWIST2 gene. This pathogenic mutation causes glutamic acid, a small negatively charged amino acid, to be substituted for a larger and positively charged lysine. The in silico protein modeling of Twist2 shows that the general 3D-structure of the protein is not affected, but the amino acid change is located inside the basic Helix-Loop-Helix domain which could disrupt dimerization and DNA binding. It has also been suggested that the phenotype heterogeneity associated with mutations on TWIST2 gene can be attributed to the interactions that this protein is capable of, and the role that it plays in the regulation of several developmental genes.

A cor da pele, olhos e cabelos são alguns dos traços fenotípicos mais aparentes quando nos referimos à identificação de características individuais. Essas características frequentemente são utilizadas na descrição de indivíduos em retratos falados usados em investigações policiais. Porém, em muitos casos as vítimas ou testemunhas não reconhecem o agressor, tornando inviável a produção desses retratos. Contudo, os vestígios biológicos deixados pelo criminoso poderiam ser utilizados na predição de suas características físicas, suprindo a falta ou complementando o retrato falado. Para que isso seja possível, é preciso conhecer as variáveis responsáveis pela formação desses fenótipos. No caso dos fenótipos de pigmentação há tanto um fator genético, quanto ambiental. Diversos genes participam da formação desses fenótipos, dentre eles está o gene MITF (Melanogenesis-associated transcription factor), um dos principais regulador da biossíntese de melanina nos melanócitos. Esse gene está fortemente associado às síndromes de Waadenburg e Tietz, as quais causam pigmentação anormal, principalmente na pele, e ao melanoma. No entanto, apesar do claro envolvimento do gene MITF na melanogênese, ainda não são conhecidas associações significativas de polimorfismos nesse gene com fenótipos de pigmentação. À vista disso, esse trabalho avaliou a relação da variabilidade do gene MITF com os fenótipos de pigmentação encontrados em uma amostra populacional do estado de São Paulo, por meio de sequenciamento de nova geração. Foram identificados 133 pontos de variação em toda a extensão do gene e sua região promotora, dos quais 21 estão associadas a pelo menos um fenótipo de pigmentação de pele, olhos ou cabelo. Adicionalmente foram encontradas associações com ao menos um fenótipo de pigmentação para 3 dos 17 haplótipos da região promotora, 7 dos 50 haplótipos da extensão que engloba a região 5UTR e codificante, e um dos 18 haplótipos encontrados na região 3UTR. Considerando os haplótipos encontrados para a extensão total do gene MITF e sua promotora, 20 dos 132 haplótipos encontrados estão associados a algum fenótipo de pigmentação. A maior parte das associações encontradas, tanto para alelos e genótipos quanto para haplótipos, são referentes a fenótipos mais escuros como cabelos castanhos escuros e pretos e pele escura. Associações com fenótipos mais claros, tais como olhos azuis e verdes e cabelos loiros e ruivos, também foram encontradas, porém envolvendo variantes e haplótipos de frequência baixa na população amostrada; tais associações, entretanto, representam achados falsos positivos. Os resultados confirmam a hipótese de que a variabilidade do gene MITF pode contribuir para a formação dos fenótipos de pigmentação de pele, olhos e cabelos dos indivíduos da população brasileira / Skin, eye and hair colors are some of the most noticeable phenotypes when referring to the identification of individual characteristics. These characteristics are often used to describe individuals in police sketches used in investigations. However, in many cases the victims or witnesses are unable to recognize the assaulter, making this sketches unfeasible. Nonetheless, biological traces left by the assaulter could be used to predict their physical characteristics, compensating or complementing these sketches. To make this possible, its necessary to know the variables responsible for the development of these traits. Pigmentation phenotype development relies on genetic and environmental aspects. A variety of genes contribute to the development of these phenotypes, among them MITF (Melanogenesis-associated transcription factor), one of the main regulators of melanin synthesis in melanocytes. This gene is strongly associated with Waardenburg and Tietz syndrome, which cause abnormal pigmentation, mostly in skin, and melanoma. Although MITFs clear involvement in melanogenesis, significant associations between this genes polymorphisms and pigmentation phenotypes are still unknown. Thus, this study evaluated the relation between MITF genetic variability and pigmentation phenotypes found in a population sample from the state of São Paulo, through next generation sequencing. There were identified 133 variation points through the whole gene and its promoter, from which 21 were associated with at least one skin, eye or hair pigmentation phenotype. Additionally, 3 of the 17 promoter haplotypes, 7 of the 50 haplotypes comprising the 5UTR and coding regions and one of the 18 3UTR haplotypes were associated with at least one pigmentation phenotype. Considering the haplotypes found for the whole gene and its promoter, 20 of the 132 haplotypes found were associated with at least one phenotype. The majority of the associations found for alleles, genotypes and haplotypes were related to darker phenotypes, like dark brown and black hair and dark skin. Associations with lighter phenotypes, like blue and green eyes and blonde and red hair, were also found, although involving variants and haplotypes with low frequencies in the studied population; these associations, however, represent false positives. The results corroborate the hypothesis that the MITF variability can contribute to the formation of pigmentation phenotypes in skin, eye and hair in the Brazilian population

