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Terpenes are one of the largest and most diverse class of natural products, produced by organisms from all kingdoms of life and with important applications in the pharma, flavor and fragrance industries. Well-known examples of terpenes are the pharmaceuticals artemisinin and taxol, the flavor and fragrance compounds menthol, santalol and sclareol, the structural material polyisoprene and the biofuel precursor farnesene. The methods and results presented in this work offer a variety of ways to modify terpene precursors for the creation of new terpene molecules. The application of these methodologies in well-established production systems could lead to the production of new substances, with applications in the industrial fields of pharmaceuticals, flavors and fragrances, and biofuels.
Global biodiversity is changing rapidly and contemporary climate change is an important driver of this change. As climate change continues, the challenge is to understand how it may affect the future of biodiversity. This is relevant to informing policy and conservation, but it requires reliable future projections of biodiversity. Biodiversity is the variety of life on Earth which includes the diversity of species. The species on Earth are linked in diverse networks of biotic interactions. Interacting species can respond differently to climate change. This can cause spatial or temporal mismatches between interacting species and result in secondary extinctions of species that lose obligate interaction partners. Yet, accounting for biotic interactions in biodiversity projections remains challenging. One way to address this challenge is the use of trait-based approaches because the impact of climate change on interacting species is influenced by species’ functional traits, i.e., measurable characteristics of the species that influence their abiotic and biotic interactions. First, species’ functional traits influence how species respond to climate change. Second, they influence whether the species find compatible interaction partners in reshuffled species assemblages under climate change. Thus, the overarching aim of this dissertation was to explore how trait-based approaches can increase our understanding of how climate change might affect interacting species. For this, I focussed on interactions between fleshy-fruited plants and avian frugivores along a tropical elevational gradient.
I investigated three principal research questions. First, I investigated how traits related to the sensitivity of avian frugivores to climate change and their adaptive capacity vary along elevation and covary across species. I combined estimates of species’ climatic niche breadth (approximating species’ sensitivity) with traits influencing species’ dispersal ability, dietary niche breadth and habitat niche breadth (aspects of species’ adaptive capacity). Species’ climatic niche breadth increased with increasing elevation, while their dispersal ability and dietary niche breadth decreased with increasing elevation. Across species, there was no significant relationship of the sensitivity of the avian frugivores to climate change and their adaptive capacity. The opposing patterns of species’ sensitivity to climate change and their adaptive capacity along elevation imply that species from assemblages at different elevations may respond differently to climate change. The independence between species’ sensitivity and adaptive capacity suggests that it is important to account for both sensitivity and adaptive capacity to fully understand how climate change might affect biodiversity.
Second, I assessed how climate change might influence the co-occurrence of interaction partners with compatible traits, i.e., the functional correspondence of interacting species. I integrated future projections of species’ elevational ranges considering different vertical dispersal scenarios with analyses of the functional diversity of interacting species assemblages. The functional correspondence of fleshy-fruited plants and avian frugivores was lowest if plant and bird species were projected to contract their ranges towards higher elevations in response to increasing temperatures. Contrastingly, if species were projected to expand their ranges upslope, the functional correspondence remained close. The low functional correspondence under a scenario of range contraction indicates that plant species with specific traits might miss compatible interaction partners in future assemblages. This could negatively affect their seed dispersal ability. These results suggest that ensuring the integrity of biotic interactions under climate change requires that species can shift their ranges upslope unlimitedly.
Third, I examined whether avian seed dispersal is sufficient for plants to track future temperature change along the elevational gradient. With a trait-based modelling approach, I simulated seed-dispersal distances avian frugivores can provide to fleshy-fruited woody plant species and quantified the number of long-distance dispersal events the plant species would require to fully track projected temperature shifts along elevation. Most plant species were projected to require several long-distance dispersal events to fully track the projected temperature shifts in time. However, the number of required long-distance dispersal events varied with the degree of trait matching and plant species’ traits. These findings suggest that avian seed dispersal is insufficient for plants to track future temperature change along the elevational gradient as woody plant species might not be able to undergo several consecutive long-distance dispersal events within a short time window, due to their long maturation times. These results also imply that the ability of bird-dispersed plant species to track climate change is associated with the specialization of the seed dispersal system and with plant species’ traits.
