BaSIC, which stands for Bioclastic and Shape-preserving Inorganic Conversion, is shape-preserving chemical conversion process of biological (or man-made) silica structures for producing complex 3-D microscale structures. This dissertation reports the BaSIC reaction of halide gases (i.e., TiF4, ZrF4, and ZrCl4) with 3-D silica structures, (i.e., diatom frustules, silicified direct-write assembly scaffolds, and Stöber silica spheres) to produce titania and zirconia replicas of the original 3-D structures. The kinetics of reaction of silica with titanium tetrafluoride gas is analyzed by using a novel HTXRD reaction chamber, nitrogen adsorption, and transmission electron microscope (TEM). The crystal structure and the temperature-induced phase transformation (from the room temperature hexagonal R-3c structure to the higher temperature cubic Pm3m structure) of polycrystalline TiOF2 that was synthesized through metathetic reaction of silica with TiF4(g) is reported. Additionally, potential applications of the converted titania diatom frustules (i.e., as a fast micron-sized ethanol sensor, and as a pesticide hydrolyzing agent) are also demonstrated in this work.

Why evaluations of the ecological outcomes of stream and river restoration have largely reported inconclusive or negative results has been the subject of much debate over the last decade or more. Understanding the reasons behind the lack of positive results is important for bettering future restoration efforts and setting realistic expectations for restoration outcomes. This thesis explores possible explanations for why researchers have failed to find clear and predictable biotic responses to stream restoration: recovery time has been too short, that restoration of habitat complexity is not clearly linked to instream biodiversity, that one monitored organism group is not representative of the entire community, that restoration effort was not intense enough to restore the potential habitat complexity of a system, and that reach-scale restoration done in the presence of catchment-scale degradation obscures restoration results. The overarching goal of this thesis is to study the holistic effect of reach-scale restoration of historic reach-scale simplification, due to timber floating in northern Swedish streams, thus avoiding the added pressure of catchment-scale degradation typically found at most restoration sites (e.g., non-point-source pollution and impervious cover). Using this model system, I was able to show that it took 25 years for riparian plant species richness at restored sites to increase above that of channelized sites. Furthermore, it was clear that restoration of these streams caused a large and rapid change in N-processing in the riparian zone and this alteration persists for at least 25 years. Additionally, multiple metrics of geomorphic complexity were needed to explain some of the more subtle responses of organism groups. Macroinvertebrates, diatoms, and macrophytes did not respond concordantly and cannot serve as surrogates or indicators for each other. I found that older best practice methods of restoration rarely restored the large-scale features needed to bring the sites up to their potential complexity because these elements were destroyed or removed from the system. Advanced restoration techniques used in more recent restorations added big boulders and instream wood and increased complexity to a level that elicited a biological response. By combining surveys of multiple metrics of structure, diversity of multiple organism groups, and process in this thesis I was able to get a holistic view of the effects of restoration of streams after timber floating. We now know that it takes at least 25 years for riparian plants and N-cycling to recover, we understand that multiple metrics of geomorphic complexity should be measured to be able to explain biotic responses, and that restored complexity should better match the potential complexity of the site in order to elicit a biological response. Finally, we know that multiple organism groups need to be assessed when evaluating the response of biodiversity to restoration.

Els sistemes aquàtics continental representen un dels ecosistemes més amenaçats a nivell mundial, com a conseqüència de l'ús intensiu quel'home en fa. La conca del Guadiana no està lliure d'aquestes pressions antròpiques. Les grans infraestructures hidràuliques i l'escorrentia provinent de l'agricultura són només exemples dels greus problemes que pateix la conca. Aquests problemes es fan especialment palesos en la zona alta de la conca, on l'escassetat d'aigua no fa més que agreujar el problema.Tot això ha generat la necessitat urgent d'avaluar l'estat de conservació d'aquests ecosistemes aquàtics continentals, poder determinar la mesura i la magnitud de les pertorbacions que els estan afectant i així proposar mesures de gestió destinades a restaurar-ne la integritat ecològica. El principal objectiu que presenta aquest és determinar els patrons de distribució de les comunitats de algals (amb una menció especial en el grup de les diatomees) i de les seves causes en la conca del Guadiana i associades, amb la finalitat d'establir i proposar eines que permetin avaluar l'estat de conservació de les masses d'aigua d'aquestes conques. / In many parts of the world freshwater ecosystems are intensely modified and degraded by human activities. Rivers have been altered since historical times, but such modifications have intensified in the last century. Water extraction and heavily regulated reservoirs are spread disturbances in the Guadiana network. Drainage from agricultural land in river systems results in large inputs of nitrates and phosphates that eutrophy river waters. Inputs of fertilizers and water extraction are especially remarkable in the North-East Guadiana watershed. The development of tools to assess ecological status of water bodies has been a key issue in the management of the freshwater ecosystems in the last decades in order to protect and restore the biological integrity of ecosystems. The present study aims to determine distributional patterns of benthic algae communities (with a special attention on diatom microflora) and their causes in Guadiana and associated watersheds in order to establish tools for assessing the conservation status of the water bodies in those watersheds.

