Understanding the genetics underlying the speciation process has been a long-standing goal of evolutionary biology. Studying inter-population crosses can elucidate the genetic architecture of reproductive isolation and, ultimately, the process of speciation. Hybridization between two species is often maladaptive and results in offspring with decreased fitness compared to the parental forms. Recently, with the development of molecular and genomic tools, it has become possible to understand how and when reproductive isolation arises and what are the underlying mechanisms in the evolution of genetic incompatibilities. Heliconius is a genus of neotropical butterfly best know for their Müllerian mimicry. Here I focus on Heliconius cydno and Heliconius melpomene, two hybridising sympatric species with low levels of inter-specific hybridisation that nonetheless results in genome-wide signatures of admixture. I show that hybrids develop ovarian tissue and, occasionally, oocytes; and use genomic approaches to examine several potential mechanisms underlying post-zygotic isolation between H. cydno and H. melpomene. Firstly, I investigate evolution by gene duplication and identify loci putatively under divergent selection that may play a role in species divergence and speciation. Secondly, I quantify sexually dimorphic expression in H. melpomene, and calculate rates of molecular evolution between H. melpomene and H. erato. Thirdly, I identify differentially expressed genes in the H. cydno x H. melpomene F1 hybrids that may be involved in the species barrier. Finally, investigate whether epigenetic silencing mechanisms could underlie post-zygotic isolation between H. cydno and H. melpomene by quantifying transposable element expression and small RNAs. Overall, I identify loci that merit further investigation for their potential in maintaining reproductive barriers between these two species. I show that different regions of the genome evolve at different molecular rates but there is no faster-Z effect, and consider how might this affect evolution of reproductive isolation. Finally, I show that aberrant epigenetic silencing, a mechanism behind hybrid sterility that is common in other species, is not correlated with post-zygotic isolation between H. cydno and H. melpomene.

Genetic variation is the raw material of evolution. The sources of this variation within a population, and its maintenance within a species, have been mysterious since the birth of the field of evolutionary genetics. In this work, I study divergently adapted freshwater and marine populations of the threespine stickleback (Gasterosteus aculeatus) as an evolutionary model to track the origin of adaptive genetic variation and to describe the evolutionary processes maintaining variation across the genome. The stickleback is a small fish with a large geographic range encompassing the northern half of the Northern Hemisphere and composed of coastal marine habitats, freshwater lakes, and river systems. Populations of stickleback adapt rapidly to changes in habitat, and fossil evidence suggests that similar adaptive transitions have been ongoing in this lineage for at least ten million years. In this work, I develop a significant extension of restriction site-associated DNA sequencing (RAD-seq) to generate phased haplotype information to estimate gene tree topologies and divergence times at thousands of loci simultaneously. I find anciently derived clades of variation associated with marine and freshwater habitats in genomic regions involved in recent adaptive divergence; some divergence times extend to over ten million years ago. This history of adaptive divergence has had profound effects on genetic variation elsewhere in the genome: chromosomes harboring freshwater-adaptive variants retain anciently derived variation in linked genomic regions, while marine chromosomes have much more recent ancestry. I present a conceptual model of asymmetric selective and demographic processes to explain this result, which will form a nucleus for future research in this species. Lastly, by incorporating genome-wide recombination rates estimated from multiple genetic maps, I describe a recombination landscape that is favorable to the maintenance of marine-freshwater genomic divergence. Low recombination rates in key chromosomal regions condense widespread divergence of the physical genome, encompassing many megabases, into a small number of Mendelian loci. Combined, my results demonstrate the interconnectedness of evolutionary processes taking place on ecological and geological timescales. The genetic variation available for adaptive evolution today is a product of the long-term evolutionary history of a species.

