Diatoms play a fundamental role in marine food webs, and significantly contribute to global primary production and carbon sequestration into the deep ocean. In many offshore areas of the open ocean, iron (Fe) input is low, and its availability often limits phytoplankton biomass. Recently, gene sequences encoding ferritin, a nearly ubiquitous iron storage and detoxifying protein, have been identified in pennate diatoms such as Pseudo-nitzschia, but not in other Stramenopiles (which include centric diatoms, brown algae and some protist plant parasites) or Cryptophyte relatives. Members of this genus readily bloom upon addition of iron to Fe-limited waters, and are known to produce the neurotoxin domoic acid. Until now, the reason for the success of pennate diatoms in the open ocean was uncertain; however, expressing ferritin would allow pennate species to store Fe after a transient input, using it to dominate Fe stimulated algal blooms. Here, the ferritin gene was cloned from the coastal pennate diatom Pseudonitzschia multiseries, overexpressed in Escherichia coli, and purified using liquid chromatography. The ferritin protein sequence appears to encode a non-heme, ferritinlike di-iron carboxylate protein, while gel filtration chromatography and SDS-PAGE indicate that this ferritin is part of the 24 subunit maxi-ferritins. Spectroscopically monitoring the addition of Fe(II) to a buffered ferritin solution shows that the P. multiseries protein demonstrates ferroxidase activity, binding iron and storing it as Fe(III) in excess of 600 equivalents per protein shell. In keeping with the typical stoichiometry of the ferroxidase reaction, oxygen (O₂) is consumed in a 2 Fe:O₂ratio while hydrogen peroxide is produced concurrently. iii Diatoms evolved from secondary endosymbiosis involving eukaryotic red algae; however, a broad phylogenetic comparison suggests that P. multiseries ferritin was likely acquired via lateral gene transfer from cyanobacteria – not from its ancestral endosymbionts. Until recently, no other ferritins have been identified in diatoms, and the protein characterized here is unique in that it seems to be derived from a prokaryotic organism yet it occurs in a marine eukaryote. These findings have direct implications for the success of pennate diatoms in both Fe rich coastal waters and upon Fe addition in the open ocean.

Diatoms play a fundamental role in marine food webs, and significantly contribute to global primary production and carbon sequestration into the deep ocean. In many offshore areas of the open ocean, iron (Fe) input is low, and its availability often limits phytoplankton biomass. Recently, gene sequences encoding ferritin, a nearly ubiquitous iron storage and detoxifying protein, have been identified in pennate diatoms such as Pseudo-nitzschia, but not in other Stramenopiles (which include centric diatoms, brown algae and some protist plant parasites) or Cryptophyte relatives. Members of this genus readily bloom upon addition of iron to Fe-limited waters, and are known to produce the neurotoxin domoic acid. Until now, the reason for the success of pennate diatoms in the open ocean was uncertain; however, expressing ferritin would allow pennate species to store Fe after a transient input, using it to dominate Fe stimulated algal blooms. Here, the ferritin gene was cloned from the coastal pennate diatom Pseudonitzschia multiseries, overexpressed in Escherichia coli, and purified using liquid chromatography. The ferritin protein sequence appears to encode a non-heme, ferritinlike di-iron carboxylate protein, while gel filtration chromatography and SDS-PAGE indicate that this ferritin is part of the 24 subunit maxi-ferritins. Spectroscopically monitoring the addition of Fe(II) to a buffered ferritin solution shows that the P. multiseries protein demonstrates ferroxidase activity, binding iron and storing it as Fe(III) in excess of 600 equivalents per protein shell. In keeping with the typical stoichiometry of the ferroxidase reaction, oxygen (O₂) is consumed in a 2 Fe:O₂ratio while hydrogen peroxide is produced concurrently. iii Diatoms evolved from secondary endosymbiosis involving eukaryotic red algae; however, a broad phylogenetic comparison suggests that P. multiseries ferritin was likely acquired via lateral gene transfer from cyanobacteria – not from its ancestral endosymbionts. Until recently, no other ferritins have been identified in diatoms, and the protein characterized here is unique in that it seems to be derived from a prokaryotic organism yet it occurs in a marine eukaryote. These findings have direct implications for the success of pennate diatoms in both Fe rich coastal waters and upon Fe addition in the open ocean.

