Molecular Mechanisms of Fragile X Syndrome Investigated by Mass Spectrometry-Based Proteomics

Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability (ID) and autism. This neurodevelopmental disorder is caused by silencing of the FMR1 gene and lack of its product, Fragile X Mental Retardation Protein (FMRP). FMRP, an RNA-binding protein, is highly expressed in the brain and plays an important role in the transport and translation of many different mRNA targets. Lack of FMRP causes disruption in synapse morphology and function, as well as disruption in synaptic plasticity. These molecular and synaptic abnormalities cause FXS symptoms like ID, autism, hyperactivity, epilepsy and anxiety. Therefore, understanding of the molecular pathogenesis of FXS is also important for other neurodevelopmental and psychiatric disorders. In last two decades, extensive research in basic neurobiology and pathophysiology led to significant advances in FXS field. However, although caused by only one gene, the molecular mechanisms of FXS are not yet well understood. Since FMRP is characterized as a translational regulator with multiple mRNA targets, it is crucial to understand the changes of the proteome in the absence of FMRP. As multiple FMRP-regulated proteins are involved in conserved neuronal signal transduction pathways, it is also necessary to study FMRP-dependent changes in the phosphoproteome dynamics. In my thesis I applied mass spectrometry-based proteomics to address several aspects of the disorder in different FXS models, such as Fmr1-KO cell lines and mice. To analyze the influence of FMRP on major signal transduction networks, I first performed a global analysis of the proteome and phosphoproteome of Fmr1- and Fmr1+ mouse embryonic fibroblast (MEF) cell lines using stable isotope labeling by amino acids in cell culture (SILAC) and high resolution mass spectrometry. In this study I detected 6,703 proteins and 9,181 phosphorylation events, and mapped 266 significantly changing proteins and 142 phosphorylation sites onto major signal transduction pathways. My results confirmed a downregulation of the MEK/ERK pathway in absence of FMRP, with decreased phosphorylation on ERK1/2. Several proteins involved in mTOR, Wnt, p53 and MAPK signaling cascades were also significantly regulated; they were previously shown to be associated with autism, but not with FXS. Additionally, I detected a significant increase of p53 and proteins linked to p53 signaling, as well as a decreased level of the major prion protein (Prp) in Fmr1- cells. Decreased p53 signaling is the likely cause for previously observed dysregulation of cell cycle control in FXS, whereas reduced levels of Prp could contribute to the cognitive deficits observed in the FXS patients. These proteins and signal transduction pathways may represent novel targets for treatment of FXS symptoms. In the second part of the thesis, I used the same MEF cell line in order to analyze potential substrates of glycogen synthase kinase 3β (GSK-3β), an emerging therapeutic target for treatment of FXS. I used two inhibitors of GSK3-β kinase, lithium and TDZD-8, to identify potential substrates of the kinase in Fmr1- and Fmr1+ cells. Lithium treatment was poorly reproducible on the proteome and phosphoproteome level, reflecting its reported low specificity for GSK3- β, whereas TDZD-8 treatment showed good reproducibility between biological replicates. Of a total of 7,285 detected phosphorylation events, 91 were significantly decreased upon TDZD-8 treatment in Fmr1+ and 146 in Fmr1- MEF cells – these phosphorylation events were likely targets of GSK-3β. Overlap of these potential substrates was poor in Fmr1+ and Fmr1- cells, pointing to different substrates of the GSK-3β kinase in Fmr1+ and Fmr1- MEF cells. Importantly, downregulation of multiple phosphorylation events on MAP1B, a well characterized GSK-3β substrate whose mRNA is a known FMRP target, was observed only in Fmr1+ MEF cell line. In healthy neurons, MAP1B is coordinating microtubule dynamics. Since abnormal axon branching is one of the leading symptoms of FXS, I postulate that the lack of regulation of MAP1B by GSK-3β is the likely cause for aberrant morphology of neurons in FXS. Functional enrichment analysis revealed an implication of the potential GSK-3β substrates in different processes in MEF cell lines. For example, in Fmr1- MEFs potential substrates seem to be more involved in cell cycle, DNA replication and RNA processing. Since downregulation of DNA damage/repair pathway in FXS patients was recently reported, this data will shed new light on differential activity of GSK-3β in Fmr1+ and Fmr1- MEF cells and deepen our understanding of molecular mechanisms regulated by this kinase. In the third part of my thesis, I postulated that increased protein synthesis in FXS is accompanied by increased protein degradation in order to maintain cellular homeostasis. Since increased protein synthesis and degradation lead to increased protein turnover, I used stable isotope labeling (“dynamic SILAC” approach) to measure protein turnover rate in mouse primary cortical neurons derived from the WT and Fmr1-KO model. Analysis showed that most of the proteins have a similar half-life in WT and Fmr1-KO, although calculated median protein half-life was higher in Fmr1-KO neurons than in WT. Functional enrichment analysis showed that proteins with the lowest turnover rates are involved in oxidative phosphorylation, Alzheimer’s, Parkinson’s and Huntington’s diseases, whereas proteins with the highest turnover rates are involved in phagosome, Wnt signaling pathway and gap junction, for both, WT and Fmr1-KO neurons. The experimental design employed did not allow for detection of low SILAC incorporation rates and proteins with very short half-lifes and further optimization of experimental conditions is needed to fully address differences in protein turnover between Fmr1-KO and WT cells. In the fourth and final part of the thesis, I investigated molecular mechanisms of the genetic “rescue” of the FXS, recently reported to occur in Fmr1-KO mice after reduction of activity of the mGluR5 receptor. To that end, I performed a proteome-wide quantitative comparison of protein levels in the hippocampi between WT, Fmr1-KO mice and Fmr1-KO/mGluR5-het cross mice (the “rescued” FXS model), obtained from the laboratory of Mark Bear (MIT). Pearson correlation of protein intensities showed good technical reproducibility between biological replicates and also showed that different genotypes are very similar to each other – of 5,238 detected proteins, only about 198 were significantly changing between genotypes. Yet, pairwise comparison of Fmr1-KO and WT revealed proteins that are known to be involved in memory, learning and long term potentiation. Moreover, the data suggests disturbed mitochondrial transcription regulation in FXS model. One of the significantly changing proteins, the major prion protein, had a lower expression level in Fmr1-KO in comparison with WT. Since I detected the same expression pattern in MEF cell lines, I postulate that Prp plays a significant role in FXS pathogenesis. In the FXS rescue model, most of the significantly changing proteins are involved in metabolic processes, with the exception of Citron Rho-interacting kinase which is functionally linked to Fmr1 gene and it is interacting with mGluR5 receptor, and therefore may be involved in the rescue mechanism. I also addressed absolute protein levels in analyzed mouse models. This approach showed no difference in the total proteome abundance between the three genotypes; however, it did show that proteins with higher upregulation levels in Fmr1-KO are more abundant that those that were found to be downregulated. This imbalance may be the cause of the overall increased protein levels previously detected in brains of FXS mice. However, a portion of changing proteins is rather small and therefore not significant and further experiments need to be performed to address this. Overall, this thesis presents an extensive proteomics analysis of the different cellular and animal models of FXS. Combined, these approaches compile a substantial source of information for the FXS research community. In addition, this data is contributing to the better understanding of FXS pathophysiology and development of a potential new treatments