Trait-based approaches are promising tools to study impacts of climate change on interacting species. The trait-based approaches that I have developed in this thesis are applicable more widely, e.g., to other types of biotic interactions, or to assess the effects of other drivers of global change. Moreover, these approaches may be further developed to model changes in biotic interactions under global change more dynamically. Taken together, I have shown how a trait-based perspective could help to account for biotic interactions in biodiversity projections. The development of such approaches and the gained knowledge are urgently needed to facilitate the conservation of biodiversity in a rapidly changing world.
Genetic and genomic tools have provided researchers with the opportunity to address fundamental questions regarding the reintroduction of species into their historical range with greater precision than ever before. Reintroduction has been employed as a conservation method to return locally extinct species to their native range for decades. However, it remains unknown how genetic factors may impact population establishment and persistence at the population and metapopulation level in the short- and long-term. Genetic methods are capable of producing datasets from many individuals, even when only low quality DNA can be collected. These methods offer an avenue to investigate unanswered questions in reintroduction biology, which is vital to provide evidence based management strategies for future projects. The Eurasian lynx (Lynx lynx) and European wildcat (Felis silvestris) are elusive carnivores native to Eurasia and have been the subject of multiple reintroduction attempts into their native range. During the 19th and 20th century, the Eurasian lynx was extirpated from West and Central Europe due to increasing habitat fragmentation and persecution. Similarly, the European wildcat was the subject of human persecution, residing in a few refugia in West and Central Europe. After legal protection in the 1950s, subsequent reintroduction projects of both species began in the 1970s and 1980s and continue to the present. Despite this large focus on species conservation, little attention has been given to the consequences these reintroductions have on the genetic composition of the reintroduced populations and if the populations have a chance of persisting in the long term. These species have not yet benefited from the large range of genetic and genomic techniques currently available to non-model organisms, leaving many fundamental aspects of their reintroduction poorly understood. In my dissertation, I investigate demography, population structure, genetic diversity and inbreeding at the population and metapopulation level in both species. In the introduction, which lays the foundation for the subsequent chapters of this PHD, I provide background on reintroduction, its role in conservation and the genetic consequences on populations, especially populations of apex and mesocarnivores. In Publication I, I investigated the reemergence of the European wildcat in a low mountain region in Germany using fine-scale spatial analysis. I found that the reintroduced population has persisted and merged with an expanding natural population. The reintroduced population showed no genetic differentiation from the natural population suggesting there is a good chance this population has retained sufficient genetic diversity despite reintroduction. In Publication II, I tracked population development and genetic diversity over 15 years in a reintroduced lynx population to determine the genetic ramifications on a temporal scale. I found slow genetic erosion after a period of outbreeding, which fits in line with other reintroduced taxa sharing similar demographic histories. I also found the number of genetic founders to be a fraction of the total released individuals, indicating that reintroduced populations of elusive carnivores may have fewer founder individuals than previously thought. In Publication III, I sampled all surviving lynx reintroductions in West and Central Europe as well as 11 natural populations to compare levels of genetic diversity and inbreeding across the species distribution. I found that all reintroduced populations have lower genetic variability and higher inbreeding than natural populations, which urgently requires further translocations to mitigate possible negative consequences. These translocations could stem from other reintroduced populations or from surrounding natural populations. The results contribute to a growing body of evidence indicating that inbreeding is likely to be more prevalent in wild populations than previously understood. Finally, in the discussion I explore how genetic methods can be applied to post-reintroduction monitoring of felid species to illuminate questions relating to genetic composition after release. The methods employed in these studies and in future work will be highly dependent on the research questions posed. Additionally, I investigate the drivers of the observed genetic patterns including founder size, source population, environmental factors, and population growth. I found that genetic diversity loss patterns across these two felid species are not clearly defined, however, management actions can be taken to mitigate the negative effects of reintroductions. These management actions include further translocation, introducing a sufficient number of released individuals and situating reintroductions adjacent to natural populations. All of these actions can minimize genetic drift and inbreeding, two factors which negatively impact small populations. This thesis further supports mounting evidence that genetic considerations should be assessed before releasing individuals, which allows for incorporation of scientific evidence into the planning process thereby increasing the overall success of reintroduction projects. Ultimately, the resources developed during this dissertation provide a solid baseline and foundation for future work regarding the consequences of reintroductions. This is especially important as an increasing number of species are at risk of extinction and reintroductions of both the European wildcat and Eurasian lynx, as well as many others, are planned in the coming years.