O registro de depósitos quaternários tem aumentado em áreas costeiras da região Amazônica. No entanto, estudos detalhados são ainda necessários visando interpretar seus ambientes de deposição, bem como reconstituir sua evolução ao longo do Quaternário. Em particular, faltam informações sobre a reconstituição das variações do nível relativo do mar ao longo da margem equatorial brasileira de modo a possibilitar a inclusão dessa área em discussões de interesse regional e global enfocando clima, tectônica e eustasia. O presente trabalho representa um esforço de integração de vários tipos de dados, i.e., sedimentologia, estratigrafia, palinologia, diatomácea, datação 14C, \'delta\'\'ANTPOT.13 C\', \'delta\'\'ANTPOT.15 N\' e C/N, objetivando reconstituir a evolução de paisagens no leste da Ilha do Marajó durante o final do Quaternário. Aspectos ligados a paleoambientes de sedimentação, padrões de vegetação, flutuações climáticas e variação do nível do mar serão abordados. Um total de 98 amostras de sedimentos argilosos e arenosos foi obtido de 85 m de testemunhos coletados com a sonda à percusão Robotic Key System (RKS). Estes testemunhos derivam de cinco poços (TSM4, TSM8, TSM10, TSM11 e TSM12) variando entre 10 e 24 m de profundidade, que foram distribuídos em um transecto proximal-distal correspondente a uma paleomorfologia em funil relacionada a um paleoestuário. As idades registradas variam entre 42.580 (±1430) anos A.P. e 3.184 (±37) anos A.P. Análises de fácies indicaram depósitos de areias grossas a finas, com estratificações plano-paralelas ou cruzadas, e argilas, maciças ou laminadas, intercaladas por camadas heterolíticas. Estas sucessões apresentam padrão granocrescente e/ou granodecrescente ascendente. Valores de \'delta\'\'ANTPOT.13 C\', \'delta\'\'ANTPOT.15 N\' e C/N sugerem matéria orgânica oriunda de fontes diversas, com contribuição de plantas terrestres (principalmente plantas \'C IND.3\'), bem como fitoplâncton marinho e de água doce, como tipicamente esperado em estuários. A combinação de dados isotópicos, C/N e associações de fácies, permitiu identificar ambientes deposicionais correspondentes a canal fluvial, planície de inundação, canal e planície de maré, bacia central, delta de maré, complexo barreira/inlet e lago. A análise palinológica apresentou mistura de táxons típicos de floresta, de vegetação aberta e de mangue. Em geral, não se detectou mudanças drásticas nos padrões de vegetação no leste da Ilha de Marajó nos últimos 40.000 anos. A assembléia polínica foi melhor representada nos testemunhos dos poços TSM8 e TSM4. Neste último, as oito amostras basais (i.e., MR248 a MR258) registraram tipos arbóreos, como Alchornea, Euphorbiaceae, Euterpe, Malpighiaceae e Moraceae/Urticaceae. Na vegetação de mangue, Rhizophora constituiu o gênero mais comum, enquanto Poacea e Cyperaceae foram os mais freqüentes entre os táxons de ervas. A proporção entre pólen de floresta e de ervas mostrou-se constante em todas as zonas inseridas no Pleistoceno Tardio. Porém, aumento significativo de tipos herbáceos, com espécies pioneiras representadas por Alchornea e Moraceae/Urticaceae, foi registrado a partir de 6.790 (±60) anos A.P. Portanto, esta é a idade considerada para o estabelecimento de vegetação aberta do leste da Ilha de Marajó. Análises de diatomáceas dos testemunhos TSM8, TSM10 e TSM11, onde não houve registro polínico, foram consistentes com o valores isotópicos e de C/N, indicando intervalos com maior contribuição de matéria orgânica derivada de fitoplâncton marinho. Diatomáceas identificadas nestes testemunhos são, em grande parte, espécies e gêneros marinhos, como Actinoptychus splendens, Paralia sulcata, Coscinodiscus radiatus, Coscinodiscus sp e Thalassiosira sp. Táxons continentais como Actinella sp1, Aulacoseira, Eunotia zygodon, Desmogonium e Pinnularia foram registrados em uma