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Zwischen Aufbruch und Wanderlust : eine Reise durch die Romantikausstellung des Museums Giersch
(2015)
Reading is an essential ability to master everyday life in our society. The ability to read is based on specific connections between brain regions involved in the reading process – so-called cortical networks for reading. These cortical networks for reading allow us to learn the correct identification of visual words. The use of visual words is based on knowledge about the orthography (lexical) and the meaning of words (semantic). This knowledge must be acquired by beginning readers (first grader), i.e. beginning readers learn in a first step to link letters to a whole word and in a second step associate this whole word with meaning. To retrieve this knowledge during visual word recognition (VWR) a cortical network for lexical-semantic process must be activated. However, it is currently unclear whether beginning readers and reading experts activate the same neuronal network during VWR. Therefore, the aim of this thesis was to investigate the question whether beginning readers (first grader, children) and reading experts (adults) use different cortical networks for the lexical-semantic processing in VWR.
To address this question we recorded electroencephalographic (EEG) activity during VWR in children and adults. Children and adults were instructed to read a visualizable word to compare this word with a following picture stimulus. The first part of this thesis is concerned with the analysis of ERPs for visual word recognition in children and adults at sensor level. For both groups we observed the typical ERP components P100 and N170 for visual word recognition. These components differed in amplitude and time course between both groups. The second part of this thesis investigated the neuronal generators (brain areas) of ERPs during VWR and possible differences between children and adults at source level. We observed a high overlap in brain areas involved during VWR in children and adults. However, the brain areas differed in activation and time course between children and adults. Finally, the third and most important part of the thesis investigated the question whether children and adults use different cortical networks for the lexical-semantic processing in VWR over time. To address this question Dynamic Causal Modeling (DCM) and Bayesian model comparison were used. We compared nine biologically plausible cortical network models underlying the ventral lexical-semantic path in VWR. In addition, increasing time intervals were used to consider possible changes of network structure during VWR. The network models included eight brain regions (four bilateral pairs) involved in the lexical-semantic processing in VWR: occipital cortex (OC), temporo-occipital part of inferior temporal gyrus (ITG), temporal pole (TP), and inferior frontal gyrus (IFG). In almost all time intervals we found evidence that children and adults use the same cortical networks for the lexical-semantic processing in VWR. However, we found differences between adults and children in the connection strengths of the favoured model. Interestingly, we found a stronger direct connection from OC to IFG in adults compared to children.
In conclusion, our results suggest that children and adults activate largely the same lexical-semantic networks during VWR over time. This supports the notion that children and adults use the same biological fiber connections for VWR. However in contrast to children, adults showed increased use of the shortcut pathway from OC to IFG. The increased use of the shortcut pathway from OC to IFG in adults can be interpreted as consequence of learning. Learning causes in accordance with the Hebbian learning rule (“neurons that fire together, wire together” (Hebb, 1949)) synaptic change. Consequently the frequent coactivation of the input and output stage of OC and IFG during the lexical-semantic process facilitates the stronger direct connection between both brain areas. The stronger direct connection from OC to IFG most likely allows adult reading experts to speed up the lexical-semantic process during VWR. Accordingly, we conclude that the stronger direct connections from OC to IFG in adults compared to children underlay the different reading capabilities in both groups.
Membrane proteins are biological macromolecules that are located in a cell’s membrane and are responsible for essential functions within an organism, which makes them to prominent drug targets. The extraction of membrane proteins from the hydrophobic membrane bilayer to determine high-resolution crystal structures is a difficult task and only 2% of all solved proteins structures are membrane proteins. Computational methods may help to gain deeper insights into membrane protein structures and their functions. This study will give an overview of such computational methods on a representative set of membrane proteins and will provide ideas for future computational and experimental research on membrane proteins.
In a first step (chapter 2), I updated an earlier, manually-curated data set of homologous membrane proteins (HOMEP) to more recent versions in 2010 (HOMEP2) and 2013 (HOMEP3) using an automated clustering approach. High-resolution structures of membrane proteins listed in the PDB_TM database were structurally aligned and subsequently clustered using structural similarity scores. Both data sets were used as a standard gold reference set for subsequent work.