Morbus Parkinson (abgekürzt als PD vom Englischen Parkinson’s disease) ist nach Alzheimer die zweithäufigste neurodegenerative Erkrankung. Die Hauptmerkmale sind Rigidität und Bradykinesie, sowie Tremor und posturale Instabilität. Im Gehirn lässt sich bei Parkinsonpatienten post mortem ein Verlust an Neuronen in der Substantia nigra feststellen, was zu den ersten beiden Anzeichen führt. Zudem gibt es intrazelluläre Einschlüsse in den betroffenen Nervenzellen – Lewy-Körperchen genannt – die aus Alpha-Synuklein und anderen Proteinen wie Ubiquitin zusammengesetzt sind. Außerdem ist der Eisenmetabolismus in Gehirnen von Parkinsonpatienten gestört und man findet Eisen-Ablagerungen, vor allem im Mittelhirn. Die Ursachen für PD sind bislang nicht abschließend geklärt. Der Großteil der Fälle ist sporadischer Natur mit unbekannter Ursache und nur bei einem geringen Anteil liegt eine Mutation in einem einzelnen Gen zugrunde. Die häufigsten Mutationen tritt in den Genen für Alpha-Synuklein (SNCA), PINK1 und PARKIN auf.
Die Serin-Threonin-Kinase PINK1 und die E3-Ubiquitin-Protein-Ligase PARKIN sind zwei Proteine, die in Stresssituationen an der Mitochondrien-Außenmembran am Abbau von alten oder nicht richtig funktionierenden Mitochondrien beteiligt sind. Dieser Vorgang nennt sich Mitophagie.
Die dieser Arbeit zugrunde liegenden Publikationen gehen den Zusammenhängen zwischen mitochondrialen Fehlfunktionen und der Pathogenese von PD nach. Da die Krankheit meist erst im hohen Alter auftritt, davon größtenteils ohne direkte Ursache, liegt der Schluss nahe, dass neben genetischen Ursachen auch Umweltfaktoren eine größere Rolle spielen könnten. Um dies näher zu analysieren, wurden experimentell verschiedene Stressoren eingesetzt.
Insgesamt wurden folgende Aspekte untersucht:
I. Welche Auswirkungen hat das Fehlen von PINK1 auf die Zelle? Gibt es einen Biomarker, der mit höherem Alter immer stärker verändert ist?
II. Welchen Einfluss haben Umweltfaktoren wie veränderte Eisen-Exposition auf die Zelle und was verändert sich beim Fehlen von PINK1?
III. Wie können mitochondriale Fehlfunktionen präferentiell das Nervensystem betreffen, wenn es nicht um respiratorische Insuffizienz geht?