An interdisciplinary approach integrating micro and macroevolution, genomic, morphometric and morphological variation, systematics, quantitative genetics, and biogeography was employed to investigate the evolutionary history of the genus Holochilus (Rodentia: Sigmodontinae). Holochilus presents poorly defined species, with nomenclatural problems and phylogenetic relationships on species level unknown. The current species number possibly does not reflect its real diversity, and no work combining genetic and morphometric evidences from all its geographic range was performed. This genus belongs to the tribe Oryzomyini, and along with other 14 genera constitute the Oryzomyini clade D, the most comprehensive generic diversity of the tribe, occupying distinct environments. The internal phylogenetic relationship within this clade is still unclear and variable. Due to its broad geographic distribution, Holochilus also represents a key piece on the study of the evolution of oryzomines of open formations of South America. Based on a comprehensive sampling, I analyzed patterns of morphometric and genomic variation within Holochilus, in order to delimit the species belonging to this genus, as well as access the phylogenetic relationship between these lineages. I investigated the sexual and ontogenetic variation in this group, comparing natural and captive populations, seeking for understand the effect of the environmental differences in the pattern of variation and ontogenetic trajectories (Chapter 1). I also evaluated and compared the genomic variation among three species of Holochilus to verify the influence of the biomes and the climatic changes in the genomic signatures (Chapter 2). I applied a model-based approach to delimit species (Chapter 3). And finally, additional investigations were made to propose the phylogenetic relationship between members of clade D, and provide date intervals for the main diversifications events, as well as the possible process responsible for the biogeographic pattern current observed related with the forest and open areas occupation (Chapter 4). Sexual dimorphism exhibited small degree of variation among populations. The greater ontogenetic variation is found in the younger age classes, but oldest individuals also show larger degree of differentiation. There are also great differences in the ontogenetic trajectories among samples, where individuals from the captive population exhibited the lower degree of variation between all age classes. The quantitative genetic analysis showed that genomic differences are observed across the taxa, and it was associated with geography. Ecological niche models revealed that biomes with larger areas of stability also presented more genomic structure, suggesting that historical dimension impacted population isolation/connectivity. Results also shows that biomes not only differ geographically and environmentally (based on past climatic conditions), but also show significant association between the environmental space and the genetic variation that is not related with geography. Eight independent lineages within Holochilus were recovered, and the phylogenetic arrangement partially corroborates previous studies. Finally, the phylogeny proposed for the clade D presented some differences in comparisons with other previously reported, and suggest that most of the cladogenetic events happened during the Pleistocene, being the expansion of open environments an important driver of diversification in this group. / Uma abordagem interdisciplinar integrando micro e macroevolução, variação genômica, morfométrica e morfológica, sistemática, genética quantitativa e biogeografia foi empregada para investigar a história evolutiva do gênero Holochilus (Rodentia: Sigmodontinae). O gênero Holochilus apresenta espécies mal definidas, com problemas nomenclaturais e relações desconhecida. O número atual de espécies possivelmente não reflete a sua diversidade real e, até o momento, não foi realizado nenhum trabalho combinando evidências genéticas e morfométricas englobando toda a distribuição geográfica desse grupo. Este gênero pertence à tribo Oryzomyini, e juntamente com outros 14 gêneros (a diversidade genérica mais abrangente da tribo) formam o clado D. A relação filogenética interna dentro deste clado ainda é variável. Devido à sua ampla distribuição geográfica, Holochilus também representa uma peça chave no estudo da evolução dos oryzomíneos de formações abertas da América do Sul. Com base em uma amostragem abrangente, analisei padrões de variação morfométrica e genômica dentro de Holochilus, a fim de delimitar as espécies pertencentes a este gênero, bem como acessar a relação filogenética entre essas linhagens. Investiguei a variação sexual e ontogenética deste grupo, comparando populações naturais e de cativeiro, buscando entender o efeito das diferenças ambientais no padrão de variação e nas trajetórias ontogenéticas (Capítulo 1). Eu também avaliei e comparei a variação genômica entre três espécies de Holochilus a fim de verificar a influência dos