Bibliotecas de produtos naturais são fontes de moléculas com ações farmacológicas, com diversas aplicações biotecnológicas. Estes compostos apresentam alta especificidade para o alvo, resultante de longo processo de seleção natural, sendo interessantes como ferramentas de estudo e princípios ativos. Porém, apesar da riqueza de estruturas presentes em tais secreções, as dificuldades em obter moléculas puras em grandes quantidades, o alto custo e o tempo de purificação, tornam-se barreiras para seu uso. Assim, cada vez mais, as técnicas ômicas são usadas como uma alternativa para produção destas toxinas obtidas em maior escala. A transcriptômica, técnica que consiste na produção de biblioteca de cDNA a partir do RNA obtido da de organismo, tecido ou célula de interesse, é altamente relevante, já que identifica o material proteico que é realmente transcrito a partir do RNA obtido em determinado tempo e situação. Sapos ainda são animais pouco estudados, quando comparados a outros animais peçonhentos e venenosos, e um dos fatores responsáveis por isso é o baixo rendimento na purificação de toxinas de seu veneno. A fim de se identificar componentes presentes nas secreções do sapo R. schneideri, foi, primeiramente, realizada extração de RNA poli A+ das secreções recém obtidas das glândulas mucosas e das secreções armazenadas por 2 anos no laboratório. A secreção armazenada há dois anos revelou qualidade e quantidade mais apropriadas para o estudo e foi, portanto, usada como material de partida para a construção do transcriptoma por metodologia tradicional. Este transcriptoma resultou em 6 clones com boa qualidade, sendo que um deles, Rs02, apresentou similaridade com a região do pró peptídeo da odorranaina. Uma vez que este transcriptoma não revelou resultados satisfatórios devido à sua baixa eficiência e visando maximizar o conhecimento sobre a secreção, um novo transcriptoma foi construído usando sequenciamento de nova geração, com sequenciador Illumina e RNA total extraído do tegumento contendo glândulas mucosas de um espécime como material de partida. O novo transcriptoma resultou em aproximadamente 131 milhões de reads brutos. Os reads foram filtrados de modo que apenas aqueles com boa qualidade (Q>20) fossem submetidos à montagem de novo. Esta etapa resultou em aproximadamente 130 milhões de reads, com média de 68 pb. Os reads foram então agrupados em contigs usando o programa SOAPdenovo2-Trans e submetidos a diversas abordagens de anotação funcional. Foram encontrados cDNAs codificantes de diversos peptídeos e proteínas com potencial aplicação biotecnológica. Além da abordagem ômica, ensaios de caracterização bioquímica, como atividade enzimática, cromatografia e eletroforese, auxiliaram a detecção de protease sem relatos prévios em secreções de sapo, uma fosfolipase A2, bem como lectina e galectina. Adicionalmente, através do transcriptoma foram identificadas cobatoxinas, mucinas e ficolinas. Portanto, este trabalho foi pioneiro no entendimento molecular do veneno da glândula mucosa de R. schneideri por métodos de vanguarda e análises bioquímicas. / Natural products libraries are known as medicine molecules sources once these molecules have a high target specificity inherited from the long natural evolutionary process. Thereby, animal, plant and microorganisms\' secretions are very important to biotechnological applications. However, despite the large number of molecules that can be found in these secretions, the difficulty on obtainment enough purification yield, high cost and long purification time, besides the small secretion amount provided by the studied organism, are factors that make this kind of study even harsher. These are the reasons why omic studies are becoming an alternative to produce these toxins in a larger scale, which allow new researches. Transcriptome is a technique that consists on the production of cDNA libraries from the secretion or gland RNA, using the transcriptase reverse enzyme, which results in a holistic poison understanding. Toads are animals that are still not widely studied, if we compare them with other venomous animals, mainly because of the insufficient purification yield. Thus, the mucous gland transcriptome from Rhinella schneideri poison, a toad that is widely found in Brazilian territory, has a great relevance on the elucidation and possibility to use several kind of molecules, especially because this gland produces unknown molecules. Thereof, aiming to unravel this poison molecules, we first compared the RNA yield from fresh and two years stored mucous gland secretion. The stored secretion has shown more suitable RNA, which was used to construct a Sanger sequencing transcriptome. It resulted in 6 clones and one of them had a good hit with odorranain pro-peptide region. In order to increase the knowledge about the secretion, we turned to Next Generation Sequencing transcriptome using Illumina technologies and one specimen integument as raw material. The new transcriptome resulted in approximately 131 million raw reads. These reads were filtered so that only those with good quality (Q>20) were used to perform the assembling. The latter step resulted in approximately 130 million reads with 68 bp average length. The reads were grouped into contigs using the assembler. Then, the resulting contigs were submitted to different functional annotation approaches. We unraveled cDNAs encoding peptides and protein with biotechnological application. Besides the omic approach, assays for biochemical characterization including chromatography, electrophoresis and proteolytic assays complemented the identification of a protease for the first time in toad secretions, phospholipase A2, as much as lectins and galectins. Thus, the transcriptome also allowed the identification of cobatoxins, mucins and ficolins. Therefore, this is the first work about the molecular composition of Rhinella schneideri mucous gland poison through avant-garde methodology and biochemical anaylsis.