Arsenic (As) undergoes extensive metabolism in biological systems involving numerous metabolites with varying toxicities. It is important to obtain reliable information on arsenic speciation for understanding toxicity and relevant modes of action. Currently, popular arsenic speciation techniques, such as chromatographic/electrophoretic separation following extraction of biological samples, may induce the alternation of arsenic species during sample preparation. The present study was aimed to develop novel arsenic speciation methods for biological matrices using surface-enhanced Raman spectroscopy (SERS), which, as a rapid and non-destructive photon scattering technique. The use of silver nanoparticles with different surface coating molecules as SERS substrates permits the measurement of four common arsenicals, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV). This speciation was successfully carried out using positively charged nanoparticles, and simultaneous detection of arsenicals was achieved. Secondly, arsenic speciation using coffee ring effect-based separation and SERS detection was explored on a silver nanofilm (AgNF), which was prepared by close packing of silver nanoparticles (AgNPs) on a glass substrate surface. Although arsenic separation using the conventional coffee ring effect is difficult because of the limited migration distance, a halo coffee ring was successfully developed through addition of surfactants, and was shown to be capable of arsenicals separation. The surfactants introduced in the sample solution reduce the surface tension of the droplet and generate strong capillary action. Consequently, solvent in the droplet migrated into the peripheral regions and the solvated arsenicals to migrated varying distances due to their differential affinity to AgNF, resulting in a separation of arsenicals in the peripheral region of the coffee ring. Finaly, a method combining experimental Raman spectra measurements and theoretical Raman spectra simulations was developed and employed to obtain Raman spectra of important and emerging arsenic metabolites. These arsenicals include monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII), dimethylmonothioarinic acid (DMMTAV), dimethyldithioarsinic acid (DMDTAV), S-(Dimethylarsenic) cysteine (DMAIIICys) and dimethylarsinous glutathione (DMAIIIGS). The fingerprint vibrational frequencies obtained here for various arsenicals, some of which have not reported previously, provide valuable information for future SERS detection of arsenicals.

The genus Clematis L. (Ranunculaceae) was used as a new model group to assess the role of the Himalayan orogeny on generation of biodiversity through investigations of its phylogeny, phylogeography and taxonomy. Although existing checklists include 28 species of Clematis from Nepal, a comprehensive taxonomic revision of available material in herbaria and additional sampling from fieldwork during this study has led to the recognition of 21 species of Clematis in Nepal, including one species (C. kilungensis) not previously recorded from Nepal. Exisiting phylogenetic and taxonomic concepts were tested with the addition of new samples from Nepal. The results highlight the shortcomings of the previous studies which were poorly resolved and indicate the need for a thorough revision of the sectional classification. Despite the increased sampling the results are still equivocal due to poor statistical support along the backbone of the phylogeny. Groups of species in well supported terminal clades are broadly comparable with results from previous studies although there are fewer clearly recognisable and well supported clades. The published dates for the evolution of Clematis were tested and the methodology of the previous study critically reappraised. The results indicate that the genus Clematis is approximately twice as old as previously reported and evolved in the middle Miocene. The phylogeny also demonstrates that, even allowing for poor support for the relationships between groups of species within Clematis, the extant Nepalese species must have multiple independent origins from at least 6 different colonisations. With their occurrence in the Pliocene and Pleistocene, these events are relatively recent in relation to the Himalayan orogeny, and may be linked more to the dispersal ability of Clematis than to the direct effects of the orogeny. Additional Nepalese samples of Koenigia and Meconopsis were added to exisiting datasets and these were reanalysed. The result from Clematis, Koenigia and Meconopsis were appraised in light of the the geocientific literature and previously published phylogeographic studies to create an overview of the drivers behind speciation in the Himalaya.

