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The main goal of this work is to contribute to the existing knowledge of soil micro-fungi in Panama and Germany. Studies about soil degradation and its influents in the soil fungi diversity have not been investigated as extensively in these countries. This is an extensive and challenging topic to examine since there is an immense phenotypic and genetic diversity in the soil fungal community and relating this community together with factors of soil degradation is an extensive task. For this reason, the present thesis studies the species identified in the study areas, in other words, the soil fungal diversity in relation to environmental factors in the Taunus Mountain range in Frankfurt, Germany, and in the Majagua valley in Chiriquí, Panama. Two complementary objectives were achieved, the first was the development of a theoretical irrigation model for degraded soils. The second was the development of a mobile application to facilitate laboratory work in the cultivation of soil micro-fungi.
The design of the methodology was based on identifying the species and relating the diversity found to soil factors. Soil samples were taken in both countries: the Taunus Mountain range was sampled eight times from January to November 2012 and the Majagua valley was sampled on three occasions between February and July 2012. In both studies, the areas included three different vegetation types (forest, grassland, and bare soil). Samples were separated for two purposes: the assessment of fungal diversity by molecular and morphological methods and soil characterization.
Soil samples used in the methodology of pyrosequencing were related to global climatic factors. Morphological identification was achieved with identification keys. Micro-fungi were cultivated in different media until obtaining pure cultures. Molecular identification was performed by getting the DNA sequences using the ITS1 and ITS4 primers and comparing the sequences with other reference sequences from GenBank. This was done considering the BLAST algorithm, which considered sequences that matched 98 % or more of maximum identity as reliable identifications.
Soil characterization was carried out to measure the soil's Physico-chemical properties; those abiotic factors were compaction, temperature, pH, moisture, and soil composition.
Species richness was calculated in each study area with the estimators Chao, Jackknife, and Bootstrap. Furthermore, the species accumulation curves were performed to observe the species discovery rate and estimate sample completeness. Estimate linear regression models correlated the influence between the soil factors (temperature, moisture, pH, soil compaction, and soil composition) and the species richness. In the same way, an analysis of ecological distance was undertaken based on the similarity in the species composition, compared across samples, and correlated with soil factors, using non-metric multidimensional scaling (NMDs).
Study of abundance showed differences between the bare soil abundances and the forest abundances in Germany and Panama; the grasslands in both countries work as transitional areas in the fungi abundance. The key stone species in Germany were Penicillium daleae, and Pochonia bulbillosa, whereas in Panama were Purpureocillium lilacinum and Trichoderma harzianum. Based on Pareto analysis, a theoretical irrigation model was developed to counteract the degradation effects on the abundance of micro-fungi in the soil.
Applications for mobile devices dealing with the cultivation of soil micro fungi were sought. Due to the small number of existing applications, a new App called Soil-Fungi-Cultures (SFC) was developed to facilitate data collection of cultivated soil micro fungi. App Inventor was the program used to design, program, test, and publish the application developed. The developed application was compared with other applications used in identifying bacteria cultures. The results showed that the new application needed more time to capture the records because it saves more information, the navigation flow was acceptable, the number of clicks was high, but it is due to the usefulness in data capture, and finally, the users rated it as a good application with an eight out of ten rating.
Pyrosequencing resulted in 204 Operational Taxonomic Units (OTUs) considering the two study areas (the Taunus Mountain range and the Majagua valley). The Pyrosequencing database was used to contribute to the most important study of fungal diversity globally based on OTUs, which surpasses any study of molecular and taxonomic diversity previously conducted. The principal result in this study was that the climatic factor is the best predictor of fungal richness and community composition on a global scale. However, the part of the research that focused on the local scale, that is to say, on the correlation patterns between the distribution of fungal species and abiotic factors, showed that the soil properties and degradation levels were not associated with fungal richness, diversity or soil composition in the study areas in Germany or Panama. The above confirms that there are exceptions to the way relationships between soil factors with fungal diversity are established at the local level.
In the case of soil samples used for morphological identification, 71 fungal species were obtained, 47 from Germany, and 32 from Panama.