Orientador: Edvaldo Aparecido Amaral da Silva / Coorientador: Olivier Leprince / Banca : Henk W.M Hilhorst / Banca: José Márcio Rocha Faria / Banca: Julia Buitink / Banca: Olivier H.L.Leprince / Resumo: Soja é uma das mais culturas oleaginosas usadas para alimentação animal e humana bem como para uma larga aplicação industrial. Dada a sua capacidade de fixar nitrogênio atmsférico, é fundamental para o desenvolvimento de uma agricultura sustentável. Produzir sementes altamente vigorosas é a chave para aumentar a eficiência da produção da cultura. Longevidade de semente é a capacidade de sobreviver no estado seco por períodos prolongados e representa uma importante característica de qualidade da semente. Nesta pesquisa o objetivo foi obter insights sobre processos moleculares que regulam a aquisição de longevidade em sementes de soja. Com o sequenciamento de nova geração da Illumina, o RNA foi sequenciado a partir de sete estágios diferentes durante a aquisição de longevidade, gerando entre 14 e 38 milhões de reads. Estes reads foram alinhados com os modelos de genes de Glycine max Wm82.a2.v1 preditos no genoma de soja. Transcritos diferencialmente expressos (DET) foram correlacionados com o aumento da longevidade. Análise de enriquecimento da ontologia do gene daqueles DET revelaram uma siginificante sobre representação de termos associados com resposta a estresse e processamento e modificação de RNA. Processo biológico fotossíntese foi relacionado à baixa longevidade. Heat Shock Factors (HSF) e vários fatores de transcrição associados com resposta a estresse biótico (WRKY e NFXL1) são genes candidatos com possíveis papéis na longevidade de semente e merecem uma caracterizaçã... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Soybean is one of the most important oil crop species used for food and feed as well as a range of industrial applications. However, producing highly vigorous seeds is a key lever to increase crop production efficiency. Low physiological seed quality, which is more prone to occur under tropical environment, leads to poor stand establishment and decreased in yields. Seed longevity is the ability to survive the dry state for prolonged periods of time and represents an important trait for seed quality. Here, the objective was to obtain insights into the mechanisms regulating the progressive acquisition of longevity. Using Illumina high-throughput sequencing, RNA was sequenced from seven different stages during the acquisition of longevity, generating between 14 and 38 million of reads. These reads were aligned to the Glycine max Wm82.a2.v1 gene model. Differentially expressed transcripts (DET) were correlated with the increase in seed longevity. Transcriptome and GO enrichment analyses of these DET revealed a significant over-representation of terms associated with response to stress and RNA processing and modification. Photosynthesis biological process was related to low seed longevity. HSF and several TF associated with biotic defense (WRKY3 and NLFX1) are candidate genes whose putative role in seed longevity deserve further characterization. We also performed the determination of the content of non-reducing soluble sugars, and we observed that the accumulation of non-reducin... (Complete abstract click electronic access below) / Résumé: Le soja est l'une des plus importantes espèces de cultures d'huile utilisée aussi bien en nourriture que dans diverses gammes d'applications industrielles. C'est pourquoi produire des graines vigoureuses est un levier essentiel pour augmenter efficacement de la production de la récolte. La qualité de graine physiologiquement faible, qui est plus à même de se produire sous un environnement tropical, mène à un pauvre établissement des plantes ainsi qu'à une diminution du rendement. La longévité d'une graine est la capacité de celle-ci à survivre à la sécheresse durant de longues périodes et représente une caractéristique importante sur la qualité d'une graine. Ici, l'objectif était d'obtenir une idée sur les mécanismes en régulant l'acquisition progressive de la longévité. En utilisant le séquençage à haut-débit, ARN a été séquencé en sept différentes étapes durant l'acquisition de longévité, générant entre 14 et 38 millions de reads. Ces reads ont été alignés sur les modeles de gene de Glycine max Wm82.a2.v1. Les transcripts différentiellement exprimés (DET) sont corrélée avec l'augmentation de la longévité de la graine. L'analise d'enrichissement via GO de ces DET ont révélé une importante surreprésentation des termes associés à la réponse au stress et traitement et modification de l'ARN. Le processu biologique Photosynthèse était liée à une faible longévité des semences. HSF (heat shock factor) et plusieurs facteurs de transcription associés à la défense biotique (WRKY 3 et ... (Résumé complet accès életronique ci-dessous) / Doutor