Die einzelnen Studien zeigten folgende Ergebnisse:
Torres-Odio/Key et al. 2017 widmete sich der Suche nach molekularen Biomarkern, wodurch PD präsymptomatisch erkannt und die Progression der Erkrankung eingeschätzt werden kann. Die Transkriptom-Analyse der Kleinhirne von Mäusen mit Pink1-/--Mutation in drei verschiedenen Altersstufen zeigte eindrücklich, dass nicht ein einzelner Faktor immer stärker verändert war, sondern, dass immer mehr Faktoren und daher auch eine steigende Zahl an
Signalwegen mit höherem Alter beteiligt waren. Diese Veränderungen betrafen inflammatorische Signalwege, insbesondere Faktoren, die mit der Erkennung und Verarbeitung von zellfremden Nukleinsäuren assoziiert sind. Aufgrund der evolutionären Herkunft von Mitochondrien als frühere Protobakterien haben mitochondriale Nukleinsäuren und Proteine zum Teil bakterielle Ähnlichkeiten, und könnten bei Fehlfunktionen ins Zytosol gelangen. Vor diesem Hintergrund lassen die Ergebnisse der Studie den Schluss zu, dass das angeborene Immunsystem in Neuronen durch eine PINK1-assoziierte mitochondriale Störung aktiviert wird.
In der Publikation Key et al. 2020 wurde Eisen als ein im täglichen Leben vorkommender Stressor eingesetzt und es wurden systematisch Faktoren des Eisenstoffwechsels bei hohen und niedrigen Eisenspiegeln im Zusammenhang mit Parkinson-Mutationen untersucht. Da Eisen für die Gesundheit von Mitochondrien eine große Rolle spielt und Eisen-Chelatoren als Therapie bei PD Patienten bereits diskutiert werden, haben die molekularen Befunde große Relevanz. Die Ergebnisse zeigen, dass unter niedrigen Eisenspiegeln Proteine reduziert waren, die am Nukleotid-Stoffwechsel beteiligt sind, sowie Faktoren, die Eisen-Schwefel-Cluster als Cofaktoren haben und wichtig für die Nukleotid-Qualitätskontrolle sind. Das Fehlen von Eisen führte zu einer Induktion von Pink1 und Prkn, was auf verstärkte Mitophagie hindeutet. Insgesamt konnte gezeigt werden, dass die mitochondriale Eisen-Schwefel-Cluster Biogenese und die post-transkriptionelle Eisenregulation entscheidend für die Pathogenese von PD, bzw. das gesunde Fortbestehen einer Zelle und letztlich auch eines Organismus sind.
In Key et al. 2019 wurde erstmalig das Gesamt-Ubiquitylom aus Gehirnen von gealterten Parkin-knockout (KO) Mäusen erhoben und analysiert, um Ubiquitylierungs-Substrate von PARKIN zu identifizieren. Hierbei zeigte sich eine veränderte Ubiquitylierung von mehreren Faktoren, die an der zellulären Calcium-Homöostase beteiligt sind. Weitere elektrophysiologische Experimente in Gehirnen von gealterten Parkin-/--KO Mäusen ergaben, dass in Nervenzellen im Locus coeruleus die Geschwindigkeit der spontanen Taktgeber erhöht, dass die langsame Nachhyperpolarisation reduziert und, dass die Dauer der Aktionspotentiale erniedrigt war, ohne Veränderung der Kaliumkanal-Ströme.
Insgesamt geht aus den drei Studien hervor, dass mitochondriale Fehlfunktionen bei dauerhaftem Bestehen weitreichende Folgen für die Gesundheit des Nervensystems haben können, denn auch kleine Veränderungen, seien es durch Mutationen oder Umweltfaktoren wie Eisen, können in einer so großen Lebensspanne wie der des Menschen über Krankheit oder Gesundheit entscheiden!
In allen drei Domänen des Lebens ist in der Translation die Initiation der geschwindigkeits-bestimmende Schritt. Die Effizienz der Translationsinitiation und ihre unterschiedliche Regula-tion ist von Translationsinitiationsfaktoren (IFs) abhängig. Bakterien enthalten nur drei IFs, während die Anzahl bei Archaeen (aIFs) und Eukaryoten (eIFs) deutlich höher ist.