RNA modification is a dynamic and complex process that involves the addition of various chemical groups to RNA molecules, contributing to their diversity and functional complexity. Among all the RNA modifications, N6-methyladenosine (m6A) is the most common post-transcriptional modification found in mRNA molecules, particularly in eukaryotic mRNA. It involves methylation of the adenosine base at the nitrogen-6 position. This modification plays a crucial role in many aspects of RNA metabolism, including splicing, stability, translation, and the cellular response to stress. With the development of m6A sequencing technologies, our knowledge of m6A has evolved rapidly over the past two decades. However, one of the most widely used m6A profiling techniques termed “m6A individual-nucleotide resolution UV cross-linking and immunoprecipitation (miCLIP)” suffers from a high unspecific background signal due to the limited antibody binding specificity.
To accurately discriminate m6A sites from the background signal in miCLIP data, in Chapter 4, I first developed different strategies to identify the true miCLIP2 signal changes that are corrected for the underlying transcript abundance changes. I performed this analysis on data that generated with an improved experiment protocol, named miCLIP2. With the best performing strategy, the Bin-based method, I detected more than 10,000 genuine m6A sites. I then used the information embedded in the genuine m6A sites to train a machine learning model - named "m6Aboost" - to enable accurate m6A site detection from the miCLIP2 data without a control dataset from an m6A depletion cell line. To allow an easy access for future users, I packaged the m6Aboost model into an R package that is available on Bioconductor.
Although previous studies have reported that m6A is involved in three different RNA decay pathways, it remains unclear how a pathway is selected for a specific transcript or m6A site. In Chapter 5, I reveal that m6A sites in the coding sequence (CDS) induce a stronger and faster RNA decay than the m6A sites in the 3’ untranslated region (3’UTR). Through an in-depth investigation, I found that m6A sites in CDS trigger a novel mRNA decay pathway, which I termed CDS-m6A decay (CMD). Importantly, CMD is distinct from the three previously reported m6A-mediated decay pathways. In terms of its mechanism, CMD relies on translation, where m6A sites in the CDS lead to ribosome pausing and subsequent destabilization of the transcript. The transcripts targeted by CMD are identified by the m6A reader protein YTHDF2, preferentially localized to processing bodies (P-bodies), and undergo degradation facilitated by the decapping factor DCP2. CMD provides a flexible way to control the expression of CDS m6A-containing transcripts which include many developmental regulators and retrogenes.
In summary, this PhD thesis introduces a novel workflow for identifying m6A sites in miCLIP data through the implementation of the m6Aboost machine learning model. Using the m6A sites identified by m6Aboost and additional data, a newly uncovered m6A-mediated mRNA decay pathway, CMD, is elucidated, providing valuable insights into m6A-mediated decay processes.
Terpene bilden mit mehr als 81.000 Verbindungen die größte Klasse der Naturstoffe. Nichtsdestotrotz wird ihre strukturelle Vielfalt durch die Isoprenregel begrenzt. Diese besagt, dass alle primären Terpensynthaseprodukte aus Bausteinen mit fünf Kohlenstoffatomen hervorgehen. Ihre Produkte sind somit kanonisch, da sie durch ein Vielfaches von fünf Kohlenstoffatomen dargestellt sind. In dieser kumulativen Arbeit wird die mikrobielle Produktion einer Vielzahl neuartiger nicht-kanonischer Terpene beschrieben und somit der chemische Strukturraum von Terpenoiden über die Grenzen der Isoprenregel hinaus erweitert. Um dies zu erreichen, wurden in verschiedenen Ansätzen die Gene des Mevalonatwege, einschließlich einer IPP-Isomerase gemeinsam mit verschiedenen Prenylpyrosphosphat-Methyltransferasen und Terpensynthasen in E. coli exprimiert und die Produktspektren der Biosynthesewege detailliert untersucht.
Ein breites Spektrum neuer C11-Terpene wurde als Nebenprodukt der bakteriellen 2-Methylisoborneol- oder 2-Methylenbornansynthasen entdeckt. Neben elf bekannten konnten 24 neuartige C11-Terpene nachgewiesen werden, die bisher noch nicht als Terpensynthase-Produkte beschrieben wurden. Vier davon, 3,4-Dimethylcumol, 2-Methylborneol und die beiden Diastereomere von 2-Methylcitronellol, konnten identifiziert werden. Außerdem wurde das C16-Terpen 6-Methylfarnesol als Produkt identifiziert.