Os insetos são capazes de sobreviver em diversos ecossistemas do planeta e, mesmo estando constantemente expostos à ameaça de infecção microbiana, permanecem livres de infecções na maior parte do tempo. Essa capacidade de sobrevivência aliada à larga distribuição dos insetos em regiões totalmente diferentes tem estimulado a pesquisa de novos agentes terapêuticos nesta classe devido à descoberta de diversos componentes de mecanismos inespecíficos de combate à infecção, sendo possível sua aplicação no controle de diversas doenças. Todavia, apesar de um grande número de moléculas de defesa ter sido identificado a partir de vários insetos, pouca informação sobre suas aplicações está disponível. Desta forma, o presente trabalho elucida o perfil transcriptômico geral e dos genes de defesa do tegumento de Podalia orsilochus durante sua fase larval. Como consequência, os transcritos e os dados obtidos permitirão o auxílio em pesquisas posteriores, seja para comparação, citação, conhecimento biológico e das respostas de defesa ou das relações de filogenia do animal. / The insects are able to survive in diverse ecosystems on earth, and even being constantly exposed to the threat of microbial infections, remain free of infection for most of the time. This survivability combined with the wide distribution of insects in totally different regions has stimulated the search for new therapeutic agents in this class due to the discovery of several components of nonspecific mechanisms to fight infection, and possible implementation in the control of various diseases. However, despite a large number of defense molecules have been identified from various insects, little information is available on their applications. Thus, this paper elucidates the general transcriptomic profile and integument of defense gene Podalia orsilochus during their larval stage. As a result, the transcripts and the data obtained will aid in further research, to compare, reference, biological knowledge and defense or animal phylogeny relationships.

As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas. / Snake venom toxins alter prey homeostasis for feeding or defense. In depth studies of venom composition are important for better antivenom production, for new drugs lead and discovery and for better understanding of biological, ecological and evolutionary processes. Research on toxins have shown the natures way of innovating, refined by evolution, diversifying functions of protein families recruited from their endogenous function to the venom gland by successive gene duplication and mutation accumulation, leading to an accelerated evolution. A myriad of available toxins and diversity of functions is still available for discovery. Combining high throughput techniques such as venom gland de novo transcriptomics and venom proteomics, one can assess and observe a more complete profile of the snake toxinome, additionally allowing an upscale in low represented and unexpected toxin detection. The aim of this project was to investigate the venom toxinome of one of the most dangerous Australian species, Pseudonaja textilis (Elapidae). The toxins identified in it venom was: protrombinase complex coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and procoagulant PLA2, neurotoxic three-finger toxins (3FTx), Kunitz-type protease inhibitor textilinin, and for the first time, a new long 3FTx, C-type lectins, CRiSPs, as well as evidences of lizard toxins from Heloderma genus and other toxin candidates calreticulin and dipeptidase 2. Metalloproteinases, little investigated in Elapidae, was cloned and detected in the venom after fractionation and immunoassay. The transcriptome revealed new toxin variants and isoforms, specially 3FTx and serine protease inhibitors, as well as transcripts from toxins not detected in the venom that deserves further investigation. Human accident symptoms are well explained by the identified toxins, however, in its natural environment, little known and undescribed toxins must have specific and important role in predation. Identifying this diversity is important to better understand toxins ways of action.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-01-06T11:37:49Z No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) / Made available in DSpace on 2017-01-06T11:37:49Z (GMT). No. of bitstreams: 1 2016 - Gabriel Alves Landulfo.pdf: 2085230 bytes, checksum: a7f7c364b23211ad81d1168cb1f6db85 (MD5) Previous issue date: 2016-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also aggressive towards humans. It inhabits some countries in the Neotropical region. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to ascertain the main molecules expressed in the gut of argasid ticks after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon. Sixty females were fed and dissected to extract the gut tissue. The transcriptome was obtained through pyrosequencing and the de novo assembly method on mRNA of the gut tissue. We identified 2,235 contigs, of which 1,729 matched database sequences, while 506 did not present any hits. The transcripts were annotated and grouped according to their biological function. Catalytic, binding and transporter activity were the most representative functions, accounting for 780, 709 and 106 contigs, respectively. The transcripts were classified into 31 categories, using both bioinformatics and data curation practices. The most representative categories were, respectively, unknown, catalytic activity and transporter channels. One hundred and three (103) digestives transcritps associated to digestion of proteins (67), carbohydrates (19) and lipid (17) were identified in the transcriptome analysis. Peptidases associated with hemoglobin digestion, such as serine, cysteine, aspartic protease and metalloenzymes, were identified in the gut of the engorged females. Genes associated with transport (hemelipoglycoprotein) and storage (ferritin) of nutrients resulting from hemoglobin digestion, such as heme, were also found in the digestive tract. The presence of a cathepsin O-like cysteine peptidase was recorded in ticks, for the first time. Two thousand and two hundred thirteen (2213) transcripts were deposited to the Transcriptome Shotgun Assembly (TSA) portal of the NCBI.The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. Genes for cathepsin L in O. mimon appear to have diverged from other more common recent ancestors. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on different datasets, such as mitochondrial genome and nuclear rRNA sequences. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity / Ornithodoros mimon ? um carrapato argas?deo parasita de quir?pteras, aves e marsupiais, al?m de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de mol?culas vitais no processo digest?o e na rela??o parasito-hospedeiro, al?m de fornecer tamb?m informa??es sobre a evolu??o dos artr?podes hemat?fagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais mol?culas expressas no intestino de uma esp?cie de carrapato argas?deo ap?s o repasto sangu?neo, atrav?s de uma an?lise transcrit?mica do intestino de f?meas ingurgitadas de O. mimon. Sessenta f?meas foram alimentadas e dissecadas para coleta o tecido intestinal. O RNAm da amostra do intestino foi extra?do, purificado e quantificado. Esse serviu de molde para s?ntese do cDNA, que foi utilizado no pirosequenciamento. O transcritoma foi obtido atrav?s do m?todo de montagem de novo do cDNA do tecido intestinal. Identificou-se 2235 sequ?ncias consensos (contigs) ou transcritos, dos quais 1729 apresentaram similaridade (hit) com sequ?ncias dos bancos de dados, enquanto que 506 n?o tiveram nenhuma similaridade. Os transcritos foram anotados e agrupados conforme as fun??es biol?gicas atribu?das as eles no processo de anota??o g?nica. Atividade catal?tica, ades?o e transporte foram as fun??es mais representativas com 780, 709 e 106 transcritos, respectivamente. Em uma an?lise n?o automatizada, os transcritos foram subcategorizados em 31 categorias. As categorias mais representativas foram desconhecido, atividade catal?tica e transportadores-canais. Identificamos 103 transcritos digestivos associados ? digest??o de prote?nas (67), carboidratos (19) e lip?dios (17). Proteinases das classes serino, ciste?ne, asp?rtica e metalo representaram as enzimas atuantes na digest?o intracelular do constituinte prote?co do repasto sangu?neo. Genes associados com o transporte (hemelipoglicoprote?na) e estocagem (ferritina) dos nutrientes resultantes da digest?o foram encontrados bem expressos no trato digestivo. Registrou-se pela primeira vez a presen?a de uma ciste?na peptidase do tipo catepsina O em carrapatos. Foram depositados no banco de dados g?nico p?blico 2213 transcritos de O. mimon. A an?lise filogen?tica das peptidases revelou que a maioria das proteinases de O. mimon ? pr?xima aos genes codificadores de proteinases de carrapatos. Transcritos de catepsinas L de O. mimon parecem ter divergido de ancestrais recentes diferentes. A infer?ncia filogen?tica baseada em conjunto de dados transcritos hom?logos tem uma resolu??o topol?gica similar a de outros conjuntos de dados, como genoma mitocondrial e sequ?ncias nuclear de RNA riboss?mico (rRNA). Os achados obtidos no presente estudo podem contribuir para compreens?o dos importantes processos dos carrapatos argas?deos, como digest?o, nutri??o e imunidade, al?m de fornecer informa??es sobre a filogenia dos carrapatos.