Das Archaeon Haloferax volcanii beispielsweise besitzt 14 Gene, die für aIFs bzw. deren Untereinheiten kodieren. Eine Deletionsanalyse ergab, dass fünf aIFs essenziell und neun aIFs nicht essenziell sind. Um einen Einblick in die Funktions- und Interaktionsbereiche der aIFs in H. volcanii zu erhalten, wurden die aIFs mit einem His-Tag versehen und überexpri-miert. Die Überexpression erfolgte in der jeweiligen Deletionsmutante. Für essenzielle aIFs fand sie im Wildtyp statt. Durch Affinitätsaufreinigungen wurden die aIFs und ihre Bindungs-partner isoliert und mittels Massenspektrometrie (MS) identifiziert. Für den Ausschluss unspe-zifischer Proteine dienten zwei stringente Kontrollen als Referenz, das Reportergen Dihydro-folatreduktase (HVO_1279) mit His-Tag und das Expressionsplasmid ohne Gen.
Die ersten Arbeiten konzentrierten sich auf den heterotrimeren Faktor aIF2. Er bindet die Ini-tiator-tRNA und ist damit für die Bildung des Präinitiationskomplexes von zentraler Bedeu-tung. Der Faktor aIF2 besteht aus jeweils einer α-, β- und γ-Untereinheit. In H. volcanii existie-ren zwei Orthologe für aIF2β. Die Überexpressionen der α-, β1-, β2- und γ-Untereinheiten führten zur Co-Isolation der jeweils anderen Untereinheiten des aIF2 (α, β1/ β2, γ).
Die Strategie der Co-Affinitätsaufreinigung und MS wurde auf alle weiteren annotierten aIFs ausgedehnt, um mögliche Funktionen zu identifizieren und ein potenzielles Interaktionsnetz-werk der aIFs zu erstellen. Für alle aIFs konnte ein unterschiedliches Muster an co-gereinigten Proteinen festgestellt werden. Mitgereinigte Proteine waren aIFs, Proteine der Translation, Transkription, Replikation und ribosomale Proteine. Auch RNA-Polymerase-Untereinheiten (RNAPUs) konnten co-isoliert werden. Mit 13 der 14 aIFs konnten andere Ini-tiationsfaktoren co-gereinigt werden. Sechs aIFs konnten zu Beginn bei keinem weiteren Initi-ationsfaktor mitgereinigt werden. Einer dieser Faktoren war aIF2β-1, der jedoch in den Affini-tätsaufreinigungen mit nachfolgender FPLC von aIF2β-2 identifiziert werden konnte. Der Fak-tor aIF1 konnte nur in der stationären Phase von aIF2α mitgereinigt werden.
Die am häufigsten co-gereinigten Proteine waren aIF2Bδ-1 und aIF5B. Für aIF2Bδ-1 kam dies überraschend, da er bereits als Translationsinitiationsfaktor ausgeschlossen wurde. Mit dem Faktor aIF2Bδ-1 selbst konnten fünf aIFs co-gereinigt werden.
Da mit den aIFs auch RNAPUs co-gereinigt werden konnten, wurden sieben RNAPUs ebenfalls mit einem His-Tag versehen und überexprimiert. Auch mit den RNAPUs konnten aIFs, sowie weitere Proteine der Translation mitgereinigt werden.
Diese Umstände legen nahe, dass es möglicherweise eine engere Verbindung der Tran-skription und Translation in H. volcanii geben könnte, als bisher angenommen.
Fatty acids in oomycetes
(2021)
The central dogma of biology is based on the concatenated transfer of information from DNA, via transcribed mRNA, to the translated protein. In eukaryotes, transcription and translation are separated locally as well as temporally by cellular compartmentalization. Prior to active export factor-dependent transport from the nucleus to the cytosol, the newly formed pre-mRNA must mature. This involves 5'capping, splicing, and endonucleolytic cleavage and polyadenylation (CPA).