Die Produktselektivität einer C11-Terpensynthasen, die 2 Methylenbornansynthase aus Pseudomonas fluorescens, wurde durch einen semirationalen Protein-Engineering-Ansatz verändert. Aminosäuren des aktiven Zentrums mit Einfluss auf die Produktselektivität wurden identifiziert. Entsprechende Varianten des Enzyms führen zu gänzlich veränderten Produktspektren. So wurden neue Einblicke in die Struktur-Funktions-Beziehung für C11-Terpensynthasen gewonnen und bisher unzugängliche nicht-kanonische Terpene produziert.
Eine IPP-Methyltranferase wurde identifiziert und charakterisiert, die den C5-Baustein der Terpenbiosynthese in eine Vielzahl von C6- und C7-Prenylpyrophosphate umwandelt. Die heterologe Expression in E. coli gemeinsam mit anderen Genen der Terpenbiosynthese erweitert das potenzielle Terpensynthase-Substratspektrum außerdem um C11-, C12-, C16- und C17 Prenylpyrosphopshate. Darüber hinaus konnten polymethylierte C41-, C42-und C43-Carotinoide synthetisiert werden. So wurde die Terpenbiosynthese durch die Modifikation ihrer Bausteine erweitert und neue ungewöhnliche Terpene produziert.
The attention on the protein PURA has increased recently following the discovery of the rare PURA Syndrome. This neurodevelopmental disorder is caused by de novo mutations in the PURA gene. Notably, our collaborators could show that the protein PURA can bind DNA and RNA in vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, I inquired on the connection of PURA-RNA binding to the cellular effect of a reduction of functional PURA as present in PURA Syndrome patients.
To investigate the binding of PURA and the impact of PURA de ciency on cellular RNA and protein expression, I performed an integrative computational analysis of multimodal data from complementary high-throughput experiments. An essential component was the examination of UV Crosslinking and immunoprecipitation (CLIP) experiments, which can query the global RNA-binding behaviour of a given protein in a cellular context. As the processing and analysis of CLIP data are rather complex, I introduce an automated command line tool for the processing of CLIP data named racoon_clip as part of this dissertation. Therefore, this dissertation comprises two major segments. Firstly, I describe the implementation and usage of racoon clip for CLIP data analysis. Secondly, I discuss my research on the protein PURA, demonstrating its global RNA-binding properties, the effects of PURA depletion and its association with neuronal functions and P-bodies, among others.
racoon_clip is a command line application that I have developed for processing of individualnucleotide resolution CLIP (iCLIP) and enhanced CLIP (eCLIP) experiments - two of the most commonly used types of CLIP experiments - in a comparable and user-friendly way.
For this, I built racoon_clip as an automated work how that encompasses all CLIP processing steps from raw data to single-nucleotide resolution crosslink events. racoon_clip is available as a command line tool that users can run with a single command. The work how is implemented with Snakemake work how management providing computational advantage tages including parallelisation, scalability and portability of the work how. The main task of racoon_clip is to extract single-nucleotide crosslink events from iCLIP, iCLIP2, eCLIP and similar data types. To strike a balance between being highly customisable and easy to use, racoon_clip supplies pre-set options for the most common types of experiments.
Additionally, it is possible for users to create a custom setup of barcode and adapter architectures, which allows them to use the software for other types of CLIP data. While accounting for the different architectures in the reads, the performed central processing steps remain the same. This leads to a high degree of comparability between the different experiment types, which I demonstrate in the exemplary processing of U2AF2 iCLIP and eCLIP data. Taken together, I am confident that racoon_clip will be beneficial to numerous researchers interested in RNA-Protein interactions as it offers easily accessible processing for CLIP data and enhances the comparability of multiple CLIP datasets across di erent experiment types.