única amostra no topo do testemunho TSM8. Informações faciológicas, juntamente com dados isotópicos, elementares, \'ANTPOT.14 C\', palinológicos e de diatomáceas, são consistentes com a existência de paleoestuário com domínio de onda no Pleistoceno Tardio e Holoceno no nordeste da Ilha do Marajó, previamente ao estabelecimento do lago Arari. As fases iniciais propostas para a interrupção do influxo fluvial para o paleoestuário Arari coincidem com a formação de vegetação aberta na ilha e a conseqüente diminuição das áreas de mangue no Holoceno médio. O efeito da tectônica regional parece ter sido de grande contribuição nas mudanças da paisagem do Marajó durante o Pleistoceno e Holoceno. Divergência do nível relativo do mar proposto para a Ilha do Marajó em relação ao padrão global, combinado com o aumento de registro de atividade tectônica no Pleistoceno Tardio e Holoceno, levaram à hipótese de que eventos transgressivos poderiam ter sido devidos à subsidência tectônica, e não à eustasia. A transgressão registrada antes de 29.000 anos A.P. ocorreu simultaneamente à forte tendência de queda eustástica após o Último Máximo Interglacial. Além disto, a transgressão holocênica no Marajó é registrada entre 9.000 e 5.000 anos A.P., portanto tendo início antes do pico transgressivo global do Holoceno médio. Durante este período de ascensão eustática, o nível relativo do mar na Ilha de Marajó começou a estabilizar, processo este que culminou com a significativa progradação da costa no final deste período. Da mesma forma, é improvável que a abertura da vegetação, com aumento significativo em espécies herbáceas, registrada a partir do Holoceno médio tenha efeito climático, uma vez que ela ocorreu simultaneamente à mudança de clima relativamente mais árido para relativamente mais úmido. A hipótese mais provável é que vegetação aberta tenha se estabelecido no leste da Ilha de Marajó em função de mudança hidrológicas, talvez promovida pela subsidência tectônica e subseqüente estabilização, culminada com a recente progradação da linha de costa. / The record of Quaternary deposits has increased in coastal areas of the Amazon region. However, detailed studies are still needed aiming to interpret their depositional environments, as well as reconstruct their evolution throughout the Quaternary. In particular, there is a lack of information about the reconstruction of relative sea level along the Brazilian equatorial margin to enable the inclusion of this area in discussions of regional and global interest focusing climate, tectonics and eustasy. The present work represents an effort to integrate vários types of data, i.e., sedimentology, stratigraphy, palinology, diatoms, \'ANTPOT.14 C\' dating, \'ANTPOT.13 C\', \'delta\' \'ANTPOT.15 N\' and C/N, aiming to reconstruct landscape evolution in eastern Marajó Island during the late Quaternary. Aspects related to sedimentary paleoenvironments, vegetation patterns, climate fluctuations and relative sea level changes will be approached. A total of 98 samples of muddy and sandy sediments was obtained from 85 m of cores collected with a percusion Robotic Key System (RKS). These cores derive from five drills (TSM12, TSM11, TSM10, TSM8 and TSM4) that reached depths ranging from 10 to 24 m, which were distributed in a proximal-distal transect corresponding to a funnel paleomorphology related to a paleoestuary. Reported ages range from 42, 580 ± (1430) \'ANTPOT.14 C\' yr B.P and 3,184 ± (37) \'ANTPOT.14 C\' yr B.P. Facies analysis showed parallel laminated or cross stratified, fine- to coarse-grained sands, massive or laminated muds and heterolithic deposits. These deposits are often arranged into coarsening and fining upward cycles. Values of \'delta\'\'ANTPOT.13 C\', \'delta\'\'ANTPOT.15 N\' and C/N suggest organic matter from several sources, with contributions from terrestrial plants (mainly \'C IND.3\' plants), as well as freshwater and marine phytoplankton, as typically expected in estuaries. Combination of isotope data, C/N and facies associations led to identify depositional settings corresponding to fluvial channel, floodplain, tidal channel/tidal flat, central basin, tidal delta, inlet/barrier complex and lagoon. Pollen analysis showed a mixture of taxa typical of forest, open vegetation and mangrove. In general, drastic changes in vegetation patterns were not detected in eastern Marajó Island in the last 40,000 years. Pollen assemblages were better represented in cores TSM8 and TSM4. In the latter, the eight basal samples (i.e., MR248 to MR258) recorded arboreal types sucha as Alchornea, Euphorbiaceae, Euterpe, Malpighiaceae e Moraceae/Urticaceae. Among mangrove vegetation, Rhizophora constitutes the most common genera, while Poacea and Cyperacea were the most frequent among the herb taxa. The proportion between forest and herb pollen was constant in all zones inserted in the Late Pleistocene. However, significant increase of herbaceous pollen types, with pioneer species represented by Alchornea and Moraceae/Urticaceae, were recorded from 6,790 (±60) \'ANTPOT.14 C\' yr B.P. Thus, this is the age considered for the establishment of the open vegetation of eastern Marajó Island. Diatom analyses of the cores TSM8, TSM10 e TSM11, which did not record pollen, were consistent with the isotope and C/N values, indicating intervals with higher contribution of organic matter derived from marine phytoplankton. Diatoms identified from these cores are, in great part, marine species and genera, such as Actinoptychus splendens, Paralia sulcata, Coscinodiscus radiatus, Coscinodiscus sp and Thalassiosira sp. Continental taxa such as Actinella sp1, Aulacoseira, Eunotia zygodon, Desmogonium and Pinnularia were recorded only in one sample from the top of the core TSM8. Facies infomration, together with isotope, elementar, \'ANTPOT.14 C\', palinological, and diatom data, are consistent with the existence of a wave-dominated paleoestuary during the Late Pleistocene and Holocene in northeastern Marajó Island, previously to the establishment of Lake Arari. The initial stages proposed for the interruption of fluvial inflow to the paleoestuary Arari coincide with the formation of open vegetation in the island and the consequent reduction of mangrove areas at the mid Holocene. The effect of regional tectonics seems to have been of a great contribution to change the Marajó landscape during Pleistocene and Holocene. Divergency of relative sea level proposed for the Marajó Island with respect to the global pattern, combined with the increased record of tectonic activity in the Late Pleistocene and Holocene, led to the hypothesis of successive transgressions linked to tectonic subsidence as opposed to eustary. The transgression recorded before nearly 29,000 yr B.P. took place simultaneously to the pronounced tendency of sea level drop after the Last Interglacial Maximum. Additionally, the Holocene transgression in Marajó is recorded between 9,000 and 5,000 yr. B.P., thus it initiated before the global transgressive peak of the mid Holocene. During this period of eustatic rise, the relative sea level in Marajó Island started to stabilize, a process that culminated with a significant coast progradation at the end of this period. It is also unlikely that the change to open vegetation recorded from the mid Holocene had a climatic response, as this change took place simultaneously to climate fluctuation from relatively more arid to relatively more humid. The most likely hypothesis is that the open vegetation became established in eastern Marajó Island in response to hydrological changes, perhaps promoted by tectonic subsidence and subsequent estabilization, culminated with the recent coastline progradation.