Diatoms play a fundamental role in marine food webs, and significantly contribute to global primary production and carbon sequestration into the deep ocean. In many offshore areas of the open ocean, iron (Fe) input is low, and its availability often limits phytoplankton biomass. Recently, gene sequences encoding ferritin, a nearly ubiquitous iron storage and detoxifying protein, have been identified in pennate diatoms such as Pseudo-nitzschia, but not in other Stramenopiles (which include centric diatoms, brown algae and some protist plant parasites) or Cryptophyte relatives. Members of this genus readily bloom upon addition of iron to Fe-limited waters, and are known to produce the neurotoxin domoic acid. Until now, the reason for the success of pennate diatoms in the open ocean was uncertain; however, expressing ferritin would allow pennate species to store Fe after a transient input, using it to dominate Fe stimulated algal blooms. Here, the ferritin gene was cloned from the coastal pennate diatom Pseudonitzschia multiseries, overexpressed in Escherichia coli, and purified using liquid chromatography. The ferritin protein sequence appears to encode a non-heme, ferritinlike di-iron carboxylate protein, while gel filtration chromatography and SDS-PAGE indicate that this ferritin is part of the 24 subunit maxi-ferritins. Spectroscopically monitoring the addition of Fe(II) to a buffered ferritin solution shows that the P. multiseries protein demonstrates ferroxidase activity, binding iron and storing it as Fe(III) in excess of 600 equivalents per protein shell. In keeping with the typical stoichiometry of the ferroxidase reaction, oxygen (O₂) is consumed in a 2 Fe:O₂ratio while hydrogen peroxide is produced concurrently. iii Diatoms evolved from secondary endosymbiosis involving eukaryotic red algae; however, a broad phylogenetic comparison suggests that P. multiseries ferritin was likely acquired via lateral gene transfer from cyanobacteria – not from its ancestral endosymbionts. Until recently, no other ferritins have been identified in diatoms, and the protein characterized here is unique in that it seems to be derived from a prokaryotic organism yet it occurs in a marine eukaryote. These findings have direct implications for the success of pennate diatoms in both Fe rich coastal waters and upon Fe addition in the open ocean. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren. / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127 / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127

No description available.

We have investigated some of the many photonic and plasmonic properties of the diatom Coscinodiscus wailesii. We start by showing that when diatom frustules are converted to high-index magnesium silicide while maintaining their structure, they exhibit a broad (1μm - 2μm) photonic bandgap that varies in wavelength according to the position and angle of the incident light on the frustule. We then demonstrate the use of the micro and nanostructured silica diatom frustule as a low-cost, easily prepared substrate for surface-enhanced Raman spectroscopy by coating the frustule in 25 nm of silver and a monolayer of thiophenol. Some potential applications of diatoms to water quality measurements are suggested, and steps are taken to image a diatom frustule and chloroplasts simultaneously in vivo using rhodamine 19 dye and fluorescence microscopy. We propose future experiments that could ascertain whether there is any biological effect of the light filtering properties of the diatom frustule, and put forth some suggestions as to how to influence the morphology and photonic properties of the frustule via chemical contaminants in the diatom seawater growth medium.