A diversidade de espécies e fenotípica pode variar consideravelmente entre grupos taxonômicos e ao longo do tempo em uma mesma linhagem. O estudo de tais variações tornou-se um dos principais objetivos da biologia evolutiva fornecendo informações importantes a respeito dos possíveis mecanismos que regulam a biodiversidade. Dessa forma, o objetivo geral da presente tese foi investigar os padrões da diversificação de espécies e da morfologia em um grupo cosmopolita de serpentes, a família Viperidae, e os potenciais processos subjacentes. Primeiramente, (1) reconstruímos as relações filogenéticas e estimamos os tempos de divergência entre as linhagens da família Viperidae utilizando uma abordagem Bayesiana. (2) Aplicando um método recentemente desenvolvido (BAMM), exploramos como as taxas de especiação e extinção variaram ao longo da radiação do grupo inferindo os possíveis processos reguladores. Por fim, (3) analisamos se a evolução do tamanho do corpo e as taxas de especiação variam nos diferentes habitats ocupados pelos viperídeos (terrestres vs arborícola). Nesta tese geramos a filogenia molecular de viperídeos mais completa até o momento utilizando sequências para 11 genes mitocondriais e nucleares abrangendo 79% das espécies viventes (264 terminais) e todos com exceção de um gênero. De maneira geral, foi possível obter relações filogenéticas robustas para o grupo com a maioria dos gêneros sendo monofilética. Os tempos de divergência obtidos indicam que os viperídeos começaram a diversificar em meados do Paleoceno tardio/meio do Eoceno inferindo idades um pouco mais tardias que o encontrado em estudos anteriores. Durante a radiação do grupo, um aumento nas taxas de especiação parece ter ocorrido durante a diversificação dos crotalíneos (pit vipers) em decorrência não só da evolução das fossetas loreais mas também como resultado de mudanças geológicas e climáticas na Ásia e da invasão do novo mundo. Após este rápido aumento inicial, as taxas de especiação desaceleraram em direção ao presente. Por fim, os resultados aqui apresentados indicam que apesar dos habitats arborícolas limitarem a evolução morfológica nos viperídeos, a evolução da arborealidade parece não afetar as taxas de especiação que permanecem similares entre linhagens arborícolas e terrestres. Isto sugere dois cenários: (1) a especiação acontece de forma independente das mudanças morfológicas nos viperídeos; ou (2) o isolamento geográfico seria um mecanismo importante na diversificação de linhagens arborícolas contrabalançando decréscimos nas oportunidades de especiação possivelmente relacionados às pressões seletivas impostas pelo ambiente arborícola. A presente tese contribui para entendermos mais sobre como evoluíram os viperídeos ao longo dos seus ∼50 milhões de anos. Além de propor cenários e hipóteses a serem futuramente explorados com os viperídeos, elaboramos uma discussão ampla e conceitual a respeito dos possíveis mecanismos por trás da diversificação de espécies e da morfologia que poderiam também ser contemplados para outros grupos de organismos. Portanto, a presente tese contribui não só para entendermos os mecanismos que geram e mantém a diversidade de serpentes, mas também para enriquecer a discussão dos mecanismos que geram e mantém a biodiversidade como um todo / Species and phenotypic diversity may vary considerably between taxonomic groups and through time for a given lineage. The study of such variation became one of the main goals of evolutionary biology and provides important information related to the possible mechanisms regulating biodiversity. The general goal of the present thesis was to investigate the patterns of species and morphological diversification in a cosmopolitan group of snakes, the family Viperidae, and the potential underlying processes. First, (1) we estimated the phylogenetic relationships and divergence times between lineages of the family Viperidae using a Bayesian approach; then we (2) applyed a recently developed method (BAMM) to explore how speciation and extinction rates varied during the radiation of the group suggesting possible underlying processes. Finally, (3) we analyzed if body size evolution and speciation rates showed distinct patterns among vipers occurring in different habitats (terrestrial vs arboreal). Herein we generated the most complete molecular phylogeny for vipers until this moment using sequences from 11 mitochondrial and nuclear genes comprising 79% of extant species (264 terminals) and all except one genus. In general, we