In the second part of this dissertation, I focus on the cellular function of the RNAbinding protein PURA. Through in-depth computational analysis of one iCLIP data set of endogenous PURA and two iCLIP data sets of overexpressed PURA in HeLa cells, I establish that PURA is a global RNA-binding protein. It preferentially binds RNAs in either the coding sequence (CDS) or the 3' untranslated region (3'UTR) of mature protein-coding transcripts by recognising a Purine-rich degenerated sequence motif. Even though overexpression of PURA results in less specific binding behaviour, the same overall binding patterns as from endogenous PURA persist. Overall characteristics of PURA binding remain similar in three distinct PURA iCLIP data sets with and without PURA overexpression.
To learn about the molecular consequences of a depletion of functional PURA in a cellular context, I used a 50% reduction of PURA in HeLa cells as a model for the heterozygous loss of PURA in PURA Syndrome and evaluated its impact on global RNA and protein expression. The results demonstrate that PURA depletion globally a ects RNA and protein expression. Additionally, I integrate PURA RNA binding with the changes in expression of RNAs and proteins in the context of PURA depletion. This reveals 234 targets of PURA that are bound by PURA and are impacted at both RNA and protein levels by the PURA protein. RNAs that are bound by PURA or change in abundance upon PURA depletion are enriched in neuronal development factors, RNA lifecycle regulators, and mitochondrial factors, among others. Consistent with a possible role of PURA in neuronal transport, there is considerable overlap between PURA bound transcripts and transcripts, that are transported to the dendritic end of neurons.
Notably, there is a link between PURA and P-bodies, as documented by the enrichment of PURA-bound RNAs in both the P-body and stress granule transcriptome. Further, PURA was found by our collaborators to be localised within P-bodies and P-body numbers were strongly reduced in cells that are depleted of PURA. This absence might be attributed to the downregulation of the proteins encoded by the PURA targets LSM14A and DDX6 as both of them were previously identified as essential for P-body formation.
Overall, the reduction of P-body numbers in PURA depletion, the neuronal function of PURA, and its association with mitochondria and RNA lifecycle regulation may indicate the cellular foundation of both PURA Syndrome and related neuronal diseases.
In summary, I present a versatile and user-friendly computational tool for the analysis of CLIP data. Subsequently, I conduct a thorough computational analysis of CLIP and other high-throughput data in the context of the RNA-binding protein PURA, which offers valuable insights into the cellular functions of PURA. These insights advance our understanding of the impact of PURA loss in PURA Syndrome and other disease contexts.
Molecular concepts for pandemic viruses : membrane fusion assays and targeting of reservoir cells
(2024)
In den letzten Jahren haben verschiedene pandemische Viren zu beträchtlichen Krankheits- und Todesfällen geführt. Um dieser ständigen Bedrohung entgegenzuwirken, ist es wichtig diagnostische Testsysteme und Therapien anzupassen oder neu zu etablieren. Diese Arbeit konzentriert sich auf die pandemischen Viren SARS-CoV-2 und HIV.
Der Zelleintritt von SARS-CoV-2 wird durch das Spike-Protein (S) ausgelöst, welches die Fusion der Virushülle mit der zellulären Membran bewirkt. Erste Studien haben gezeigt, dass das S-Protein eine hohe Fusionsaktivität aufweist. Aus diesem Grund sollten in dieser Arbeit neue Fusionstests etabliert werden, um potenzielle Inhibitoren der Zellfusion zu evaluieren. Im ersten Teil dieser Thesis wird die Etablierung von quantitativen Tests zur Evaluierung der Zell-Zell und Partikel-Zell Fusionsaktivität, welche durch S bewirkt wird, demonstriert.
Trotz jahrelanger Forschung können HIV-Patienten nicht geheilt werden und Virusinfektionen treten weiterhin weltweit auf. Das größte Problem bei der Entwicklung eines Heilmittels ist die frühe Bildung von Reservoirzellen während einer Infektion. Um diese Reservoirzellen zu identifizieren, wurde der Oberflächenmarker CD32a vorgeschlagen. Die Nutzung von Cas9-Nukleasen zur Inaktivierung von HIV ist in vitro erfolgreich, aber der effiziente Transfer in Reservoirzellen bleibt weiterhin herausfordernd. Im zweiten Teil dieser Thesis werden Rezeptor-gerichtete Adeno-assoziierte Vektoren (AAVs) für die HIV-Gentherapie präsentiert, die CD4 und CD32a für den Zelleintritt nutzen.