Subsequently, I have updated and applied the sequence alignment program AlignMe to determine protein descriptors that are suitable for detecting evolutionary relationship between homologous a-helical membrane proteins. Single input descriptors were tested alone and in combination with each other in different modes of AlignMe by optimizing gap penalties on the HOMEP2 data set. Most accurate alignments and homology models on the HOMEP2 data set were observed when using position-specific substitution information (P), secondary structure propensities (S) and transmembrane propensities (T) in the AlignMe PST mode. An evaluation on an independent reference set of membrane protein sequence alignments from the BAliBASE collection showed that different modes of AlignMe are suitable for different sequence similarity levels. The AlignMe PST mode improved the alignment accuracy significantly for distantly related proteins, whereas for closely-related proteins from the BAliBASE set the AlignMe PS mode was more suitable. This work was published in March 2013 in PLOS ONE. In order to allow also an easier usage of the AlignMe program, I have implemented a web server of AlignMe (chapter 4) that provides the optimized settings and gap penalties for the AlignMe P, PS and PST modes. A comparison to other recent alignment web server shows that the alignments of AlignMe are similar or even more accurate than those of other methods, especially for very distantly related proteins for which the inclusion of membrane protein information has been shown to be suitable. This work was published in the NAR web server issue in July 2014.
Although membrane-specific information has been shown to be suitable for aligning distantly related membrane proteins on a sequence level, such information was not incorporated into structural alignment programs making it unclear which method is the most suitable for aligning membrane proteins. Thus, I compared 13 widely-used pairwise structural alignment methods on an updated reference set of homologous membrane protein structures (HOMEP3) and evaluated their accuracy by building models based on the underlying sequence alignments and used scoring functions (e.g., AL4 or CAD-score) to rate the model accuracy (chapter 5). The analysis showed that fragment-based approaches such as FR-TM-align are the most useful for aligning structures of membrane proteins that have undergone large conformational changes whereas rigid approaches were more suitable for proteins that were solved in the same or a similar state. However, no method showed a significant higher accuracy than any other. Additionally, all methods lack a measure to rate the reliability of the accuracy for a specific position within a structure alignment. In order to solve these problems, I propose a consensus-type approach that combines alignments from four different methods, namely FR-TM-align, DaliLite, MATT and FATCAT and assigns a confidence value to each position of the alignment that describes the agreement between the methods. This work has been published 2015 in the journal “PROTEINS: structure, function and bioinformatics”.
Consensus alignments were then generated for each pair of proteins of the HOMEP3 data set and subsequently analyzed for single evolutionary events within membrane spanning segments and for irregular structures (e.g., 310- and p-helices) (chapter 6). Interestingly, single insertions and deletions could be observed with the help of consensus alignments in the conserved membrane-spanning segments of membrane proteins in four protein families. The detection of such single InDels might help to identify crucial residues for a proteins function.
Acinetobacter baumannii virulence is mediated by the concerted action of three phospholipases D
(2015)
Acinetobacter baumannii causes a broad range of opportunistic infections in humans. Its success as an emerging pathogen is due to a combination of increasing antibiotic resistance, environmental persistence and adaptation to the human host. To date very little is known about the molecular basis of the latter. Here we demonstrate that A. baumannii can use phosphatidylcholine, an integral part of human cell membranes, as sole carbon and energy source. We report on the identification of three phospholipases belonging to the PLD superfamily. PLD1 and PLD2 appear restricted to the bacteria and display the general features of bacterial phospholipases D. They possess two PLDc_2 PFAM domains each encompassing the HxKx4Dx6GS/GGxN (HKD) motif necessary for forming the catalytic core. The third candidate, PLD3, is found in bacteria as well as in eukaryotes and harbours only one PLDc_2 PFAM domain and one conserved HKD motif, which however do not overlap. Employing a markerless mutagenesis system for A. baumannii ATCC 19606T, we generated a full set of PLD knock-out mutants. Galleria mellonella infection studies as well as invasion experiments using A549 human lung epithelial cells revealed that the three PLDs act in a concerted manner as virulence factors and are playing an important role in host cell invasion.
Tectonin β-propeller containing protein 2 (TECPR2) was first identified in a mass- spectrometric approach as an interactor of GABARAP, an ATG8-family protein playing a role in autophagy. The mammalian ATG8 protein family consists of seven members, namely MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2. All share an ubiquitin-like core and possess two additional N-terminal α-helices, which are important for the distinct functions of the proteins. First determined in various organelles the ATG8 proteins are shown to be involved in autophagy, supporting the formation and cargo recruitment of autophagosomes, the vesicles transporting cargo for autophagic degradation.