were able to recover well supported phylogenetic relationships with most genera being monophyletic. Divergence time estimates suggested that vipers started to diversify around the late Paleocene/middle Eocene finding older ages than previous studies. During the group radiation, an increase in speciation rates seems to have occurred during the diversification of crotalines (pit vipers) not only due to the evolution of loreal pits but also as a result of climatic and geological changes in Asia and the invasion of the New World. After this rapid initial increase, speciation rates decelerated toward the present. Lastly, the results presented here suggest that although arboreal habitats constrain morphological evolution in vipers the evolution of arboreality does not seem to affect speciation rates, which remain similar among arboreal and terrestrial lineages. Our results suggest two distinct scenarios: (1) speciation could be independent of morphological evolution in vipers; or (2) geographic isolation would be an important mechanism underlying species diversification in arboreal lineages offsetting decreases in speciation opportunities potentially related to the selective pressures imposed by the arboreal environment. The present thesis contribute to increase our understanding about how vipers evolved during their ∼50 million years. In addition to providing scenarios and hypotheses to be further explored with vipers, we elaborated a broad and conceptual discussion about the possible mechanisms underlying species and morphological diversification that might apply to other groups of organisms. Therefore, this thesis comprises a contribution that goes beyond the understanding of mechanisms generating and maintaining the diversity of snakes, but will hopefuly enrich the discussion of mechanisms that generate and maintain biodiversity as a whole

Na presente Tese foram usados métodos de inferência filogenética e de filogeografia buscando identificar os processos históricos de diversificação do gênero Conopophaga na América do Sul, em especial na Mata Atlântica. O monofiletismo do gênero e a estrutura filogeográfica das espécies distribuídas no sudeste da Mata Atlântica (Conopophaga lineata e C. melanops), foram testados utilizando seqüências de DNA mitocondrial. Para a filogenia foram utilizadas duas matrizes, sendo uma de 2270 pb (941 pb da subunidade 2 da NADH desidrogenase (ND2), 343 pb do ND3 e 986 pb do citocromo b) e outra de 878 pb (461 pb do ND2 e 417 pb do cit b). Nas análises de filogeografia de C. lineata e C. melanops foram utilizadas seqüências da região controladora de 472 pb e 439 pb, respectivamente. Os resultados demonstraram que o gênero Conopophaga é monofilético e que provavelmente uma rápida radiação ocorreu nesse gênero depois da especiação de C. melanogaster e de C. melanops. Dessa radiação, foram recuperados dois grupos: (1) Um grupo que se distribui somente na Amazônia e mantém a característica ancestral da coloração negra da mandíbula e (2) um grupo distribuindo-se na Amazônia e também na Mata Atlântica e que possui a mandíbula branca. Nesse último grupo, C. l. cearae não se agrupou com C. lineata, demonstrando que essa espécie não é uma espécie monofilética. A relação entre as espécies que apresentam a mandíbula branca parece indicar a ocorrência de uma conexão entre o leste da Amazônia e a Mata Atlântica no passado. O estudo filogeográfico de C. lineata revelou a existência de possíveis eventos de vicariância: (1) na região compreendida pelo Vale do Rio Paraíba do Sul e (2) à oeste de São Paulo e Paraná, separando as populações mais ao sul. Apesar de as inferências filogenéticas realizadas em C. melanops e C. lineata não serem totalmente concordantes, é possível que exista um padrão de vicariância nessa região. Concluindo, a ocorrência desses eventos vicariantes, tais como eventos geológicos e ciclos de alterações climáticas tenham influenciado na diversificação da família Conopophagidae. Além disso, eventos de dispersão e/ou seleção também podem auxiliar no entendimento da história biogeográfica do grupo, bem como de outros grupos na América do Sul. / In order to identify the historical processes of diversification of the gender textitConopophaga in South America, especially in the Atlantic forest, methods of phylogenetic and phylogeography inference were used in the present thesis. The genus phylogeny and the phylogeographic structure of two species ( textitConopophaga lineata and textitC. melanops) which occurs in the Southeast of the