High-throughput sequencing is a powerful tool to study diverse aspects of biology and applies to genome, transcriptome, and small RNA profiling. Ever increasing sequencing throughput and more specialized sequencing assays demand more sophisticated bioinformatics approaches. In this thesis, I present 4 studies for which I developed computational methods to handle high-throughput sequencing data to gain insights into biology. The first study describes the genome of High Five (Hi5) cells, originally derived from Trichoplusia ni eggs. The chromosome-level assembly (scaffold N50 = 14.2 Mb) contains 14,037 predicted protein-coding genes. Examination and curation of multiple gene families, pathways, and small RNA-producing loci reveal species- and order-specific features. The availability of the genome sequence, together with genome editing and single-cell cloning protocols, enables Hi5 cells as a new tool for studying small RNAs. The second study focuses on just one type of piRNAs that are produced at the pachytene stage of mammalian spermatogenesis. Despite their abundance, pachytene piRNAs are poorly understood. I find that pachytene piRNAs cleave transcripts of protein-coding genes and further target transcripts from other pachytene piRNA loci. Subsequently, systematic investigation of piRNA targeting by integrating different types of sequencing data uncovers the piRNA targeting rule. The third study describes computational procedures to map splicing branchpoints using high-throughput sequencing data. Screening >1.2 trillion RNA-seq reads determines >140,000 BPs for both human and mouse. Such branchpoints are compiled into BPDB (BranchPoint DataBase) to provide a comprehensive branchpoint catalog. The final study combines novel experimental and computational procedures to handle PCR duplicates that are prevalent in high-throughput sequencing data. Incorporation of unique molecular identifiers (UMIs) to tag each read enables unambiguous identification of PCR duplicates. Both simulated and experimental datasets demonstrate that UMI incorporation increases the reproducibility of RNA-seq and small RNA-seq. Surveying 7 common variables in high-throughput sequencing reveals that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine the PCR duplicate frequency. Finally, I show that removing PCR duplicates without UMIs leads to substantial bias into data analysis. / 2020-12-11T00:00:00Z

As toxinas de veneno de serpentes têm como principal função alterar a homeostase das presas para fins de alimentação ou defesa. O estudo aprofundado da composição do veneno de serpentes é importante para a produção de soros antiofídicos mais eficientes, para a descoberta de novos fármacos e na compreensão de processos biológicos, ecológicos e evolutivos. As pesquisas com toxinas têm mostrado uma versatilidade natural, refinada pela evolução, na diversificação de funções em famílias de proteínas recrutadas de suas funções endógenas, por meio de sucessivas duplicações e acumulo de mutações levando a uma evolução acelerada. A miríade de toxinas disponíveis e sua diversidade de funções ainda não foram completamente descritas. A combinação das análises em larga escala do transcriptoma de novo da glândula de veneno e do proteoma do veneno permite elaborar um perfil mais completo do toxinoma do veneno, permitindo inclusive um aumento na sensibilidade da detecção de toxinas pouco representadas e inesperadas nos venenos. O objetivo geral deste estudo foi analisar o toxinoma do veneno de uma das mais perigosas espécies australianas, a Pseudonaja textilis (Elapidae). Foi possível identificar no veneno as toxinas: fatores de coagulação de veneno do complexo protrombinase, subunidades de fosfolipases A2 (PLA2) da neurotoxina textilotoxin e PLA2 de atividade procoagulante, neurotoxinas tipo three-finger toxin (3FTx), inibidores de protease do tipo-kunitz textilinin, e pela primeira vez, uma nova variante de 3FTx, lectinas tipo C, CRiSP além de indícios de toxinas de lagarto Heloderma e outras proteínas candidatas a toxinas como calreticulin e dipeptidase 2. Metaloproteinases, pouco estudadas em Elapidae, foram clonadas e detectadas no veneno por ensaios de fracionamento e imunoreatividade. A análise do transcriptoma identificou novas isoformas e variantes de toxinas, principalmente das 3FTx e dos inibidores de serinoproteases, assim como transcritos de toxinas que não foram detectadas no veneno e que merecem mais investigações. O quadro de sintomas com acidentes em humanos é bem explicado pelas toxinas identificadas, porém, em seu habitat natural, as toxinas pouco conhecidas e até então não descritas devem ter funções importantes e específicas na predação. Identificar esta diversidade de variantes é importante para entender o modo de ação das toxinas. / Snake venom toxins alter prey homeostasis for feeding or defense. In depth studies of venom composition are important for better antivenom production, for new drugs lead and discovery and for better understanding of biological, ecological and evolutionary processes. Research on toxins have shown the natures way of innovating, refined by evolution, diversifying functions of protein families recruited from their endogenous function to the venom gland by successive gene duplication and mutation accumulation, leading to an accelerated evolution. A myriad of available toxins and diversity of functions is still available for discovery. Combining high throughput techniques such as venom gland de novo transcriptomics and venom proteomics, one can assess and observe a more complete profile of the snake toxinome, additionally allowing an upscale in low represented and unexpected toxin detection. The aim of this project was to investigate the venom toxinome of one of the most dangerous Australian species, Pseudonaja textilis (Elapidae). The toxins identified in it venom was: protrombinase complex coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and procoagulant PLA2, neurotoxic three-finger toxins (3FTx), Kunitz-type protease inhibitor textilinin, and for the first time, a new long 3FTx, C-type lectins, CRiSPs, as well as evidences of lizard toxins from Heloderma genus and other toxin candidates calreticulin and dipeptidase 2. Metalloproteinases, little investigated in Elapidae, was cloned and detected in the venom after fractionation and immunoassay. The transcriptome revealed new toxin variants and isoforms, specially 3FTx and serine protease inhibitors, as well as transcripts from toxins not detected in the venom that deserves further investigation. Human accident symptoms are well explained by the identified toxins, however, in its natural environment, little known and undescribed toxins must have specific and important role in predation. Identifying this diversity is important to better understand toxins ways of action.

