Editorial: Controversies and solutions in environmental sciences: Addressing toxicity of sediments and soils

This is the author's accepted manuscript. The final published article is available from the link below. Copyright @ 2007 Ecomed Publishers

Identification of a possible role of thymine DNA glycosylase (TDG) in epigenome maintenance

Thymine DNA glycosylase (TDG) was discovered as an enzyme capable of removing uracil (U) and thymine (T) from G/U and G/T mispairs, respectively. Owing to this ability, TDG was proposed to initiate restoration of C/G pairs at sites of cytosine or 5-methycytosine (5-meC) deamination. In addition to products of base deamination, the substrate spectrum of TDG covers a wide range of DNA base damages resulting from base oxidation and alkylation. TDG was also found to engage in physical and functional interactions with transcription factors, and more recent evidence supports additional interactions with the de novo DNA methyltransferases Dnmt3a and 3b in the context of gene transcription. Together with its biochemical properties, these observations suggest that TDG might be targeted to gene regulatory sequences as part of a macromolecular assembly to control their functional integrity. TDG may counteract the mutagenic effects of C and 5-meC deamination in CG-rich regions and/or be involved in the maintenance of CpG promoter methylation patterns. A tight regulation of CpG methylation at gene regulatory regions is critical for accurate gene expression, proper cellular differentiation and embryonic development. A somewhat surprising but in this context consistent finding was that, in contrast to other DNA glycosylases, TDG is essential for proper fetal development since a targeted knockout of the gene leads to embryonic lethality. To gain insights into the biological functions of TDG, we aimed to establish and apply biochemical fractionation procedures for high affinity purification and structural and functional characterization of TDG containing proteins complexes. The first part of the thesis was concerned with biochemical characterization of the protein interaction network of TDG in living mammalian cells. To this end, I applied different approaches allowing high affinity isolation of protein complexes from mammalian cells, such as the tandem affinity purification (TAP) method as well as immunoprecipitation of endogenous protein and of the TDGa isoform from TdgA overexpressing embryonic stem (ES) cells. These efforts, however, did not reveal any TDG interacting partners in subsequent mass spectrometry (MS) analyses. These results were surprising, as TDG was previously reported to interact with transcription factors and DNA methyltransferases. Remarkably, however, all previously identified protein interactors of TDG were discovered in screen with the respective partner proteins, and under conditions of simultaneous overexpression of both interacting proteins. The only proteins ever identified in screen with TDG were Sumo1 and Sumo3, which turned out to covalently modify the glycosylase. For this reason, we decided to pursue our search with classical cell fractionation experiments. We first did gel filtration experiments from total cell lysates and showed that TDG is indeed able to form distinct multiprotein complexes in undifferentiated mouse embryonic stem cells that may also contain the RNA helicase p68. Further subcellular fractionation experiments then revealed that TDG is present in all cell compartments, with a significant fraction of nuclear TDG being associated with chromatin, together with p68 and de novo DNA methyltransferases. Together with published findings, these results suggested that protein complexes containing TDG might act in a chromatin-associated context, at gene regulatory regions. The developmental phenotype of Tdg-/- knockout mice and the interactions of TDG with factors involved in developmental gene regulation (e.g. retinoic acid receptors RAR/RXR) implicate a function of TDG during early development and cell differentiation, at times governed by dynamic changes in gene expression, DNA methylation and histone modifications. Such changes have been studied using a well-established during in vitro differentiation of ES cells to lineage committed neuronal progenitors (NPs). We thus aimed to address the function of TDG as part of chromatin associated protein complexes during the process of retinoic acid induced differentiation