Atlantic forest were tested using sequences of mithocondrial DNA. Two matrixes were used to perform the phylogenetic analyses. The first one comprising 2270 bp (941 bp of ND2, 343 bp of ND3 and 986 bp of cytochrome b) and the second one comprising of 878 bp (461 bp of ND2 and 417 bp of cytochrome b). The phylogeography analyses of textitC. lineata and textitC. melanops were done using sequences from the control region consisting of 472 bp and 439 bp, respectively. The results demonstrated that the genus textitConopophaga is monophyletic and probably after textitC. melanogaster and textitC. melanops speciation, a rapid diversification had occurred in this genus. Following this event two distinct groups were recovered: (1) a group distributed in Amazonian, which maintains the ancestral characteristic of black jaw and (2) a group possessing white jaw occurring in the Amazonian and also in the Atlantic forest. In the last group, the subspecies C. l. cearae did not grouped with textitC. lineata demonstrating that this species is not monophyletic. Moreover, the distribution pattern of species presenting white jaw indicates a plausible a connection between the east of the Amazonian and the Atlantic forest in the past. The phylogeographic study of textitC. lineata revealed the existence of possible vicariant events: (1) in the area of Vale do Rio Paraíba do Sul and (2) in the west of São Paulo and Paraná, separating the southern south populations. Although the phylogeographic structure observed in textitC. melanops and in textitC. lineata are not in total agreement, the occurrence of vicariant events still remains as a possible explanation for the phylogeographic patterns in this region. Finally, the occurrence of these vicariant events like, geological events and climatic oscilations, may have influenced the diversification of the family Conopophagidae. Moreover, dispersion events and/or selection should also be considered for the understanding of biogeographic history of this group and also other ones in South America.

O ferro é um nutriente essencial para quase todas as espécies vivas e desempenha muitos papéis essenciais nos sistemas biológicos, como transporte de oxigênio, respiração, metabolismo energético, destruição de peróxidos e síntese de DNA. A deficiência de ferro no organismo pode acarretar diversas desordens fisiológicas, como por exemplo, a anemia ferropriva, que está relacionada com os níveis de hemoglobinas no sangue. Uma das maneiras de se obter as necessidades diárias desse nutriente e, dessa forma evitar as desordens fisiológicas ocasionadas pela deficiência do mesmo, é o consumo de frutas, hortaliças e carnes na dieta, uma vez que esses alimentos são ricos nesse elemento. Dessa forma, foram feitas determinações para avaliar as quantidades totais e a bioacessibilidade (através de métodos in vitro) de ferro, e de outros macro e micronutrientes, em diferentes tipos de alimentos. Além disso, visando buscar correlações com os resultados obtidos, a composição centesimal e o conteúdo de ácido fítico dos alimentos analisados também foram determinados. A concentração de ferro heme foi determinada para as amostras de fígado bovino. As quantidades totais e bioacessíveis dos elementos permitiram constatar que dentre todos os alimentos analisados, o espinafre foi o alimento que apresentou a maior quantidade de ferro total, porém verificou-se que a bioacessibilidade deste nutriente é muito baixa, sendo inferior a 20%. Isto pode estar relacionado ao elevado teor de oxalatos e fitatos (que possuem capacidade complexante) presente neste tipo de alimento. Ainda com relação a bioacessibilidade deste nutriente, o fígado bovino cru foi o alimento que apresentou a maior quantidade de ferro bioacessível. Porém, quando o mesmo é submetido ao processo de cocção, o valor da bioacessibilidade deste nutriente diminui consideravelmente, o que está correlacionado com a quebra do complexo de ferro heme presente em maior parte neste alimento, transformando-se em ferro iônico, que possui uma menor biodisponibilidade no organismo humano, pois é absorvido de forma menos eficaz que o ferro heme. Além disso, com relação aos alimentos de origem vegetal, os resultados mostraram que o potássio (K) possui as maiores quantidades totais e bioacessíveis para os alimentos analisados, resultado que se encontra em concordância com o fato deste elemento ser fundamental no crescimento de qualquer espécie vegetal. A determinação do conteúdo de ácido fítico (fitatos) permitiu evidenciar que