Expression of OCT-4 and SOX-2 in Bone Marrow-Derived Human Mesenchymal Stem Cells during Osteogenic Differentiation

AIM: Determine the levels of expression of pluripotency genes OCT-4 and SOX-2 before and after osteogenic differentiation of human mesenchymal stem cells (hMSCs).METHODS: Human MSCs were derived from the bone marrow and differentiated into osteoblasts. The analyses were performed on days 0 and 14 of the cell culture. In vitro differentiation was evaluated due to bone markers – alkaline phosphatase (AP) activity and the messenger RNA (mRNA) expression of AP and bone sialoprotein (BSP). The OCT-4 and SOX-2 expression was evaluated at mRNA level by real-time qPCR and at protein level by immunocytochemistry.RESULTS: In vitro cultures on day 14 showed an increase in AP activity and upregulation of AP and BSP gene expression. OCT-4 and SOX-2 in undifferentiated hMSCs on day 0 is detectable and very low compared to tumor cell lines as a positive control. Immunocytochemistry detected OCT-4 in the cell nuclei prior (day 0) and post differentiation (day 14). On the same time points, cultures were negative for SOX-2 protein.CONCLUSION: Messenger RNA for pluripotency markers OCT-4 and SOX-2 isolated from hMSCs was less present, while OCT-4 protein was detected in cell nuclei prior and post differentiation into osteoblast lineage