biomas e das mudanças climáticas nas assinaturas genômicas das espécies (Capítulo 2). Em seguida eu apliquei uma abordagem baseada em modelos para delimitar as espécies (Capítulo 3). Finalmente, investigações adicionais foram realizadas para propor as relações filogenéticas entre os membros do clade D, fornecendo datas para os principais eventos de diversificação, e inferências sobre possíveis processos responsáveis pelo padrão biogeográfico atual, relacionado os mesmos com a ocupação florestal e áreas abertas (Capítulo 4). O dimorfismo sexual apresentou pequeno grau de variação entre as populações. A maior variação ontogenética é encontrada nas classes etárias mais jovens e mais velhas. Há também grandes diferenças nas trajetórias ontogenéticas entre as amostras, onde indivíduos da população cativeiro exibiram o menor grau de variação entre todas as classes etárias. A análise genética quantitativa mostrou que diferenças genômicas são observadas em todos os táxons e essa diferença está associada à geografia. Modelos de nichos ecológicos revelaram que os biomas com maiores áreas de estabilidade também apresentaram maior estruturação genômica, sugerindo que uma dimensão histórica impactou o isolamento/conectividade entre as populações. Os resultados também mostram que os biomas não só diferem geograficamente e ambientalmente (baseado em condições climáticas passadas), mas também mostram associação significativa entre o espaço ambiental e a variação genética que não está relacionada com a geografia. Adicionalmente, foi recuperado oito linhagens independentes dentro de Holochilus, e o arranjo filogenético parcialmente corrobora estudos anteriores. Finalmente, a filogenia proposta para o clado D apresentou algumas diferenças em comparação com outros estudos, e sugeriu que a maioria dos eventos cladogenéticos ocorreram durante o Pleistoceno, sendo a expansão dos ambientes abertos um importante motor de diversificação neste grupo.

A espécie de cianobactéria Cylindrospermopsis raciborskii pertence a ordem Nostocales e tem a capacidade de realizar a fixação biológica do N2 atmosférico, sintetizar cianotoxinas e também de proliferar em águas doces formando florações de cianobactérias nocivas. A população de C. raciborskii encontrada no Brasil é conhecida por produzir derivados de saxitoxina (STX), enquanto cilindrospermopsinas (CYN), as quais são detectadas em isolados de outros países, não têm sido detectada em isolados brasileiros. Neste estudo, a produção de CYN por dois isolados brasileiros de C. raciborskii, CENA302 e CENA303, submetidos a diferentes condições nutricionais, foi avaliada usando o teste imunológico ELISA específico para detecção de CYN. Em seguida, genes evolvidos na biossíntese de CYN foram buscados no genoma dessas duas linhagens usando amplificações por PCR com iniciadores específicos e posterior sequenciamento Sanger, bem como sequenciamento genômico na plataforma HiScan SQ. Além disso, foram realizadas buscas nos dois genomas obtidos pela plataforma HiScan SQ de genes envolvidos com a síntese de algumas outras moléculas já descritas em cianobactérias, bem como aqueles relacionados com a fixação biológica de N2 e diferenciação do heterócito. Apesar da utilização de diferentes condições nutricionais, não foi detectada a produção de CYN no ensaio de ELISA em ambos os isolados de cianobactérias. No sequenciamento Sanger, dos nove genes sintetases de CYN buscados, quatro foram amplificados e sequenciados na linhagem C. raciborskii CENA302 e três na linhagem CENA303. Estas sequências de nucletídeos foram traduzidas em aminoácidos, e as funções das proteínas e os domínios preditos confirmaram a sua identidade como genes sintetase de CYN. O sequenciamento dos genomas completos das duas linhagens apresentou altos valores de qualidade de bases e elevada cobertura genômica. A busca por genes sintetase de CYN nos dois genomas foi realizada pelo mapeamento por referência das leituras, mostrando que algumas porções do agrupamento estavam contempladas no genoma, representando aproximadamente 10% do agrupamento de CYN. A anotação das regiões entre os genes descritos como flanqueadores do agrupamento CYN mostrou uma inversão gênica indicando a ocorrência de eventos que podem ter levado a perda ou dispersão no genoma deste agrupamento. Os genes anotados relacionados com fixação biológica de N2 e diferenciação do heterócito nas linhagens CENA302 e CENA303 apresentaram altas identidades com genes homólogos descritos em C. raciborskii. A ausência de alguns nucleotídeos no gene hetP envolvido na formação do heterócito possivelmente levou a perda de sua função na C. raciborskii CENA303, impedindo a diferenciação das células vegetativas em heterócitos, uma vez que não foi observado a presença dessa célula diferenciada em nenhuma das condições nutricionais avaliadas. Todos os genes de moléculas bioativas conhecidas de cianobactérias buscados pelo mapeamento por referência das leituras não foram encontrados nos genomas das linhagens C. raciborskii CENA302 e CENA303. Os resultados obtidos neste estudo revelaram a possibilidade de estar ocorrendo eventos evolutivos que estão causando a perda dos genes responsáveis pela síntese de CYN nos isolados brasileiros de C. raciborskii, demonstrando divergência alopátrica. / The cyanobacterial species Cylindrospermopsis raciborskii belongs to the order Nostocales and has the ability to perform biological nitrogen fixation, synthesize cyanotoxins and also to proliferate in freshwaters