os mesmos possuem diferentes intensidades de ligação com os cátions dos nutrientes presentes nos alimentos avaliados, e desse modo a influência dos fitatos na bioacessibilidade dos elementos analisados é maior para alguns e menor para outros, dependendo da estabilidade dos complexos formados entre os minerais e os fitatos. As concentrações de ferro heme obtidas para as amostras de fígado bovino demonstraram que o aquecimento provoca uma diminuição considerável na porcentagem de ferro heme presentes nas amostras, variando entre 33 e 43 %. Esta diminuição está relacionada à clivagem oxidativa do anel porfirínico em que o Fe está coordenado, e essa redução é relativamente alta uma vez que a liberação do ferro do complexo heme só ocorre em temperaturas elevadas, de 85 a 100 °C. / Iron is an essential nutrient for almost all living species and plays essential roles in many biological systems, such as oxygen transport, respiration, energy metabolism, destruction of peroxides, and DNA synthesis. Iron deficiency in the body may cause various physiological disorders, such as anemia, which is related to hemoglobin levels in blood. One way to obtain the daily requirement of this nutrient and thus avoid the physiological disorders caused by its deficiency is to ingest fruits, vegetables, and meat, because they are rich in this element. This work evaluated the bioaccessibility (by in vitro methods) and the total amounts of iron and other macro and micronutrients in different types of food. Seeking correlations with the results, the percent composition and phytic acid content of the analyzed foods were also determined, and the concentration of heme iron in samples of bovine liver was analyzed. The total and bioaccessibles amounts of elements allowed to observe that among all foods, spinach contained the highest amount of total iron, but the bioaccessibility of this nutrient was low, below 20%, probably due to the high levels of oxalates and phytates (which have complexing capacity) in this type of food. Still regarding the bioaccessibility of this nutrient, bovine liver was the food with the highest amount of bioaccessible iron. However, cooking considerably reduced bioaccessibility of this nutrient. This decrease is correlated with the breakdown of the heme iron complex present in bovine liver, wich generated the less bioavailable and the less efficiently absorbed ionic iron. Concerning plant species, the results showed that potassium (K) had the highest total and bioaccessible amounts for the analyzed foods, a result that agreed with the fact that this element is essential for the growth of any kind vegetable. Determination of phytic acid (phytates) content showed that they correlated with the cations of the nutrients in the tasted foods differently; i. e., phytates affected the bioaccessibility of the analyzed elements to different degrees, depending on the stability of the complexes formed between the minerals present in a given food and phytic acid. The heme iron concentrations in bovine liver samples revealed that heating significantly lowered the percentage of heme iron present in the samples, which ranged from 33 to 43%. This decrease was related to the oxidative cleavage of the porphyrin ring. The reduction in heme iron was relatively large, since the release of heme iron complex only occurs at elevated temperatures from 85 to 100 ° C.

Alguns dos padrões ecológicos mais consistentemente encontrados na natureza, como as relações espécie-área e as distribuições de rank-abundância, podem ser previstas por uma classe de modelos neutros. Nesse contexto, neutralidade quer dizer que há equivalência demográfica entre os indivíduos de todas as espécies. Para os modelos dessa classe, extinções causadas por flutuações demográficas são contrabalanceadas por algum mecanismo de especiação. Cada modo de especiação deixa uma marca nos padrões ecológicos emergentes. Foi mostrado que um modelo com uma implementação mecanística de especiação gera padrões de diversidade que dependem de limites geográficos. Eu usei simulações baseadas em indivíduos com uma implementação mecanística de especiação para investigar se padrões espaciais intrínsecos das comunidades poderiam transformar os padrões de biodiversidade. Eu descobri que existe uma transição de fase no modo de especiação que depende da estrutura espacial da comunidade. Uma gama extensa de padrões encontrados na natureza puderam ser unificados em um único modelo dada essa transição de fase. Relações entre riqueza e idade de um