La méthylation de l'ADN est l’une des marques épigénétiques les plus étudiées et est largement conservée. Mes travaux de thèse présentent le premier méthylome d'une diatomée marine P. tricornutum qui appartient à la famille des Stramenopiles. P. tricornutum présente une méthylation d’environ 6% qui est présente en mosaïque sur l’ensemble du génome. Une méthylation importante a été retrouvé chez les éléments transposables, en particulier les éléments amplifiés récemment de type Copia. L’analyse met en évidence plus de 320 gènes méthylés dans trois contextes génomiques différents : à proximité des éléments transposables, en grappes de gènes méthylés, et dans des gènes uniques. En outre, les gènes largement et complètement méthylés ont été trouvé fortement corrélés avec le silencing transcriptionnel et l'expression différentielle dans des conditions spécifiques. Enfin, il a été constaté que les gènes susceptibles d’avoir été acquis par transfert horizontal de gènes bactériens étaient préférentiellement insérés dans des régions riches en éléments transposables, ce qui suggère un mécanisme par lequel l'expression de gènes étrangers peut être tamponnée à la suite de leur insertion dans le génome. En résumé, P. tricornutum a une faible méthylation de l'ADN et une methylation relativement importante des éléments transposables et seulement quelques gènes méthylés. Ce premier méthylome d’une diatomée Stramenopile ajoute de manière significative à notre compréhension de l'évolution de la méthylation de l'ADN chez les eucaryotes. En ce qui concerne les modifications des histones, la distribution des marques H3K4me2, H3K9me2 et H3K27me3 a été examinée chez P. tricornutum. H3K4me2 est principalement associée à des gènes alors que les deux marques H3K9me2 et H3K27me3 ciblent principalement des éléments transposables. La répartition de H3K27me3 est inhabituelle et différente de ce qui a été observé chez les espèces modèles étudiées à ce jour. Les gènes marqués par H3K27me3 ont tendance à être faiblement exprimés et de façon différentielle. H3K27me3 et H3K9me2 ont tendance à co-marquer non seulement les éléments transposables méthylés, mais aussi des gènes fortement méthylés, ce qui semble être important pour le maintien du silencing des gènes différentiellement exprimés. L'analyse combinatoire de différentes marques d’histones et la méthylation de l'ADN nous a donné un aperçu du paysage de la chromatine chez les diatomées, et aidera à définir les caractéristiques structurales et fonctionnelles conservées. / DNA methylation is the most extensively studied and widely conserved epigenetic mark. Here the first whole genome methylome from a stramenopile, the marine model diatom P. tricornutum is reported. In P. tricornutum, around 6% of the genome was methylated in a mosaic landscape. Extensive methylation in transposable elements (TEs), especially in recently amplified Copia-like elements was found. Over 320 genes were found methylated occurring in three different genomic contexts: in the proximity of TEs, in clusters of methylated genes, and in single genes. Furthermore, genes extensively and completely methylated correlated strongly with transcriptional silencing and differential expression under specific conditions. Finally, it was found that genes likely acquired by horizontal gene transfer from bacteria were preferentially inserted within TE-rich regions, suggesting a mechanism whereby the expression of foreign genes can be buffered following their insertion in the genome. In general, P. tricornutum has low DNA methylation with relatively extensive DNA methylation on TEs and a few methylated genes. This first Stramenopile methylome adds significantly to our understanding of the evolution of DNA methylation in eukaryotes. As for the histone modifications, genome wide distribution of H3K4me2, H3K9me2 and H3K27me3 were examined in P. tricornutum. H3K4me2 is mainly associated with genes while both H3K9me2 and H3K27me3 marks target mainly transposable elements (TEs). The distribution of H3K27me3 is unusual and different from what have been profiled in model species so far. The genes marked by H3K27me3 tend to be lowly and differentially expressed. H3K27me3 and H3K9me2 tend to co-mark not only methylated TEs but also heavily methylated genes, which appears to be important for maintaining the silencing of differentially expressed genes. The combinatorial analysis of different histone marks and DNA methylation gave us an overview of diatom chromatin landscapes, and will help to define conserved structural and functional features.

Cyanobacterial blooms and their toxins are a major water quality and potential health risk around the world. This thesis developed an analytical method for microcystin congeners in sediments in order to examine their fate in lakes and establish the history of toxin-producing cyanobacteria in relation to environmental change using lake sediments. A novel method for both intra- and extracellular microcystins in lake sediments was developed, consisting of accelerated solvent extraction, hydrophilic-lipophilic balance solid phase extraction and multiple reaction monitoring-based HPLC-MS/MS quantitation. The method achieved comparable recoveries of intra- and extracellular cyanotoxins based on nine microcystins and nodularin (marine analogue). The analytical method was validated using surficial and deeper sediments from seven lakes of diverse geography and trophic state. To study the fate of microcystins, a multi-year, whole lake study of Microcystis blooms was conducted to obtain both in situ and in vitro half-life estimates of microcystin-LA (MC-LA), an understudied, but increasingly reported microcystin. MC-LA appeared to undergo slower rates of decomposition and persist longer than the more frequently studied MC-LR. Experimentally, high light intensity increased in vitro decomposition of dissolved MC-LA while high temperature enhanced decomposition in the particulate phase. Sediment deposition measurements and estimates of sediment-pore water distribution coefficients, sediment accumulation rates, and diffusive fluxes indicated that microcystin congeners differ in their fate. Notably, MC-LA preferentially distributed into pore water and remobilized (by diffusion) from sediments and into overlying water while MC-RR adsorbed more strongly to sediment particles. Finally, the sediment record of an eutrophic lake of major recreational importance was examined to identify possible drivers of toxigenic cyanobacteria and determine if the perceived increase in toxigenic cyanobacteria could be corroborated. Microcystins were detected to the bottom of the core (early 1800s), indicating that toxigenic cyanobacteria were present prior to the first permanent settlements. Microcystins were significantly correlated with changes in diatom-inferred nutrients (DI-TP and DI-TKN) within the sediment core as well as with specific algal pigments. Sediment microcystins in the upper layers also significantly correlated with a 20-year monitoring record for water column microcystins suggesting that sediment microcystins can be used as a proxy for past surface water conditions.

