Mn was found to reduce the toxicity of Cu to the marine diatom Thalassiosira pseudonana (clone 3H) in the chemically well defined medium Aquil (Morel et al., 1979), verifying the results of Sunda and Huntsman (1983). A cation-exchange resin technique developed by Zorkin (1983) was modified for use in natural seawater samples to estimate the biologically active Cu and Mn. Seawater samples taken from the bottom waters of a local fjord were found to support better growth of the test organism than samples from shallow waters, although the concentration of the biologically active Cu as estimated by the resin technique was similar for samples collected from all depths. The biologically active Mn concentration was found to be higher in the bottom water samples, indicating that the bioassay organism was probably responding to the interaction between the ionic forms of Cu and Mn, rather than to changes in the biologically active Cu concentration. / Science, Faculty of / Earth, Ocean and Atmospheric Sciences, Department of / Graduate

The main goal of this study was to assess the impact that nitrate and light have on the relationship between the gene expression of the enzyme nitrate reductase and the incorporation of nitrate in the cosmopolitan diatom Thalassiosira pseudonana, both in laboratory experiments and in natural environments. Continuous cultures were grown at different nitrate (NO3-) concentrations (i.e., 60, 120, and 400 µM) to evaluate their effects on the expression levels of different genes of the nitrogen metabolic pathway (i.e., nitrate and ammonium transporters, nitrate and nitrite reductases, glutamine synthetases II and III). Semi-continuous cultures were grown under different irradiances (i.e., 50, 110, 200, and 320 µmol photon cm-2 s-1) to assess the influence of light intensity (irradiance) on the relationship between the expression of those genes, uptake, and assimilation of nitrate. The expression of all of the genes that were tested decreased significantly (p < 0.05) at the highest concentration of NO3- (i.e., 400 µM), with nitrate transporters showing the most pronounced change from 27.97 to 0.59 fold change cell-1 x 10-6, at 60 and 400 µM NO3- concentrations respectively. Ammonium transporters were detected at all concentrations of NO3-, suggesting that cells are always ready to metabolize ammonium. Growth was limited (µ = 0.99 d-1) by the low irradiance treatment, was maximum (µ = 2.04 d-1) at 200 µmol photon cm-2 s-1 and was inhibited (µ = 1.54 d-1) at the highest irradiance. These trends were reflected in gene expression and uptake rates, with minimum values at the lowest and highest irradiance levels. However, results from the enzymatic assay did not show any significant differences between treatments (p > 0.05). The trends observed in the enzymatic rates could be explained by the gene expression of NO3- reductase and the uptake and growth rates in a multiple regression analysis (R2 = 0.66, p < 0.05). The results of this study show that uptake is independent of gene expression, probably because of a decoupling between transcription and protein synthesis. Not all of the newly synthesized transcripts will inevitably be translated into proteins. And even if they were, there could be post-translational mechanisms preventing the enzymes to become active. This indicates that uptake can be independent of the expression. It was attempted to measure the expression of T. pseudonana genes involved in the metabolism of NO3- in natural diatom assemblages. The use of gene expression as a proxy for metabolic processes carried out by a phytoplankton assemblage in the field is limited and depends on environmental factors, since the current methods of assessing expression rely on genomic sequences that are particularly variable in phytoplankton. The assessment of gene expression provides a useful insight into physiological studies of phytoplankton, and it should be complemented with other measurements, such as the biomass and taxonomic composition of the assemblage for a more complete picture of marine ecosystem nutrient dynamics. / Graduate

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.

Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name ‘soluble silicome proteins’ (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.