clado podem ser melhor compreendidas considerando-se o efeito previsto de desaceleração crítica da diversificação. Uma nova interpretação foi dado ao efeito \"Clado Morto Andando\", característico dos períodos seguintes a extinções em massa. Uma redefinição objetiva e biologicamente razoável para especiação alopátrica é explorada, graças às propriedades da transição de fase descrita. Eu proponho a existência de um \"crédito de especiação\", e exploro suas possíveis implicações para a conservação a longo prazo da biodiversidade / Some of the most consistent ecological patterns encountered in nature, such as species-area relationships and rank-abundance distributions, can be predicted from a class of neutral models. In this context, neutrality means demographic equivalence between individuals of all species. Within this class of neutral models, species extinction by demographic fluctuations is counterbalanced by some speciation mechanism. Each particular speciation mode leaves an imprint in the resulting patterns. A model with a mechanistic speciation implementation was shown to generate patterns dependent on geographic constraints. I used individual based simulations with a mechanistic speciation implementation to investigate whether the intrinsic spatial patterning of organisms could transform biodiversity patterns. I found out that there is a phase transition on speciation modes that is dependent on the spatial structure of the community. An extended range of the biodiversity patterns found in nature can be unified into a single model because of this phase transition. Clade richness and age relationships may be understood by the predicted critical slowdowns in diversification. A new interpretation is given to the post mass extinction \"Dead Clade Walking\" effect. An objective and biologically reasonable redefinition of allopatric speciation is explored by exploiting the phase transition. I propose the \"speciation credit\" effect, and its potential implications for long term biodiversity conservation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this work, the aim of this study is to evaluate sample preparation procedures and liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) and LC coupled to chemical vapor generation (CVG) and ICP-MS (LC-CVG-ICP-MS) for identification and quantification of As species in mollusks collected in BTS. Conditions related to As species extraction were assessed with the use of microwave assisted extraction (MAE) and ultrasound-assisted extraction (UAE). The influence of the main parameters involved in extraction of As species such as the type and concentration of extraction solution, sample mass, temperature and extraction time were evaluated. The extraction was possible by using water at 80 °C (0.2 g sample and 6 mL of w ater) for 6 min using MAE. For the chromatographic separation the type of mobile phase [(NH4)2CO3 and (NH4)2HPO4] concentration (1 to 20 mmol L-1), pH (5.0 to 9.0), flow rate (1.05 to 1.45 mL min-1) and elution mode (isocratic and gradient) were evaluated. The volume of sample injected into the chromatograph was fixed to 200 μL. For the separation of As species an anion exchange column (Hamilton PRP-X100, 250 x 4.1 mm) was used. Six As species were detected in the mollusks being identified arsenobetaine (AsB), As(III), dimethylarsenic acid (DMA) and ρ- arsanilic acid (ρ-ASA). The best separation of the As species was obtained by gradient elution mode and using the following program: 0 to 1.4 min using 6 mmol L-1 (NH4)2HPO4 to 1.4 to 2.7 min using 20 mmol L-1 (NH4)2CO3 and 2.7 to 15 min using 6 mmol L-1 (NH4)2HPO4). The mobile phase flow rate was set at 1.25 mL min-1. CVG parameters were evaluated and the concentrations of NaBH4 and HCl, and carrier gas flow rate were set to 1.0% (m/v) 1.0 mol L-1 and 1.15 L min-1, respectively. The extraction procedures and determination of total As were evaluated using certified reference materials BCR 627 (Tuna Fish Tissue), DORM-2 (Dogfish Muscle) and NIST 1566b (Oyster tissue). The agreement of the results obtained was 95 ± 3 to 101 ± 5%, respectively. The sum of the concentrations of As species determined by LC-ICP-MS and the total As concentration in digested samples and extracts measured, determined by ICP OES were also in good agreement on a confidence level of 95% (Student t-Test). The developed methods by employing the LC-ICP-MS and LC-CVGICP- MS for As speciation analyses in bivalves mollusks are considered suitable. / Neste trabalho foi avaliado o preparo