Kieselalgen (Diatomee) sind eukaryotische einzellige Algen die hochspezifische Proteine (sogenannte Silaffine) erzeugen, um ‘nanopatterned’ Silica-Zellwände herzustellen. Diese Proteine zeigen geringe oder gar keine Homologie innerhalb der Diatomeen Gattung und sind ausgiebig (extensiv) posttranslatorisch modifiziert. Zum Unterschied zu konventioneller Modifikation (z.B. Phosphorylierung und Glykosylierung) weisen Lysinreste von Silaffinen einige Polyaminketten mit sehr heterogenen molekularen Strukturen auf. Diese Modifikationen sind spezifisch für Kieselalgen und spielen somit hypothetisch eine Rolle in der Biosilica-Synthese. Allerdings sind Lysin Polyamin Modifikationen, modifizierte Proteine und modifizierte Stellen kaum charakterisiert. Um diese Frage zu beantworten entwickelten wir eine Methode Polyamine zu quantifizieren und die Position von Polyamin-Modifikationen in engverwandte Proteine zu identifizieren (in morphologisch unterschiedliche Diatomeen Thalassiosira pseudonana, T. oceanica und Cyclotella cryptica). Wir zeigten, dass das Gesamtmuster von Polyaminender phylogenetischen Nähe dieser Kieselalgenarten folgt und dass diese Polyaminmodifikationen an Konsensusstellen sogar in Proteinen auftraten, die keine Sequenzähnlichkeit zeigten.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung / Diatoms are eukaryotic unicellular algae that employ highly specialized proteins called silaffins for making nanopatterned silica-based cell walls. These proteins share little or no homology across diatom species and are extensively post-translationally modified. Apart from conventional modifications (e. g., phosphorylation and glycosylation) lysine residues of silaffins bear polyamine chains with highly heterogeneous molecular structure. The latter appear to be specific for silicifying organisms and therefore hypothesized to play a key role in biosilica synthesis. However, polyamine modifications of lysines, modified proteins, and modification sites remain poorly characterized. To address these questions, we developed a method to quantify polyamines and identify sites of polyamine modifications in proteins from phylogenetically closely related, yet morphologically distinct diatoms Thalassiosira pseudonana, T. oceanica, and Cyclotella cryptica. We demonstrated that the overall pattern of polyamines followed the phylogenetic proximity across these diatom species and showed that polyamine modifications occurred at consensus sites even in proteins showing no sequence similarity.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung

The aim of this work is to test the proposed approach of algal scavenging as a driver of sediment mercury (Hg) on different time scales and to gain a better understanding of the mechanisms of Hg accumulation in lake sediments. A 3000-year sediment record from Nylandssjön in northern Sweden was analysed for this purpose, as well as a 20-year sediment record from the seasonal sediment traps of this lake. The diatom proxy biogenic silica (bSi) was determined by Fourier transform infrared spectroscopy (FTIRS) and chlorophyll-a (Chl-a) as a proxy for primary productivity by non-destructive visible near-infrared reflectance spectroscopy (VNIRS). Silica, normalised to minerogenic matter by aluminium (Si/Al) as an indirect diatom proxy and other geochemical parameters were analysed by the non-destructive method of X-ray fluorescence spectroscopy (XRF). The Hg content in the sediment was determined using the the thermal decomposition atomic absorption spectrophotometers (TD-AAS) method. Over the different time scales, organic matter (OM) is an important control factor for Hg, which in turn was strongly associated with primary productivity. Hg was normalised against OM by determining the Hg/LOI or Hg/C ratios. No positive correlation was found between the normalised Hg ratios and the proxies of primary productivity (bSi, Chl-a and Si/Al). Negative correlations between OM and minerogenic elements coinciding with human-induced erosion events and increasing Hg levels in the sediment were found. This is true, both for the long-term record in the sediment cores and the high-resolution data from the sediment traps. Furthermore, in the seasonal sediment record of the sediment traps, in-lake processes like lake turnover in spring and autumn could be linked to precipitation of iron oxyhydroxides (FeOOH) and increasing sedimentary Hg. This may be supported by the parallel sediment accumulation of other metals like nickel (Ni) and zinc (Zn) at the time of the lake turnover.