O estudo de três testemunhos rasos (até 2,5m de profundidade) coletados na região de Jaguaruna, litoral centro-sul de Santa Catarina, permitiu reconstituir parte da história de evolução sedimentar holocênica na área. Para isso, foi feita a análise integrada de quatro tipos de variáveis: sedimentológicas, diatomológicas, palinológicas e geoquímicas (Ctotal, Ntotal, 13C e 15N). Os dados de micropaleontologia foram tratados por métodos de estatística mutivariada (análise fatorial de correspondência, análise de componentes principais e classificação ascendente hierárquica), que evidenciaram agrupamentos significativos entre os microfósseis e, desse modo, auxiliaram nas interpretações. A ca. 5000 anos AP, a área de estudo deveria ser ocupada por um conjunto de lagunas interconectadas, cuja existência pôde ser atestada pelos sedimentos de fundo lagunar, na base de um dos testemunhos, em zona atualmente colonizada por mata de restinga. A ligação da laguna com o mar, nesta época, é verificada pela presença de diatomáceas marinhas e pelo sinal isotópico e elementar da matéria orgânica preservada nos sedimentos, indicativo de origem algácea, com valores de 13C de fitoplâncton marinho. A perda da conexão da laguna com o oceano ocorreu em diferentes momentos nos três testemunhos, sendo o último registro de desconexão da laguna observado a ca. 2740-2370 anos cal AP. Variações nas assembléis de diatomáceas no registro da fase lagunar (de 5500 até 2740-2370 anos cal AP) indicaram pelo menos dois períodos de mudanças maiores na salinidade da paleolaguna. Estes períodos puderam ser comparados a dados de paleoprecipitação, publicados em trabalhos anteriores, obtidos para o Estado de Santa Catarina. Observou-se boa correlação entre períodos com maior precipitação e períodos de diminuição na salinidade das águas da laguna, o que seria relacionado à sua diluição por maior aporte fluvial, sem excluir, no entanto, a possibilidade de menor influência marinha devido a dinâmica de abertura e fechamento das conexões da laguna com o oceano. O registro da vegetação através da analise palinológica auxiliou na elaboração do modelo evolutivo da área e permitiu reconstituir parte da história da vegetação no contexto de preenchimento da bacia lagunar. O desenvolvimento da vegetação de restinga em áreas antes ocupadas por vegetação característica de borda lagunar reforçaria a idéia de progradação das fácies costeiras assoreando por completo a paleolaguna na área. A síntese dos dados palinológicos entre os três testemunhos permite interpretar que, pelo menos desde 4000 anos cal AP, a vegetação na área de estudo não sofreu grandes modificações, o que sugere clima semelhante ao presente. A análise integrada dos diferentes indicadores paloambientais sugere que a dinâmica sedimentar deve ser a maior responsável pelas mudanças observadas nos três testemunhos, com evolução dos ecossistemas ligada, principalmente, a variações no regime e substrato deposicionais, relacionadas à progradação costeira. / Sediments from three cores collected in Jaguaruna (south Brazilian coast, Santa Catarina state) were analyzed, in order to reconstruct the Holocene sedimentary evolution of the area. The palaeoenvironmental reconstruction was based on microfossil proxies (diatoms, pollen and spores), analysis of sedimentary organic matter (13C, 15N and C/N ratios), and sedimentological analysis. From at least 5000 cal yr AP, the site was occupied by a system of interconnected lagoons, whose existence is verified in the core sediments by the record of paleolagoon sediments in area occupied by restinga forest nowadays. The existence of this lagoon and of its connection with the sea is indicated by the presence of marine diatoms and by the isotopic and elementary composition of the sedimentary organic matter, which is indicative of algal origin, with 13C values of marine phytoplankton. The end of the connection between sea and lagoon was registered in different moments in the three cores and the last record of disconnection is found in ca. 2740-2370 cal yr AP. The variations of the diatom assemblages from the lagoonal phase (between 5000 and 2740-2370 cal yr AP) indicated at least two periods of major changes in the paleolagoon salinity. These periods could be compared with the published paleoprecipitation data for the Santa Catarina state, which showed a good correlation between higher precipitation and reduction in the lagoon water salinity. This correlation would be related to dilution of water salinity by the greater fluvial discharges. However, it can not be exclude the possibility of a lower sea influence related with the dynamics of opening and closing connections between lagoon and ocean. The pollen analysis allowed the reconstruction of part of the vegetation history in the context of lagoon basin sedimentary filling. The development of the restinga forest in areas previously colonized by open vegetation around the lagoon suggested the progradation of coastal facies that completely filled paleolagoon in the area. The synthesis of the palinological data showed that no major changes of the mainland vegetation ecosystem took place at least since 4000 cal yr AP. The vegetation in the study area did not suffer great modifications; with no record of climatic changes. The multi-proxies analysis suggests that the sedimentary dynamics must be responsible for the greater changes observed in the core sediments. The evolution of ecosystems could be related to variations of the depositional process and modification in the character of the substratum, related to the coastal progradation.