da amostra e o uso da cromatografia a líquido acoplada à espectrometria de massa com plasma indutivamente acoplado (LC-ICP-MS) e LC acoplado ao sistema de geração química de vapor (CVG) e ICP-MS para a identificação e quantificação das espécies de As presentes em tecidos de moluscos bivalves. Condições relacionadas à extração das espécies de As foram avaliadas com o emprego de extração assistida por micro-ondas (MAE) e ultrassom (UAE). A influência dos principais parâmetros envolvidos na extração das espécies de As, como o tipo e concentração da solução extratora, massa de amostra, temperatura e o tempo de extração por MAE e o tempo de sonicação por US foram avaliados. Extração de As foi possível usando água a 80 °C (0,2 g de amostra e 6 mL de água) durante 6 min com MAE. Para a separação cromatográfica foram avaliados o tipo de fase móvel [(NH4)2CO3 e (NH4)2HPO4], a concentração (1 a 20 mmol L-1), o pH (5,0 a 9,0), a vazão (1,05 a 1,45 mL min-1) e o modo de eluição (isocrático e gradiente). O volume de amostra injetada no cromatógrafo foi fixado em 200 μL. Para a separação das espécies de As foi utilizada uma coluna de troca aniônica (Hamilton PRPX100, 250 x 4,1 mm). Foram detectadas seis espécies de As nos moluscos, sendo identificadas somente a arsenobetaína (AsB), o As(III), o dimetilarsênio (DMA) e o ácido ρ- arsanílico (ρ-ASA). A melhor separação das espécies de As foi obtida por eluição gradiente utilizando as seguintes condições: 0 a 1,4 min empregando 6 mmol L-1 de (NH4)2HPO4, 1,4 a 2,7 min empregando 20 mmol L-1 de (NH4)2CO3 e 2,7 a 15 min utilizando 6 mmol L-1 de (NH4)2HPO4. A vazão da fase móvel foi fixada em 1,25 mL min-1. Os parâmetros de CVG avaliados foram a concentração de NaBH4 e HCl e a vazão do gás de arraste, as quais foram fixadas em 1,0 % (m/v), 1,0 mol L-1 e 1,15 L min-1, respectivamente. Os procedimentos de extração e determinação de As total foram avaliados utilizando os materiais de referência certificados BCR 627 (Tuna Fish Tissue), DORM-2 (Dogfish Muscle) e NIST 1566b (Oyster tissue). A concordância entre os resultados obtidos foi de 95 ± 3 a 101 ± 5%, respectivamente. Os valores entre a soma das concentrações das espécies de As obtidos por LC-ICP-MS e As total nos digeridos e extratos obtidos, determinado por ICP OES também foram concordantes em um nível de confiança de 95% (Teste t-Student). Desta forma, o método desenvolvido empregando LC-ICP-MS e LC-CVG-ICP-MS foi considerado adequado para determinação de espécies de As em moluscos bivalves.

Abstract This thesis is focused on the hydrolysis of aluminium, the polymerisation of the hydrolysis products, and how these can be monitored by mass spectrometric methods. The main aim of this research is to figure out how the aqueous speciation of aluminium changes as a function of pH (3.2–10), concentration (1–100 mM), reaction time (1s–14d), and counter anion (Cl-, SO42-, HCOO-). The method used was electrospray mass spectrometry. The results showed more variable speciation than those suggested earlier. The main species were Al2, Al3, and Al13, which were found in all of the conditions under scrutiny. The effect of pH was the most remarkable of all the parameters researched. The formation of large highly charged complexes was strongly dependent on it. Also the Al-concentration in the bulk solution had a clear effect on speciation: in dilute solutions there were more protonated ligands and less attached counter anions. This could mean that the species in more diluted bulk solutions had fewer different states of charge. Reaction time caused only minor changes to speciation in the initial pH: there was slightly more variation of a certain sized species in the aged solution. In elevated pH, the birth of important Al13 oligomers was time dependent. The effect of the counter anion was tremendous. In a chloride environment the speciation was rich and diversified. With sulphate the speciation was limited to solid- like compounds, and the variation of single-sized species was almost lacking. The formate as a counter anion caused most surprising results; the charge of aluminium in some studied complexes was lowered from the common 3+ to 1+. If this reaction also occurs in natural circumstances, the uses of aluminium formate would be wide. The results can be utilised in following the progress of dissolution, the mobilization and toxicity of aluminium in natural waters, as well as in water purification, and in reaching minimal chemical contamination levels in sludge as well as in aqueous waste.