Universiẗat, Diss., 2002--Bremen.

Thesis (doctoral)--Universität Potsdam, 2002. / "April 2003"--P. [2] of cover. Lebenslauf. Includes bibliographical references (p. 118-133). Also available via the World Wide Web.

Techn. University, Diss., 2005--München.

Universiẗat, Diss., 2003--Frankfurt (Main).

Nanoparticle (NP) implementation in industry has increased during the last years. However, the consequences of NP release to the environment have not been fully understood yet and long-term effects are hardly predictable. Information regarding a possible NP uptake by organisms is urgently needed. Here, we investigate the uptake of presynthesized AuNPs of different diameters by living diatoms. To better understand the influence of NPs on diatoms, cells were monitored in vivo using Surface Enhanced Raman Spectroscopy (SERS). By 3D Raman imaging, AuNPs with diameters ≥50 nm could clearly be localized inside the cells, whereas smaller AuNPs were never detected in the cell interior. This indicates a size-dependent uptake mechanism that comes along with different toxicities. As diatoms present an essential source of the marine food chain, this observation is relevant not only for diatoms themselves but also for higher organisms.

Diatoms are unicellular, eukaryotic microalgae inhabiting nearly all aquatic habitats. They are famous for their micro- and nanopatterned silicabased cell walls, which are envisioned for various technologic purposes. Within this review article, we summarize recent in vivo modifications of diatom biosilica with respect to the following questions: (i) Which metals are taken up by diatoms and eventually processed into nanoparticles (NPs)? (ii) Are these NPs toxic for the diatoms and––if so––what factors influence toxicity? (iii) What is the mechanism underlying NP synthesis and subsequent metabolism? (iv) How can the obtained materials be useful for materials science?

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

Since the 18th-century scientists are studying diatoms, fascinated by their beauty and diversity. Their nano- and micropatterned biosilica cell walls are outstanding examples of biologically controlled mineral formation. Although the knowledge about diatom cell wall formation increased over the last 60 years, the process is still far away from being completely understood. Diatom cell walls exhibit highly interesting material properties, making them appealing to material scientists. Due to those properties, diatom cell walls are on the brink of becoming powerful tools in nanotechnology. However, the production of tailored silica structures for nanotechnology requires a much better understanding of the processes and components involved in cell wall morphogenesis. Recent studies set the focus on insoluble organic matrices as important parts of this process, suggesting that they act as templates in silica morphogenesis. Therefore, in this study, the occurrence of insoluble organic matrices and their possible silica precipitation activity was analyzed in the three diatom species T. pseudonana, T. oceanica and C. cryptica. For all three species girdle band and valve derived insoluble organic matrices could be identified. The extracted insoluble organic matrices exhibited structural features present in the corresponding biosilica cell walls. The highest similarities were found in the valve derived matrices of C. cryptica. Accessibility studies showed that the biosilica associated insoluble organic matrices of T. pseudonana were only partially accessible, arguing for an entrapment of insoluble organic matrices in the silica, rather than an attachment to the surface of the cell wall. All examined insoluble organic matrices of the three species exhibited intrinsic silica precipitation activity. The most intriguing structures were formed by the insoluble organic matrices of C. cryptica, yielding a porous silica pattern. The addition of biosilica derived soluble components or long-chain polyamines promoted this process and moreover lead to the reconstitution of biosilica-like hierarchical silica pore patterns. The generated silica structures were templated by the underlying structure of the insoluble organic matrix. The result presented in this thesis make this the first study reporting the in vitro generation of diatom biosilica-like hierarchical silica pore patterns using all natural cell wall components. It supports the hypothesis of microplates acting as templates for biosilica morphogenesis and introduces an interesting experimental setup for silica-based in vitro studies on the mechanism of pore formation in diatoms.