Zur Charakterisierung der Fusionsaktivität von SARS-CoV-2 wurden drei quantitative Fusionstests etabliert, welche Partikel- und Zell-Zell Fusionen berücksichtigen. Für den Partikel-Zell Fusionstest wurden lentivirale Vektoren (LV) verwendet, welche das S-Protein auf ihrer Oberfläche präsentierten. Die Transduktionseffizienz von S-LV erreichte auf Zellen, die den SARS-CoV-2 Rezeptor ACE2 exprimieren, ein Signal-zu-Hintergrund Verhältnis von über 2000. Durch die Präsentation von S auf leeren LV-Partikeln konnte die Fusion von benachbarten Zellen detektiert und quantifiziert werden („fusion-from-without“ (FFWO)). Für die Quantifizierung wurde ein Reporter-Komplementationstest etabliert. Hierbei wurden die Alpha- und Omega-Fragmente der β-Galaktosidase getrennt in zwei Zielzellpopulationen exprimiert, die beide ACE2 exprimierten. Durch die Zugabe von S-Partikeln kam es zur Fusion der Zielzellen und zur Komplementation der Alpha- und Omega-Fragmente. Die resultierende β-Galaktosidase-Aktivität konnte anschließend quantifiziert werden. Unter optimalen Versuchsbedingungen erreichte dieser Assay ein Signal-zu-Hintergrund Verhältnis von 2,7 Größenordnungen. Anschließend wurde der Komplementationstest für die Messung der Zell-Zell Fusion verwendet. In diesem Test exprimierten Effektorzellen S und das Alpha-Fragment, Zielzellen ACE2 und das Omega-Fragment. Obwohl die S-Expression auf den Effektorzellen sehr gering war, konnte dennoch eine signifikante Fusion nachgewiesen werden. Auch hier konnte unter optimalen Versuchsbedingungen ein hohes Signal-zu-Hintergrund Verhältnis von 2,9 Größenordnungen festgestellt werden. Nach der Etablierung der Testsysteme wurden S-spezifische Inhibitoren verwendet. Im Gegensatz zu Partikel-Zell-Fusionen wurde die Fusionsaktivität von S auf Zellen nur mäßig inhibiert. Dies deutet daraufhin, dass das Eindringen von Partikeln in Zellen wirksamer verhindert werden kann als die Ausbreitung durch Zell-Zell Fusionen.
Um AAVs spezifisch an HIV-Reservoirzellen zu binden, wurden CD4- und CD32a-spezifische DARPins („designed ankyrin repeat proteins“) in Rezeptor-verblindete AAVs eingebaut. Ebenso wurden beide DARPins gleichzeitig auf dem Kapsid präsentiert, um eine höhere Spezifität für doppelt-positive Zellen zu erreichen. Wenn diese Partikel einer Zellmischung aus CD4-, CD32a- und CD4/CD32a-exprimierenden Zellen zugesetzt wurden, transduzierten die bispezifischen Vektoren vorzugsweise doppelt-positive Zellen. Diese Präferenz war am höchsten in Zellkulturen, die stark unterrepräsentierte CD4/CD32a-exprimierende Zellen enthielten. Unter diesen Voraussetzungen erreichten bispezifische Vektoren eine bis zu 66-fach höhere Transduktionseffizienz auf CD4/CD32a-positive Zellen im Vergleich zu CD32a-exprimierenden Zellen. Darüber hinaus zeigten bispezifische AAV eine präferentielle Bindung und Transduktion von isolierten Primärzellen und Zellen in Vollblut. Selbst nach systemischer Injektion in humanisierte Mäuse wurden doppelt-positive Zellen effizienter von bispezifischen als von monospezifischen AAVs transduziert. Schließlich zeigten die generierten Vektoren, welche die Cas9 Nuklease transferierten, eine effiziente Inhibition der HIV-Replikation.