The distinctiveness of brain structures and circuits depends on interacting gene products, yet the organization of these molecules (the "transcriptome") within and across brain areas remains unclear. High-throughput, neuroanatomically-specific gene expression datasets such as the Allen Human Brain Atlas (AHBA) and Allen Mouse Brain Atlas (AMBA) have recently become available, providing unprecedented opportunities to quantify molecular neuroanatomy. This dissertation seeks to clarify how transcriptomic organization relates to conventional neuroanatomy within and across species, and to introduce the use of gene expression data as a bridge between genotype and phenotype in complex behavioral disorders. The first part of this work examines large-scale, regional transcriptomic organization separately in the mouse and human brain. The use of dimensionality reduction methods and cross-sample correlations both revealed greater similarity between samples drawn from the same brain region. Sample profiles and differentially expressed genes across regions in the human brain also showed consistent anatomical specificity in a second human dataset with distinct sampling properties. The frequent use of mouse models in clinical research points to the importance of comparing molecular neuroanatomical organization across species. The second part of this dissertation describes three comparative approaches. First, at genome scale, expression profiles within homologous brain regions tended to show higher similarity than those from non-homologous regions, with substantial variability across regions. Second, gene subsets (defined using co-expression relationships or shared annotations), which provide region-specific, cross-species molecular signatures were identified. Finally, brain-wide expression patterns of orthologous genes were compared. Neuron and oligodendrocyte markers were more correlated than expected by chance, while astrocyte markers were less so. The localization and co-expression of genes reflect functional relationships that may underlie high-level functions. The final part of this dissertation describes a database of genes that have been implicated in speech and language disorders, and identifies brain regions where they are preferentially expressed or co-expressed. Several brain structures with functions relevant to four speech and language disorders showed co-expression of genes associated with these disorders. In particular, genes associated with persistent developmental stuttering showed stronger preferential co-expression in the basal ganglia, a structure of known importance in this disorder.

Freshwater sculpins (Cottus) are common throughout temperate regions of the Northern Hemisphere. Their broad distribution in the Western North America makes them a good model for understanding phylogeographic relationships among western fishes. Within much of the interior west three lineages, C. bairdii, C. confusus, and the C. beldingii complex, are most prevalent. The distribution of these three overlap in the Snake River Basin. All occur below Shoshone Falls on the Snake River. However, only two currently reside in the Upper Snake River above the falls. An exception are the Lost River streams of central Idaho. While these streams are technically part of the Upper Snake River Basin, they do not directly connect with the Snake River. Preliminary studies with a single mitochondrial DNA (mtDNA) gene suggested multiple pathways for Cottus introduction into the Lost River stream complex. Here, three mitochondrial and five nuclear genes were examined to investigate the phylogenetic relationships of these three lineages. Sequences were obtained from 71 different populations in the Lost River streams and surrounding basins. Maximum Likelihood (ML) phylogenies were constructed using these data. Our data indicate that relationships among populations within these species are complex and that no single invasion into the Lost River streams and surrounding regions can account for the phylogenetic signals detected. Instead, it appears that multiple invasions in an evolving landscape played a significant role in the modern distribution of species in this region.

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