Urkundenübersetzen und -dolmetschen: Abbildung versus Anpassung

Diese Masterarbeit setzt sich mit dem Thema Urkundentranslation auseinander und hatte zum Ziel, der Frage auf den Grund zu gehen, ob denn Behauptungen, wie sie in der Literatur beispielsweise bei Stolze (vgl. 1999: 1666) zu finden sind, ein Urkundentranslat könne ausschließlich die Funktion einer Verständishilfe erfüllen, sei nur in Verbindung mit seinem jeweiligen Ausgangstext gültig und müsste somit stets dokumentarisch übersetzt werden, tatsächlich so bestätigt werden können, oder ob es auch solche Translationssituationen in der Praxis gibt, die danach verlangen einen an die Ziel(rechts-)kultur, -textleser und -textkonventionen angepassten Zieltext zu erstellen, welcher in dem jeweiligen neuen, zielkulturellen Verwendungskontext als ein eigenständiges Instrument funktionieren solle und nicht sofort als eine Übersetzung erkennbar sein solle. Anhand unterschiedlicher theoretischer - translationswissenschaftlicher und rechtlicher – Ansätze sowie der Analyse textexterner und textinterner Faktoren sechs ausgewählter Urkundenübersetzungsbeispiele – je drei dokumentarischen und drei instrumentellen Charakters – im Sprachenpaar Kroatisch-Deutsch konnte gezeigt werden, dass auch für das Urkundenübersetzen unterschiedliche Übersetzungsmöglichkeiten in Frage kommen und stets der jeweilige Auftrag, der wiederum Aufschluss über die zielkulturellen Translationsfaktoren, sprich die die jeweilige Translationssituation bestimmenden Faktoren, also im Einzelnen die Zieltextsprache, die für den Zieltext geltende Rechtsordnung, Zieltexttyp beziehungsweise Zieltextfunktion und die Zieltextrezipienten, über die jeweils einzusetzende(n) Translationsstrategie(n) entscheiden. Sie sind es die letzten Endes dem jeweiligen Translator helfen können bei einer Urkundentranslaterstellung zwischen den ausgangskulturorientierten oder den zielkulturorientierten makro-und mikrostrukturellen Translatgestaltungselementen zu wählen und sich somit entweder für die dokumentarische oder instrumentelle Übersetzungsweise zu entscheiden. Dabei allerdings - und das zählt zu den wohl wesentlichsten Schlussfolgerungen, die aus der vorliegenden Arbeit gezogen werden konnten – handelt es sich bei der dokumentarischen und instrumentellen Übersetzungsstrategie, sprich bei der Dichotomie Verfremdung versus Einbürgerung, um zwei tendenzielle, übergeordnete Translationsweisen, die die zu verwendenden Mittel für eine Translatgestaltung lediglich grob festlegen und von denen beide auf den unterschiedlichen Ebenen der Translatgestaltung, sowohl mikro- also auch makrostrukturell gesehen also, unterschiedlich ausfallen können. Das bedeutet also, dass ein Translator im Zuge der Translatgestaltung beziehungsweise Produktion trotz gewählter übergeordneter Strategie noch eine ganze Reihe weiterer Entscheidungen treffen muss. So muss er beispielsweise eigenständig und im Idealfall bewusst darüber entscheiden, ob er den gesamten Text, sprich alle makro- und mikrostrukturellen Textelemente des Translats an die des jeweiligen Ausgangstextes angleichen wird, oder doch für bestimmte mikrostrukturelle Aspekte wie beispielsweise die rechtlichen Standardformeln im konkreten Fall an die zielkulturellen Konventionen anpassen wird. All diese Tatsachen berücksichtigend kann man folglich die Urkundentranslation zusammenfassend als eine Tätigkeit im Spannungsfeld zwischen der ‚bloßen‘ Abbildung des Ausgangstextes und der zielkulturentsprechenden Neuverfassung beschreiben. Diese fordert von Translatoren eine Vielzahl an unterschiedlichen Kompetenzen. Neben ausgezeichneten Sprachkenntnissen und sonstigen translatorischen Kompetenzen müssen sie außerdem auch noch über ein fundiertes Wissen über die jeweiligen Konventionen der Rechtssprachen beziehungsweise Rechtskulturen, mit denen sie arbeiten, verfügen sowie zumindest Grundkenntnisse aus dem Rechtsbereich besitzen.This master thesis deals with the topic translation of documents and pursued the target to analyse if thesis a document translation could exclusively have the particular function as an aid to understanding, is only valid in combination with its source text and should therefore be translated exclusively in a documentary way is true or whether there are as well translation circumstances which require the production of a target text which should be adjusted to the conventions of the target (legal) culture, target readers and target language, which would work as an independent instrument in the new application context of the target culture and would not be recognized immediately as a translated text. Based on different theoretical approaches - to translation and to legal texts – and the analysis of extratextual and intratextual factors of six chosen document translation examples – three documentary and three instrumental – with the language pair Croatian – German it could be shown that for the translation of documents as well different translation possibilities exist and that the task which informs about the translation factors of the target culture i.e. about those factors, which influence the translation situation, that is the target language, the legal system which is valid for the target text, the text-type of the target text or its text function and the target recipients, decides about the translation strategy or strategies which should be used. Those are the factors which in the end can help a translator to choose between either macro- and microstructural elements for the production of the target text which are adapted to the source culture or those which are adapted to the target culture and therefore help him to choose between the documentary and the instrumental way of translation. At this point, however, there is one of the most important conclusions that could be drawn from this master work. The documentary and instrumental translation strategy/ies i.e. the dichotomy Alienation versus naturalization need to be considered as only two general ways of translation which roughly determine the elements which should be used for the production of the target text, and which on different levels of the target-text-production i.e. both the macrostructure and the microstructure can vary. This means that a translator during the production of the target text in spite of the chosen translation general strategy still has to make a lot of additional decisions. For example he has to decide on his own and hopefully with awareness if he will adapt the whole text i.e. all macro- and microstructural text elements of the target text to those from the source text or whether he will adapt some of the microstructural aspects like for example the standard wordings to the conventions of the target culture. Considering all those facts we can describe the translation of documents in a summarizing way as a profession in the tense atmosphere in between of the ‚bare‘ copy of the source text and a target-cultural-adapted reproduction. This fact demands a lot of different skills from the translator. Beside excellent language skills and any other translational skills they have to have a well-founded knowledge of the legal languages or legal cultures from those countries they are working for and at least a basic knowledge of the law as well