Autophagy is the process of recycling cytoplasmic contents by degradation of misfolded proteins or damaged organelles in order to supply nutrients. Also clearance of pathogens can be achieved via autophagy. Importantly, LC3B is incorporated into the autophagosomal membrane and is therefore used as the main marker for autophagosomes. Previous studies exhibited that depletion of TECPR2 leads to a loss of LC3B-positive structures in cells, which suggests TECPR2 to positively regulate autophagic processes.
A frame shift deletion in the gene encoding for TECPR2 causes the generation of a premature stop codon and subsequent an unstable version of the protein, which is then degraded. Mutation in the TECPR2 gene triggers a neurodegenerative disorder termed hereditary spastic paraparesis (HSP). HSPs are a diverse group of neurodegenerative diseases that are characterized by spasticity in prevalent lower extremities and were mediated by a loss of axonal integrity of the corticospinal motor neurons. In the context of HSP more than 50 gene loci were identified by now. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy causing a form of HSP, the exact function of TECPR2 is unknown.
This study primarily focused on the determination of TECPR2’s binding mode to ATG8 proteins in vitro and in cells. The association of TECPR2 to all ATG8-family proteins was confirmed in in vitro pulldown experiments. Following fragment-based binding and peptide array experiments, the LC3-interacting region (LIR) of TECPR2 could be verified with mutants of TECPR2 lacking the LIR motif. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) were conducted to gain deeper insights into the binding preference to the different ATG8-family members. Moreover, the crystal structure of TECPR2-LIR was solved. In cells colocalization studies with overexpressed ATG8 proteins unraveled a preferential binding to the LC3-subfamily.
Further, mass spectrometric analysis revealed novel association partners of TECPR2: SEC24D, HOPS and BLOC-1, all of those participating in different endomembrane trafficking pathways. Interaction and colocalization of TECPR2 with these components was validated with several immunoprecipitation experiments and the N-terminal part of the protein comprising the WD40-domain could be defined as the binding site for all three of the association partners. In further approaches, the requirement of the LIR-motif and the necessity of the availability of LC3 protein for the particular interactions were determined. Interestingly, in the absence of LC3C the binding of TECPR2 to SEC24D was completely disrupted whereas a loss of LC3B only resulted in a decreased association. Notably, the binding proteins were not subjected to autophagosomal degradation, indicating that TECPR2 may operate as a multifunctional scaffold protein. While depletion of TECPR2 destabilized HOPS and BLOC-1, the autophagy defect observed in TECRP2-deficient cells could not be attributed to functional impairment of these two complexes.
Moreover, loss of TECPR2 led to a decline in protein levels of SEC24D and of its heterodimer partner SEC23A. Thus, TECPR2 is required to regulate the protein levels of SEC23A and SEC24D and subsequently the formation of the heterodimers. Together, SEC24D and SEC23A form the inner coat of COPII vesicles. These vesicles are responsible for the anterograde transport of cargo from the ER toward the Golgi compartment. COPII-coated vesicles are secreted form ER at distinct sites, termed ER exit sites (ERES). The small GTPase SAR1A maintains the vesicle budding, coating and secretion at the ERES. Together with SEC13, SEC31 forms the outer coat of the COPII vesicles and therefore serves as a general ERES marker.
Consistent with a defect in COPII coat assembly, the number of ERES diminished in the absence of TECPR2. These phenotypes could be rescued by the wildtype TECPR2 protein but not by the LIR-mutant. Intriguingly, these results were mimicked by depletion of LC3C, which localized to ERES. By monitoring the release of various cargos from ER in dependency of TECPR2 or LC3C, a role of both proteins in ER export was determined. These facts indicated that TECPR2 cooperates with LC3C to facilitate COPII assembly, ERES maintenance and ER export. Notably, fibroblast derived from a HSP patient carrying mutated TECPR2 showed diminished SEC24D protein levels and delayed ER export.
Concurrent with emerging evidence for a role of ERES in autophagosome formation, depletion of TECPR2 or LC3C or overexpression of a constitutive inactive SAR1 mutant reduced puncta formation of the early autophagosomal protein WIPI2.