Haslea ostrearia est une diatomée cosmopolite pouvant proliférer en milieu naturel et dont les efflorescences sont particulièrement fréquentes dans les claires à huîtres de la côte Atlantique française. Cette diatomée produit la marennine, un pigment bleu-vert hydrosoluble connu pour induire le verdissement des branchies des huîtres, augmentant ainsi leur valeur marchande. La marennine présente des activités allélopathiques, antioxydantes ou encore antibactériennes in vitro et son utilisation en aquaculture a été envisagée suite à la mise en évidence d’effets prophylactiques chez des bivalves. Les objectifs de ce doctorat étaient 1) d’identifier les effets de ce pigment sur la croissance des bactéries du genre Vibrio, fréquemment impliquées dans la mortalité massive de bivalves et 2) de s’assurer de son innocuité sur des organismes marins (e.g. mollusques, échinodermes). Nos résultats sur la croissance bactérienne ont montré que les relations dose-effet ne présentaient pas systématiquement une tendance linéaire et également que la marennine pouvait totalement inhiber ou bien stimuler la croissance de Vibrio en fonction de la souche testée. De plus, des concentrations écologiquement pertinentes ont induit des effets négatifs sur la survie et le développement d’animaux marins. Ces effets indésirables n’ont cependant été observés que chez des stades fragiles de développement tels que les embryons et les larves. Enfin, nos résultats questionnent l’utilisation de H. ostrearia en aquaculture et soulignent la nécessité d’établir des doses seuils d'exposition afin de prévenir tout effet indésirable, tout en bénéficiant des effets prophylactiques de la marennine. / Blooms of the diatom Haslea ostrearia are recorded worldwide in marine environments and occur frequently in oyster ponds in the Western French coast. This diatom produces a blue-green water soluble pigment named marennine that is known to turn oyster gills green, which provides a higher market value to the bivalves. Although H. ostrearia has been identified centuries ago, little is known about the ecological significance of the blue pigment. Marennine displays allelopathic, antioxidant or antibacterial activities in vitro and its use in aquaculture was considered as some prophylactic effects were demonstrated on farmed shellfish. The aims of this thesis were thus 1) to further identify the effects of this pigment on the growth of bacteria from the genus Vibrio that are frequently involved in bivalve mass mortality and 2) to ensure its safety on marine organisms (e.g. molluscs, crustaceans, echinoderms). Our results on bacterial growth demonstrated that the dose-response curves did not systematically present a linear pattern (e.g. “U shape”, hormetic responses) and that marennine could either totally inhibit the growth of Vibrio or stimulate it depending on the strain tested. Also, ecologically relevant doses could impair the survival and the development of the marine organisms tested. Such noxious effects seemed to only target early and fragile developmental stages as embryos and larvae, while adults appeared unaffected. Finally, our results question the exploitation of H. ostrearia in aquaculture and highlight the need to set exposure threshold doses to prevent any adverse effects but to benefit from the prophylactic strategy resulting from the use of marennine.

The Sierra Nevada snowpack is vital to the water supply of California, the world’s sixth largest economy. Though tree ring and instrumental records show the dramatic influence of environmental change on California’s hydroclimate over the last millennium, few proxy archives assess winter precipitation variability farther back in time. Here, we use diatoms from a ~3,200 yr. old sediment core to reconstruct the paleolimnology of June Lake, a hydrologically closed glacial lake in the eastern Sierra Nevada. We test the hypothesis that limnologic and climatic changes control diatom assemblages at June Lake. Fossil diatom assemblages from June Lake sediments chiefly consist of the planktic genera Stephanodiscus and Lindavia; their relative abundances in sediments are controlled by lake response to changes in the length of the winter season. We establish a Lindavia:Stephanodiscus index to infer winter length; our results indicate three periods where winter seasons are longer than average: ~3.2-2.9 ka, ~2.2-1.7 ka, and ~0.6 ka-0.05 ka. Over the last ~100 yrs., June Lake has experienced stronger water column stratification and an expansion of the available benthic diatom habitat, indicating significantly warmer winters and lower lake levels. It is possible that this change is the result of anthropogenic climate warming.