In almost all parts of the world the industrialisation grows continuously and thus, the chemical pollution of natural waters has become a major public concern. A major consequence and one of the key environmental problems we are facing today is the increasing contamination of freshwater systems with chemicals. The chemicals are detected in wastewater, surface (river) water, ground water and drinking water ubiquitously in natural waters and not only in industrialised areas. The main point sources for water pollution and the release of these synthetic organic substances of human origin, so called micropollutants (MPs), are wastewater treatment plants (WWTPs). These MPs such as pharmaceuticals, personal care products, disinfectant chemicals, chemicals used in the industry and in households, contraceptives, hormones, food additives, artificial sweeteners, pesticides, biocides, and many emerging contaminants are only incompletely removed by the existing conventional wastewater treatment technologies. The MPs end up in the water cycle and have adverse effects on wildlife aquatic ecosystems and human health even at very low concentrations. Therefore, advanced wastewater treatment (AWWT) technologies, such as ozonation, treatment with activated carbon, biofiltration, membrane bioreactors (MBRs) or exposure to ultraviolet light are investigated as options to upgrade conventional WWTPs. However, several studies show that especially the ozonation of wastewater generates diverse transformation products (TPs) with unknown properties. These TPs could be more toxic than the mother compound. Thus, a post-treatment after the ozonation process is required.
The present thesis was part of the BMBF-funded TransRisk project dealing with “the characterisation, communication, and minimisation of risks of emerging pollutants and pathogens in the water cycle”. One main objective was the investigation of conventional treated wastewater after a full-scale ozonation with four post-treatments (each non-aerated and aerated granular activated carbon (GAC) filtration and biofiltration) in comparison to a MBR treatment of raw (untreated) wastewater separately and in combination with an additional ozonation on a pilot WWTP. For this purpose, the wastewater samples were characterised with a comprehensive battery of in vitro and in vivo bioassays. The in vitro bioassays were performed to detect endocrine activities (such as (anti)estrogenic and (anti)androgenic activities), genotoxicity, and mutagenicity. The results showed a decreased estrogenic activity due to the conventional wastewater treatment as well as the ozonation, but a distinct increase of the anti-estrogenic activity and the mutagenicity in the ozonated wastewater, possibly caused by new formed TPs, that were reduced after the post-treatments whereas the GAC filtration performed better than the biofiltration. The in vivo bioassays included for example the impact of the wastewater on mortality, reproduction, development, and energy reserves of the test organisms. The in vivo on-site tests with the mudsnail Potamopyrgus antipodarum and with the amphipod Gammarus fossarum indicated a major impact of conventional treated wastewater, ozonated wastewater, and MBR treated wastewater. The flow channel experiments in the laboratory with Gammarus pulex pointed to a serious impact of an estrogenic effluent on life-history traits of the amphipod. Finally, an ozonation of the wastewater with subsequent GAC filtration represented the most promising option. In addition, chemical analyses of 40 selected MPs, so called tracer substances, performed in parallel to the in vitro and in vivo bioassays underlined this assumption.
A second main objective was the optimisation of the preparation of water and wastewater samples for ecotoxicological in vitro bioassays because common sample preparation techniques are predominantly adapted for chemical analyses. Therefore, the impact of sample filtration, long-term acidification with following neutralisation as well as the enrichment with solid phase extraction (SPE) in combination with short-term acidification were investigated using amongst others raw (untreated) wastewater, hospital wastewater, conventional treated and ozonated wastewater, surface water, and ground water. Overall, eleven in vitro bioassays were performed for the detection of endocrine activities, genotoxicity, and mutagenicity. The results show that sample filtration and acidification/neutralisation significantly affected the outcome of the bioassays especially the anti-estrogenic activity and the mutagenicity whereas the sample filtration had a minor impact than the acidification. Thus, the testing of untreated (waste)water samples is advisable because the sample is minimally processed. Furthermore, the SPE extracts showed in parts high cytotoxic effects whereby no conclusions on the results of the bioassays were possible. However, the enrichment of endocrine activity and mutagenicity was predominantly effective but depended on the used SPE cartridge and the pH value of the (waste)water samples. Based on the results the use of a Telos C18/ENV cartridge and an acidified sample is recommendable. In the end, there is a need to optimise the sample preparation for in vitro bioassays to reach their maximum outcome for the best possible assessment of the water quality.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Research on the human and animal microbiome has become increasingly important in recent years. It is now widely accepted the gut microbiome is of crucial importance to health, as it is involved in a large number of physiological processes. The term ‘microbiome’ refers to the all living microorganisms including their genes and metabolites in a defined environment, while the specific composition of microorganisms consisting of bacteria, archaea and protozoa is referred to as the ‘microbiota’ (Lane-Petter, 1962; Lederberg and McCray, 2001).