In summary, this study uncovered a role for TECPR2 in ER export at ERES through interaction and stabilization of SEC24D, a COPII coat protein. This process also depended on ATG8-family protein LC3C, which is localized at ERES. Both proteins are required for correct COPII-mediated secretion. Moreover, the presence of TECPR2 and LC3C on ER allows development of omegasomes, membranous structures budding ER to form autophagosomes, by stabilization of WIPI2 and therefore contribute to autophagosome formation.
Part VI of our series on ISIS : "Blogforum 'Kalifat des Terrors: Interdisziplinäre Perspektiven auf den Islamischen Staat".
In 2014, two insurgency organisations stood out by their expansion, success and brutality: The Islamic State (IS) and Boko Haram (BH). The former emerged from the conflicts in Syria and Iraq and became a major actor in the Middle East, its influence reaching beyond the borders of its self-proclaimed “caliphate”, while the latter spread its violence throughout north-eastern Nigeria, spilling over into Cameroon, Chad and Niger. Because of their still growing success, many wonder about a possible partnership between IS and BH. To this I answer that there is a connection, but no partnership. Currently, any evidence suggesting a partnership is circumstantial at best...
Be it in the case of opening a website, sending an email, or high-frequency trading, bits and bytes of information have to cross numerous nodes at which micro-decisions are made. These decisions concern the most efficient path through the network, the processing speed, or the priority of incoming data packets.
Despite their multifaceted nature, micro-decisions are a dimension of control and surveillance in the twenty-first century that has received little critical attention. They represent the smallest unit and the technical precondition of a contemporary network politics – and of our potential opposition to it. The current debates regarding net neutrality and Edward Snowden’s revelation of NSA surveillance are only the tip of the iceberg. What is at stake is nothing less than the future of the Internet as we know it.
Background aims: Immunomagnetic enrichment of CD34+ hematopoietic “stem” cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products.
Methods: Granulocyte-colony stimulating factor–mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms.
Results: Nine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells.
Conclusions: The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.
Allergy against birch pollen is among the most common causes of spring pollinosis in Europe and is diagnosed and treated using extracts from natural sources. Quality control is crucial for safe and effective diagnosis and treatment. However, current methods are very difficult to standardize and do not address individual allergen or isoallergen composition. MS provides information regarding selected proteins or the entire proteome and could overcome the aforementioned limitations. We studied the proteome of birch pollen, focusing on allergens and isoallergens, to clarify which of the 93 published sequence variants of the major allergen, Bet v 1, are expressed as proteins within one source material in parallel. The unexpectedly complex Bet v 1 isoallergen composition required manual data interpretation and a specific design of databases, as current database search engines fail to unambiguously assign spectra to highly homologous, partially identical proteins. We identified 47 non-allergenic proteins and all 5 known birch pollen allergens, and unambiguously proved the existence of 18 Bet v 1 isoallergens and variants by manual data analysis. This highly complex isoallergen composition raises questions whether isoallergens can be ignored or must be included for the quality control of allergen products, and which data analysis strategies are to be applied.
We present an extensive experimental study of the recently predicted pygmy quadrupole resonance (PQR) in Sn isotopes, where complementary probes were used. In this study, (α,α' γ ) and (γ , γ') experiments were performed on 124Sn. In both reactions, Jπ = 2+ states below an excitation energy of 5 MeV were populated. The E2 strength integrated over the full transition densities could be extracted from the (γ , γ') experiment, while the (α,α'γ ) experiment at the chosen kinematics strongly favors the excitation of surface modes because of the strong α-particle absorption in the nuclear interior. The excitation of such modes is in accordance with the quadrupole-type oscillation of the neutron skin predicted by a microscopic approach based on self-consistent density functional theory and the quasiparticle-phonon model (QPM). The newly determined γ -decay branching ratios hint at a non-statistical character of the E2 strength, as it has also been recently pointed out for the case of the pygmy dipole resonance (PDR). This allows us to distinguish between PQR-type and multiphonon excitations and, consequently, supports the recent first experimental indications of a PQR in 124Sn.