of ES cells to NPs. In the second part of the thesis we made use of a this well-established in vitro differentiation system to examine the genome-wide localization of TDG to chromatin by TDG chromatin immunoprecipitation (ChIP) and to correlate TDG association to chromatin with gene expression and DNA methylation changes linked to cellular differentiation. TDG ChIP combined with high throughput sequencing showed that TDG associates with high preference to CpG islands in promoters of actively transcribed genes or genes poised for transcriptional activation. Such CpG rich sequences are normally unmethylated in mammalian genomes. Interestingly, we found TDG to localize to promoters of many genes controlling pluripotency (e.g. Oct4, Nanog) and developmental processes (e.g. Sfrp2, Tgfb2, Gata6), thus, supporting a function of TDG in cell differentiation and/or embryonic development. As different lines of circumstantial evidence have associated TDG with changes in CpG methylation following activation of hormone responsive gene promoters, we went on to further test genome-wide promoter methylation in Tdg+/- and Tdg-/- NPs making use of a combination of methylated DNA immunoprecipitation (MeDIP) and microarray technology. This showed that the loss of TDG does not affect global promoter DNA methylation. Nevertheless, there were a number of significant differences, suggesting that TDG might affect the CpG methylation pattern at some promoters. Also, owing to the limited resolution of the MeDIP method, however, we could not exclude an involvement of TDG in the control of DNA methylation of specific promoter CpGs. Additional bisulfite sequencing of promoters of TDG bound developmental genes (e.g. Sfrp2, Tgfb2) in NPs and differentiated mouse embryonic fibroblasts (MEFs) have indeed proved that loss of TDG affects local changes in DNA methylation at particular CpGs. Subsequent analysis of genome-wide gene expression profiles of ES cells and differentiated Tdg+/- and Tdg-/- NPs revealed that a limited number of genes (229) are differentially regulated in ES, whereas substantial differences in gene expression in were observed in NPs (1022 genes). This implicated a specific function of TDG in the regulation of cell differentiation triggered gene expression changes. Detailed analysis of the expression of the Pax6 gene, accurate regulation of which is essential for proper neuron development, showed that its promoter is bound by TDG and that its transcription is inappropriately regulated upon further differentiation of Tdg-/- NPs into the neuronal lineage. Whereas Tdg+/- NPs efficiently downregulated Pax6 (50x) and further differentiated into neuron-like cells, Tdg-/- NPs only partially downregulated Pax6 gene expression (6x) and underwent apoptosis at day 2 after plating in neuronal medium. This phenotype was complemented by expression of TDGa, clearly implicating TDG in the regulation of Pax6 expression during differentiation of ES cells to terminal neurons. We further observed misregulation of pluripotency genes (e.g. Oct4) regulated by TDG bound promoters during early differentiation of ES cells. In the absence of TDG, ES cells showed the tendency to enter spontaneous and/or RA induced differentiation, suggesting a role for TDG in the regulation of pluripotency. During RA induced differentiation we further observed the activation of the neuron specific gene Lrrtm2 exclusively in TDG proficient cells. In addition, ChIP experiments showed that transcription factors involved in the activation of the Lrrtm2 gene (e.g. COUP-TFI, RAR) are not recruited to the respective promoter in Tdg-/- cells, suggesting that TDG might act passively as a scaffold factor important for the recruitment of transcription factors to promoter regions. I set out to clarify the biological function of TDG by investigating its molecular interactions in mammalian cells. I found that TDG, as a DNA repair enzyme, associates tightly with chromatin, where it localizes with high preference to CpG island promoters of active genes and genes poised to be expressed. I also found that the loss of TDG causes misregulation of genes during cell differentiation and that this appears to be related to a function of TDG in establishing and/or maintaining CpG methylation pattern in gene regulatory sequences. These discoveries implicate a novel function of DNA repair, in the maintenance not only of the genome, but also the epigenome