to form harmful cyanobacterial blooms. The C. raciborskii population from Brazil is known to produces saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from other countries, has thus far not been detected in Brazilian strains. In this study, the production of CYN by two Brazilian isolates of C. raciborskii CENA302 and CENA303, under different nutritional conditions, was evaluated using ELISA immunoassay specific for the detection of CYN. Then, genes involved in the biosynthesis of CYN were searched in the genome of these two strains using PCR amplification with specific primers and subsequent Sanger sequencing, as well as genomic sequencing on the platform HiScan SQ. Furthermore, searches were performed in the two genomes obtained by the platform HiScan SQ of genes involved in the synthesis of several other molecules already described in cyanobacteria, as well as those related to the biological N2 fixation and heterocyte differentiation. Despite the use of different nutritional conditions, no CYN production was detected by the ELISA assay for both cyanobacterial isolates. In the Sanger sequencing, of the nine CYN synthetase gene searched, four were amplified and sequenced in the C. raciborskii strain CENA302 and three in the strain CENA303. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. The complete genome sequencing of the two strains showed high values of bases quality and high genomic coverage. The search for CYN synthetase genes in the two genomes was performed by mapping reads to reference, showing that some portions of the cluster were contemplated in the genome, representing approximately 10% of CYN cluster. The annotation of regions between the genes described as flanking the CYN cluster showed a gene inversion indicating the occurrence of events that may have caused the loss or dispersion in the genome of this cluster. The annotated genes related to biological N2 fixation and heterocyte differentiation in the CENA302 and CENA303 strains showed high identities with homologous genes from other strains of C. raciborskii. The absence of a few nucleotides in the hetP gene involved in the heterocyte formation perhaps led to the loss of its function in the C. raciborskii CENA303, preventing the differentiation of vegetative cells into heterocyte, since it was not observed the presence of this differentiated cell in any of the nutritional conditions evaluated. All genes of known cyanobacterial molecules searched by mapping reads to reference were not found in the genomes of the C. raciborskii strains CENA302 and CENA303. The results of this study revealed the possibility of being occurring evolutionary events that are leading to loss of the genes responsible for the synthesis of CYN in the C. raciborskii Brazilian isolates, demonstrating allopatric divergence.

Pre-mRNA (precursor messenger RNA) splicing is a fundamental process in eukaryotic gene expression. In order to catalyse the excision of the intervening intronic sequence between two exons, the spliceosome is assembled stepwise on the pre-mRNA substrate. This ribonucleoprotein machine is extremely dynamic: both its activation and the progression through the catalytic stages require extensive compositional and structural remodelling. The first part of this thesis aims at understanding how the spliceosome is activated after assembly. When this work was started, the GTPase Snu114 was thought to activate the helicase Brr2 to unwind the U4/U6 snRNA duplex, which ultimately leads to the formation of the spliceosome active site. To explore the role of Snu114, a complex built from Snu114 and a part of Prp8 was expressed and analysed in its natural context, bound to U5 snRNA. However, before I was able to obtain highly diffracting crystals, the structure of Snu114 was determined in the context of a larger spliceosomal complex by electron cryo-microscopy by competitors. Regardless, the role of Snu114 in spliceosome activation remains elusive. In a short section of this thesis, genetic and biochemical analysis suggest Snu114 to be a pseudo-GTPase, precluding a role for Snu114-catalyzed GTP hydrolysis in activation. The second and larger part of the thesis describes the development of a novel, biochemical method to analyse spliceosome remodelling events that are caused by the eight spliceosomal helicases. Purified spliceosomes assembled on a defined RNA substrate are analysed by UV crosslinking and next-generation sequencing, which allows for the determination of the RNA helicase binding profile at nucleotide resolution. In vitro spliceosome iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) was initially developed targeting the helicase Prp16 bound to spliceosomal complex C. The obtained binding profile shows that Prp16 contacts the intron, about 15 nucleotides downstream of the branch in the intron-lariat intermediate. Our finding supports the model of Prp16 acting at a distance to remodel the RNA and protein interactions in the catalytic core and thereby it promotes the transition towards a conformation of the spliceosome competent for second step catalysis. Control experiments, which locate SmB protein binding to known Sm sites in the spliceosomal snRNAs, validated the method. Preliminary results show that in vitro spliceosome iCLIP can be adapted to analyse additional spliceosomal helicases such as Prp22. Finally, I performed initial experiments that give promising directions towards time-resolved translocation profiles of helicases Brr2 and Prp16.