2004 erschütterten Terroranschläge in London und Madrid die Welt. Die Reaktion der EU/EG im seinerzeit schnellsten Gesetzgebungsverfahren seit Bestehen: Verabschiedung der Vorratsdatenspeicherungsrichtlinie. 2009 sah der EuGH die Richtlinie trotzdem als Binnenmarktregelung an, mit der Harmonisierung auf dem Informationsmarkt herbeigeführt werden sollte. Äußerungen zum Inhalt – quasi als obiter dictum möglich gewesen – versagte er sich. 2010 beurteilte dann das BVerfG die bundesdeutsche Umsetzung, aber – ganz dem Verhältnis von EuGH und BVerfG entsprechend – nur den die europäischen Mindestvorgaben überschießenden Teil. Dieses Gesetz hatte national schon für reichlich Sprengstoff im Rechtsstaat gesorgt und u.a. den Rücktritt der beharrlich widersetzlichen Justizministerin bewirkt. ...
Im Wirtschaftsregulierungsrecht treten immer häufiger Mehrpersonenverhältnisse auf: Die Regulierungsentscheidung der Regulierungsbehörde betrifft nicht nur den Adressaten, sondern hat mittelbar auch Wirkungen auf die Ausgestaltung der Wettbewerbsfreiheit der Konkurrenten. Materielles und Verfahrensrecht erlauben aber bisher kaum eine Beteiligung der Interessen des Dritten. Verwaltungsrecht und Verwaltungswissenschaft sind aufgefordert, hierfür Problemkonstellationen zu identifizieren und Lösungsvor-schläge zu unterbreiten.
Der Beitrag analysiert zunächst Dreiecksverhältnisse mit besonderem Blick auf das Wirtschaftsverwaltungsrecht und schlägt als einen möglichen Weg zur Bewältigung daraus resultierender Probleme in Instrumenten der Kooperation vor, wie sie etwa im Gesundheitsrecht mit dem Gemeinsamen Bundesausschuss (GBA) existieren, auch wenn dieses Rechtsgebiet (zu Unrecht) als wenig als wegweisend wahrgenommen wird.
Sowohl bezüglich der Modellierung der Sprachkompetenz bei mehrsprachigen Kindern als auch hinsichtlich der Bestimmung der hierfür notwendigen Indikatoren herrscht nach wie vor Forschungsbedarf. Die entsprechenden Erkenntnisse sind aber für eine valide Sprachstandsdiagnostik, auf Basis welcher auch eine Sprachförderung stattfinden kann, unerlässlich. In der vorliegenden Dissertation werden daher mit Hilfe von Verteilungsstatistiken, konfirmatorischen Faktoranalysen und Strukturgleichungsmodellen zunächst ausgewählte Sprachstandsindikatoren sowie das Konstrukt Sprachkompetenz von Kindern mit Deutsch als Zweitsprache zu Beginn der 1. Klasse modelliert und im Anschluss ihr Einfluss auf die Orthographiekompetenz dieser Schülerinnen und Schüler am Ende der 2. Klasse untersucht. Zusätzlich wird das phonologische Arbeitsgedächtnis als ein weiterer Prädiktor in das Modell eingebunden. Es wird der Fragestellung nachgegangen, ob die für die Modellierung der Sprachkompetenz verwendeten Indikatoren, wie bspw. Bildbenennung, Kasus, Syntaxerwerbsstufen und die mittlere Äußerungslänge (MLU) für die Gruppe der mehrsprachigen Kinder in der Schuleingangsphase geeignet bzw. valide sind. Dabei werden die jeweiligen Streuungen, die Korrelationen untereinander (konvergente Validität) sowie die Leistung der Indikatoren für die jeweiligen sprachlichen Kompetenzbereiche anhand der Höhe der Faktorladungen betrachtet. Ebenfalls wird die prognostische Validität der Indikatoren hinsichtlich der Rechtschreibkompetenz beleuchtet. Des Weiteren wird geprüft, ob sich die Sprachkompetenz bei DaZ-Kindern in eine semantische, eine morphologische und eine syntaktische Fähigkeit unterteilen lässt und ob die Sprachkompetenz als Faktor zweiter Ordnung modelliert werden kann. Weitere Fragestellungen betreffen die Modellierung der Rechtschreibkompetenz, also die Anzahl und Art der latenten Variablen, die dieses Konstrukt abbilden, sowie den Einfluss des phonologischen Arbeitsgedächtnisses und der einzelnen sprachlichen auf die orthographischen Teilkompetenzen.