Analysis of the environmental and economic impacts of a Plečnik bench made of mineralized wood using LCA and LCC methodology

Les je v krožnem biogospodarstvu prepoznan kot obetavna surovina, saj ima sposobnost skladiščenja ogljika, ki pa je pogojena z življenjsko dobo izdelka. Podaljšanje življenjske dobe lesa lahko dosežemo z ustrezno zaščito, pogosto z uporabo kemikalij, ki so lahko okolju in ljudem nevarne. Mineralizacija lesa s hidroksiapatitom predstavlja potencial za okolju prijazno zaščito lesa. Na primeru Plečnikove parkovne klopce, mineralizirane s hidroksiapatitom, sta bili izvedeni LCA in LCC analizi. Analiza LCA je temeljila na standardih EN ISO 14040 in EN ISO 14044, analiza LCC je bila prilagojena po vmesniku Evropske komisije za javna naročila. Pri primerjavi mineraliziranega lesa z življenjsko dobo 16 oz. 24 let z referenčnim (nemineraliziranim) lesom so rezultati pokazali, da je mineraliziran les z življenjsko dobo 24 let okolju najprijaznejša in hkrati najcenejša izbira. Največje negativne vplive na okolje je predstavljal mineraliziran les z življenjsko dobo 16 let, najdražja alternativa je bil referenčni les.Wood is considered a promising raw material for the circular bioeconomy and has the ability to store biogenic carbon, and this is one reason why we want to extend the service life of wood. Toxic chemicals are often used for wood preservation, but hydroxyapatite seems to be an environmentally friendly solution for wood mineralization. LCA and LCC analyses were performed on a case study of a Plečnik bench, comparing mineralized wood with a service life of 16 and 24 years to a non-mineralized reference variant. LCA was based on EN ISO 14040 and EN ISO 14044, while LCC was adapted from the European Commission’s LCC tool for public procurement. When comparing the results, mineralized wood with a service life of 24 years proved to be the most environmentally friendly and cost-effective option. The mineralized wood with a service life of 16 years had the greatest negative environmental impact, while the most expensive option was the reference wood

Deep Coverage of the Escherichia coli Proteome Enables the Assessment of False Discovery Rates in Simple Proteogenomic Experiments

Recent advances in mass spectrometry (MS) have led to increased applications of shotgun proteomics to the refinement of genome annotation. The typical “proteo-genomic” workflows rely on the mapping of peptide MS/MS spectra onto databases derived via six-frame translation of the genome sequence. These databases contain a large proportion of spurious protein sequences which make the statistical confidence of the resulting peptide spectrum matches difficult to assess. Here we performed a comprehensive analysis of the Escherichia coli proteome using LTQ-Orbitrap MS and mapped the corresponding MS/MS spectra onto a six-frame translation of the E. coli genome. We hypothesized that the protein-coding part of the E. coli genome approaches complete annotation and that the majority of six frame-specific (novel) peptide spectrum matches can be considered as false positive identifications. We confirm our hypothesis by showing that the posterior error probability distribution of novel hits is almost identical to that of reversed (decoy) hits; this enables us to estimate the sensitivity, specificity, accuracy, and false discovery rate in a typical bacterial proteo-genomic dataset. We use two complementary computational frameworks for processing and statistical assessment of MS/MS data: MaxQuant and Trans-Proteomic Pipeline. We show that MaxQuant achieves a more sensitive six-frame database search with an acceptable false discovery rate and is therefore well suited for global genome reannotation applications, whereas the Trans-Proteomic Pipeline achieves higher specificity and is well suited for high-confidence validation. The use of a small and well-annotated bacterial genome enables us to address genome coverage achieved in state-of-the-art bacterial proteomics: identified peptide sequences mapped to all expressed E. coli proteins but covered 31.7% of the protein-coding genome sequence. Our results show that false discovery rates can be substantially underestimated even in “simple” proteo-genomic experiments obtained by means of high-accuracy MS and point to the necessity of further improvements concerning the coverage of peptide sequences by MS-based methods