In recent years, research has focused on various of these communities in the soil (Fierer, 2017), water (Sunagawa et al., 2015), air (Leung et al., 2014) and especially in the human gut. However, this topic is also becoming increasingly relevant for the conservation of endangered species. In the face of global mass extinctions and the listing of over 42,000 animal species as ‘critically endangered’, conservation breeding programmes are more important than ever (Díaz et al., 2019; IUCN, 2022). The responsibility for these tasks lies with zoological institutions, which are dedicated to animal conservation and the continuous monitoring of animal welfare. Microbiome research offers a non-invasive method to support species conservation. By analysing faecal samples, microbial markers can be identified that provide important information about the health status and reproductive cycle of animals (Weingrill et al., 2004; Antwis et al., 2019). Zoological facilities also provide an ideal research environment for comparing individuals from different habitats. In addition, all necessary metadata such as age, sex, kinship or medical treatment are documented and can be used for the analysis.
This is the starting point for this thesis. In order to identify such microbial markers, it is necessary to understand the microbiome of a variety of animal species. The first aim is therefore to characterise the faecal microbiota of 31 mammalian species, focusing on herbivores and carnivores. It could be shown that they differ significantly in terms of both microbial diversity and microbiota composition. Herbivorous species express a very diverse microbial composition, consisting mainly of cellulose-degrading taxa of the families Fibrobacteraceae or Spirochaetaceae. In contrast, the microbiota of carnivorous species is less diverse and is dominated by protein-degrading Fusobacteriaceae and Clostridiaceae. In addition, this thesis proves that the microbiota of herbivorous species is highly consistent, whereas the microbiota of carnivorous species is highly variable. The results of this study provide important insights for the sampling scheme of future projects. Especially when analysing carnivorous species, single samples are not sufficient to capture the full variability of the microbiome.
These results lead to the question of whether this variability can be explained by daily fluctuations in the individual microbiome and whether this can be used to distinguish between species or individuals. Using individual longitudinal data and a combined approach of clustering algorithms and dynamic time warping, it is shown that such a distinction is possible at the species and individual level. This was confirmed for both a carnivorous (Panthera tigris) and a herbivorous (Connochaetes taurinus) species. These results confirm the influence of the host individual on the faecal microbiota, in addition to the often described influence of diet (Ley et al., 2008a; Kartzinel et al., 2019).
Based on the knowledge gained from these studies, a methodology has been developed that will enable the conservation of species in the field to be supported by microbiome research in the future. The focus here lays on the identification of host-specific metadata based on the faecal microbiota. The developed regression model is able to distinguish between carnivorous, herbivorous and omnivorous hosts with up to 99% accuracy. In addition, a more accurate phylogenetic classification of the family (Canidae, Felidae, Ursidae, Herpestidae) can be made for carnivorous hosts. For herbivorous hosts, the model can predict the respective digestive system with up to 100% accuracy, distinguishing between ruminants, hindgut fermenters and a simple digestive system. The acquisition of host-specific metadata from an unknown faecal sample is an important step towards establishing microbiome research in species conservation. Field studies in particular will benefit from such new methods. Usually, costly microsatellite analysis and high-quality host DNA are required to obtain host-specific information from faecal samples. The newly developed method offers a less costly and labour-intensive alternative to conventional techniques and opens up a more accessible field for microbiome research in the field.
Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2.
Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes.
To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance.
To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences.
In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.