A Systematic Review of Research on Moderators in Asynchronous Online Discussions

Background: The term moderator was first used to describe a leadership role in online educational discussions over 40 years ago. Over multiple decades of research, the term and the roles it describes have been defined inconsistently, with four conceptual frameworks offering differing positions on the responsibilities and functions of a moderator. Purpose: This three-paper dissertation examined the usage of the term moderator in asynchronous online discussions (AODs) and the associated definitions of roles, situating usage of the term in current literature and providing insight into trends and impact areas related to the role of moderators in online educational discussions. The findings could be valuable to researchers investigating ways to support students in online courses, policymakers creating guidelines for online course designs, and practitioners seeking best practices in planning and implementing moderated AODs in courses. Methods: The three papers present portions of a systematic review study. The first paper addresses the scoping process, including systematic review protocols. The second paper is a reflective analysis of researcher experience through conducting the systematic review. The third paper reports the systematic review’s key findings. Results: The first paper reports a review of 76 sources in which disparate perspectives regarding moderator definitions, identities, roles, duties, and training programs were observed. Using a comparative analysis of four conceptual frameworks on moderation, a taxonomy was generated that delineates moderator duties into managerial, monitoring, pedagogical, technical, and social roles. The first paper concludes with definitions of the protocols that could be used for a systematic review on this topic. The second paper highlights the need for a robust scoping process that precedes a systematic review and offers benefits of mitigating unexpected issues that can arise relating to search results and sources. It also offers guidance regarding the refinement of protocols during a systematic review study. The third paper reports results from the systematic review of 52 sources. Key findings include the following: (1) nearly half of the 52 papers did not cite a conceptual framework focused on moderation; (2) the field is discordant, lacking consensus in themes for research designs, outcomes, foci, and questions; (3) half of the 52 reviewed papers involved case studies or similar small study designs; (4) the majority of papers collected data on students in higher education, but there was a lack of consistency in the reporting of demographic information; (5) research foci tended toward investigating peer moderators or the role of the instructor as a moderator; (6) research questions tended to focus on strategies of moderators or student performance and discussion quality; (7) most definitions or expectations of a moderator included discussion and social management duties. Conclusion: With continued expansion of online education, there is a growing need for instruction and frameworks to assist practitioners in implementing effective moderated AODs in their curricula. This systematic review illuminates the need for continued research on the topic and provides direction for future research that can contribute to refinement of discussion moderation techniques and implementation

Umweltchemie 2003

Neben der Beobachtung von Chemikalien in den Umweltkompartimenten, insbesondere fern der Emissionen, sind Fortschritte bei Probennahme, Analytik und physikalischer Messtechnik die wichtigsten Impulsgeber umweltchemischer Forschung. In der Atmosphärenchemie gewinnen Satelliten als messtechnische Plattformen an Bedeutung. Aerosole und Wolken werden verstärkt untersucht. Sensationell ist, dass Polymerisationsreaktionen in den Partikeln des atmosphärischen Aerosols Teil der natürlichen Luftchemie sind

Erfassung sedimentgebundener Schadstoffe in einem abwasserbelasteten Flußstausee (Speichersee) mit einer Biotestbatterie

Henschel T, Kalbfuß W, Ahlf W, Steinberg C, Traunspurger W. Erfassung sedimentgebundener Schadstoffe in einem abwasserbelasteten Flußstausee (Speichersee) mit einer Biotestbatterie. Vom Wasser. 1997;88:295-307

Chemical mobility and bioavailability of sediment-bound heavy metals influenced by salinity

The transfer of metals from contaminated sediments to algal cell walls (Scenedesmus quadricauda) and organisms from various trophic levels (euryhaline osmoconform hydroid Cordylophora caspia and algae Brachiomonas submarina) was studied with a multichamber device. The system consists of a central chamber which contained the mud suspension and six external chambers containing the different biological indicators. The solids in the central and external chambers are separated by 0.45 µm-diameter membranes which allow diffusion of the mobilized, dissolved metal compounds. Experiments were performed with dredged sediments at various salinities (0.5, 1.0, 1.5, and 2.0 percent, respectively) and the kinetic of re-adsorption was obtained by taking samples after different time intervals. High enrichment of Cd was found in the living alga Brachiomonas submarina, but on the other side only a weak influence of salinity on re-adsorption could be observed. Model experiments with ionic Cd showed a clear dependency on Cd-sorption on the algae, Cd-concentration in solution, and salinity. These results indicate that the transfer of metals mainly depends on the specific surface properties of the substrates and on the specific chemical form of the dissolved mobilized metal