Pour pallier le manque de connaissances sur la biogéographie des diatomées nordiques, le recueil d'un maximum d'informations est encouragé. Rassembler les données paléolimnologiques régionales permet d'effectuer des analyses écologiques et environnementales dans un contexte géographique plus large. La Circumpolar Diatom Database (CDD), un projet initié en 1997 par le Laboratoire de Paléoécologie Aquatique (LPA), tente de répondre à ces besoins. Toutefois, cette première version de la CDD comporte certaines lacunes, dont l'insertion de données de type « Z » (niveau de carottage) et l'implantation dans un système d'information géographique (SIG). L'objectif principal de cette étude est de développer une nouvelle structure de base de données relationnelle particulièrement adaptée aux données paléolimnologiques favorisant l'archivage, la consultation et la représentation cartographique des données à l'échelle circumpolaire. En second lieu, ce projet vise à tester le potentiel d'implantation de la CDD dans un SIG et d'entrevoir sa diffusion au sein de la communauté scientifique. Les étapes méthodologiques de développement d'un système d'information ont mené à la création d'un modèle de base de données relationnel composé par langage SQL. Une liste de références taxonomiques a été réalisée par l'unification de plusieurs listes existantes. Une interface utilisateur a été développée sous forme de formulaires MS Access facilitant la consultation des résultats. L'utilisation de la CDD a été testée dans ArcGIS par une connexion OLE DB et par conversion en « Personal Geodatabase ». La collecte de données a permis de tripler le volume d'informations de la nouvelle CDD. Elle renferme maintenant quelque 572 sites d'échantillonnage, 39 350 données d'occurrence et 15 000 données limnologiques. Sa couverture s'étale sur huit régions circumpolaires arctiques réparties sur trois continents : Amérique du Nord, Europe et Asie. Jusqu'à maintenant, la couverture spatiale de la CDD permet difficilement d'effectuer des analyses sur une vaste échelle. Il est donc primordial d'encourager la communauté paléolimnologique à contribuer davantage au projet. Ce projet a généré un système d'information exploitable, et les phases d'implantation et de distribution pourront être entreprises en fonction de l'avenir souhaité pour la CDD et des ressources disponibles.

Este estudo foi desenvolvido no Parque Estadual da Ilha do Cardoso (PEIC) e no Parque Estadual Carlos Botelho (PECB), na região sudeste do Brasil. A área é caracterizada pela presença de vegetação nativa de Floresta Atlântica de Encosta e no caso do PEIC, também por Restinga e vegetação de manguezal. Foram coletadas amostras de solos a partir de trincheiras ou tradagens, e amostras em dois mangues, Sítio Grande e do rio Jacariu. Análises isotópicas (12C, 13C, 14C, 14N, 15N) e biológicas (palinologia, diatomologia) foram utilizadas nesse estudo para entender a história da paleovegetação da região durante do Pleistoceno tardio e o Holoceno, assim como suas relações com as mudanças climáticas. Os dados isotópicos (\'delta\'13C, 14C) da matéria orgânica dos solos (MOS) em dois pontos do PEIC mostraram valores de \'delta\'13C característicos de plantas C3 (aproximadamente -27,0%o), indicando a presença de vegetação de floresta nos últimos 10.800 anos AP. Em três pontos (CB1, CB2 e TURVI) do PECB os dados isotópicos (\'delta\'13C) da MOS indicaram a presença de plantas C3 em todo o perfil analisado. Nos pontos TOR e FAZ os valores de \'delta\'13C foram mais enriquecidos (entre -20,9%o e -23,5%o) nas camadas mais profundas, indicando a presença de uma vegetação menos densa que a atual, com uma provável presença de plantas C3 e C4, no período de aproximadamente 25.000 a 15.000 anos AP, sugerindo a presença de um clima mais seco. De aproximadamente 15.000 anos AP até o presente um empobrecimento isotópico (até -28,0%o) foi observado indicando a expansão da floresta, provavelmente associada à presença de um clima mais úmido que o período anterior. No mangue Sítio Grande os valores de \'delta\'13C foram mais empobrecidos (até -28,0%o) entre aproximadamente 40.000 e 19.000 anos AP. Os valores mais empobrecidos de \'delta\'13C associados com altos teores de carbono (até 40%) e da razão C/N (até 130) indicaram a presença de uma floresta na atual área do mangue e o abaixamento do nível relativo do mar. A presença de pólen de Ilex, Alchornea, Weinmannia, Rapanea, Symplocos, Drimys e Podocarpus sugerem a presença de uma floresta característica de clima mais frio e úmido que o atual entre 40.000 e 19.000 anos AP. Entre 19.000 e 2.200 anos AP observou-se um hiato sedimentar (provavelmente erosivo), atribuído a prováveis atividades neotectônicas ocorridas na região. Os valores mais enriquecidos de \'delta\'13C (aproximadamente -26,0%o e -24,0%o) associados com menores valores de COT (entre 0,3% e 6,0%) e razão C/N (entre 2 e 27), juntamente com a presença de diatomáceas marinhas, indicaram a presença do mangue e o retorno da linha de costa na posição atual na região desde pelo menos 2.200 anos AP. No mangue do rio Jacariu os resultados de \'delta\'13C apresentaram valores entre -24,0%o e -26,0%o. Os valores de COT e da razão C/N associados com os valores de \'delta\'13C indicaram uma mistura de plantas C3 e fitoplâncton na composição da matéria orgânica. As diatomáceas encontradas foram características de ambiente estuarino-lagunar e estiveram presentes aproximadamente nos últimos 1.500 anos AP / This study was developed in the Parque Estadual da Ilha do Cardoso (PEIC) and Parque Estadual Carlos Botelho (PECB), in the southeastern region of Brazil. The area is characterized by the presence of native vegetation of Atlantic Forest and in the case of PEIC, also for Restinga and mangrove vegetation. Soil samples were collected from trenches and by drilling. Were collected two cores in the Sítio Grande and Jacariu mangroves. Isotope (12C, 13C, 14C, 14N, 15N) and biological (pollen and diatoms analysis) tools were used in the study to understand the paleovegetation history of the region during the late Pleistocene and Holocene and its relation to climatic changes. The soil isotope data (\'delta\'13C) of soil organic matter (SOM) at two locations in the PEIC area showed values characteristics of C3 plants (approximately -27,0%o), indicating the presence of forest vegetation during the last 10.800 years BP. In three locations (CB1, CB2 and TURVI) in the PECB area the isotope data (\'delta\'13C) of SOM indicated the presence of C3 plants in the whole profile. In the locations TOR and FAZ the values were more enriched (from -20,9%o e -23,5%o) in the deeper horizons, indicating the presence of a less dense vegetation than the present, with a probable mixture of \'delta\'13C, 14C plants, in the period of approximately 25.000 to 15.000 years BP, suggesting the presence of a drier climate. From 15.000 years BP to the present, a isotopic depletion (até -28,0%o) was observed and associated with the expansion of the forest, probably associated to the presence of a more humid climate than the previous period. In the Sítio Grande mangrove the \'delta\'13C values were more depleted (approximately -28,0%o) from 40.000 to 19.000 years BP. The higher values of total organic carbon (40%) and C/N ratios (130), associated with more depleted \'delta\'13C values observed during this period indicated the predominance of C3 land plants in the location at present occupied by the mangrove and the decrease of the relative sea level. The presence of pollen of Ilex, Alchornea, Weinmannia, Rapanea, Symplocos, Drimys and Podocarpus during the period of 40.000 to 19.000 years BP suggest the presence of a colder and humid climate than today. From 19.000 to 2.200 years BP a sedimentary hiatus (probably erosive) was observed, attributed to a neotectonic activity in the studied area. The presence of mangrove since at least 2.200 years BP in its present position and the return of the marine cost line is associated with the values of COT (from 0,3% to 6%) and C/N ratio (from 2 to 27), more enriched \'delta\'13C values (from approximately -26,0%o e -24,0%o) and the presence of marine diatoms. In the Jacariu mangrove the \'delta\'13C results associated with the smallest values of total organic and C/N ratios, probably indicated a mixture of marine phytoplankton and C3 land plants in the composition of the organic matter. The marine diatoms were present approximately in the last 1.500 years BP and they are characteristics the estuarine site