The subject matter of this article is Axel Honneth’s theory of recognition as it has been exposed in his more recent book, Das Recht der Freiheit. Throughout the paper his attempts to describe injustices within modern capitalist societies using the notions of pathologies and anomie will be analyzed and criticized, especially from the viewpoint of their inability to deal with processes and contexts of disrecognition (Aberkennung). With help of this category, Honneth’s diagnosis regarding the moral progress in modern societies, as well as his notion of second order disorders, as injustices will be confronted and, hopefully, complemented.
Die Substitution von klassischen, mit der Nahrungsmittelproduktion in Konkurrenz stehenden, Substraten wie Glukose durch alternative Kohlenstoffquellen in der Biotechnologie ist sowohl aus ethischer, als auch aus ökonomischer Sicht erstrebenswert. Diese Arbeit beschreibt die Synthese von Bulkchemikalien in Form zweier Dicarboxylsäuren und einer Feinchemikalie in Form eines Sesquiterpens aus dem alternativen Substrat Methanol mit Hilfe genetisch veränderter Stämme des methylotrophen α-Proteobakteriums Methylobacterium extorquens.
Mesacon- und (2S)-Methylsuccinsäure sind Dicarboxylsäurederivate der CoA-Ester Mesaconyl- und (2S)-Methylsuccinyl-CoA, die als Intermediate im Ethylmalonyl-CoA- Weg (EMCP) vorkommen. M. extorquens nutzt den EMCP für die Regeneration von Glyoxylat, das für das Wachstum auf C1-Substraten wie Methanol obligatorisch ist. In dieser Arbeit konnte erstmals Mesacon- und (2S)-Methylsuccinsäure de novo durch die Expression einer für die Vorstufen Mesaconyl- und (2S)-Methylsuccinyl-CoA aktiven Thioesterase produziert werden. Ein kobaltlimitiertes Wachstum von M. extorquens führte aufgrund mangelnder Cofaktorversorgung zweier Vitamin-B12-abhäniger Mutasen im EMCP zu einer Akkumulation der beiden CoA-Ester-Vorstufen, womit eine Produktion von 0.65 g/l Mesacon- und (2S)-Methylsuccinsäure erreicht wurde. Weitergehende Untersuchungen belegten außerdem einen positiven Effekt eines ausgeschalteten PHB-Zyklusses auf die Produktion der beiden EMCP- Dicarboxylsäurederivate.
Diese Arbeit beinhaltet zusätzlich grundlagenwissenschaftliche Untersuchungen zur Substitution der EMCP-katalysierten Glyoxylatregeneration durch einen heterologen Glyoxylatzyklus in EMCP-negativen M. extorquens-Stämmen. Dabei konnte erstmals ein methanolverwertendes, methylotrophes Bakterium identifiziert werden, das einen Serin-Zyklus in Kombination mit dem Glyoxylat-Zyklus zur Kohlenstoffassimilation verwendet, ohne dabei zusätzliche Stoffwechselwege zur CO2-Fixierung wie den EMCP, RuMP oder CBB-Zyklus zu verwenden.
Die Präsenz einer nativen C30-Carotinoidbiosynthese, ausgehend von der Vorstufe Farnesylpyrophosphat (FPP), empfiehlt M. extorquens als Produktionsorganismus für (Sesqui-)Terpene. In dieser Arbeit wurde mit Hilfe einer induzierbar gesteuerten Expression einer Terpensynthase in Form einer α-Humulen-Synthase, einer FPP-Synthase und eines prokaryontischen Mevalonatweges, erstmals die de novo Synthese eines Terpens aus Methanol am Beispiel des α-Humulens etabliert. Durch optimierte Expressionen der Terpensynthase, FPPS und einzelner MVA-Gene mit Hilfe angepasster Translationsinitiationsraten der jeweiligen ribosomalen Bindestellen und der Verwendung eines in der nativen Carotinoidbiosynthese inhibierten M. extorquens-Stammes wurden finale Produkttiter von bis zu 1.65 g/l α-Humulen in Fed-Batch-Fermentationen erreicht.
Diese kumulative Dissertation beinhaltet außerdem einen Reviewartikel, in dem der verwendete Mikroorganismus M. extorquens in mikrobiologischer, genetischer, biochemischer und auch biotechnologischer Hinsicht ausführlich beschrieben wird. Zudem gibt ein Buchkapitel eine Übersicht über die Verwendung von Methanol in der Biotechnologie.