Transcription of a new pre-mRNA is terminated by hydrolytic cleavage in the 3'-UTR, and the newly formed 3'-end is protected from premature degradation by synthesis of a poly(A) tail. These processes are catalyzed by four multi-protein complexes (CFIm, CFIIm, CPSF, and CsTF) and poly(A) polymerase (PAP). CPA is sequence-specific and dependent on RNA-binding proteins (RBPs). APA-specific sequences include the poly(A) motif ('AAUAAA' and certain motif variants), the UGUA motif, and U/GU-rich sequences upstream and downstream of the poly(A) signal, respectively. About 70% of mammalian genes have more than one polyadenylation site (PAS) and express transcripts of different lengths by a mechanism called alternative polyadenylation (APA). This can affect the length of the 3'UTR (3'UTR-APA) or the coding sequence of the transcript (CDS-APA) if the alternative PAS is upstream of the STOP codon. The length of the 3'UTR affects the stability, export efficiency, subcellular localization, translation rate, and local translation of the nascent transcript. 3'UTR-APA is regulated in the interplay of the cis-elements (poly(A) motif, UGUA and U/GU) and trans-elements (expression of CPA factors). In this context, the functions of the individual cis and trans elements have been extensively studied, yet the regulation of alternative polyadenylation-the decision whether to use the proximal or distal PAS-is less deciphered and requires additional study.
In murine P19 cells, we were able to demonstrate for the first time a direct link between 3'UTR-APA and nuclear export of mature mRNA by the splicing factors SRSF3 and SRSF7 and decipher the mechanism. At the core here is the direct recruitment of the export factor NXF1 by SRSF3 and SRSF7 to transcripts with 3'UTRs of different lengths.
The primary goal of the thesis presented here was to decipher the function of SRSF3 and SRSF7 in the regulation of 3'UTR-APA and to determine the basic mechanism. For this purpose, various genome-wide methods, such as RNA-Seq, MACE-Seq, and iCLIP-Seq, were integrated and the findings were supported by reporter gene and mutation studies.
Initial determination of the poly(A)-tome in P19 cells by MACE-Seq yielded approximately 16,000 PAS and showed that slightly less than 50% of all genes used two or more PAS and expressed alternative 3'UTR isoforms. Further DaPARS analyses after knockdown of Srsf3 or Srsf7 confirmed that SRSF3 affected more transcripts than SRSF7 and led primarily to the expression of long 3'UTRs, whereas SRSF7 promoted the expression of short 3'UTRs. Integration of SRSF3- and SRSF7-specific iCLIP data suggested a possible competition between SRSF3 and SRSF7 at the proximal PAS (pPAS), which could thus act as a hotspot of 3'UTR regulation.
Experiments with intron-free reporter genes revealed that SRSF3- and SRSF7-dependent regulation of 3'UTR-APA is independent of splicing. With respect to SRSF7, a concentration dependence was demonstrated. Mutation experiments involving the SRSF3- and SRSF7-specific binding motifs in the 3'UTR also confirmed the hypothesis of competition between the two SR proteins.
Extensive Co-IP experiments clearly demonstrated that only SRSF7, but not SRSF3, can interact with CFIm and FIP1 (a subunit from the CPSF complex) in an RNA-independent manner. In addition, we showed that these interactions exhibited some phosphorylation dependence, such that the interaction to FIP1 arose primarily in the semi- to hypophosphorylated state of SRSF7. Whereas the interaction to CFIm was mainly detected in the hyperphosphorylated state. The differential affinity between SRSF3 and SRSF7 for polyadenylation factors could be attributed to two SRSF7-specific domains in subsequent mutation experiments: A CCHC-type Zn finger between the RRM and the RS domain, and a hydrophobic 27 amino acid long region in the middle of the RS domain. Together, this suggested that SRSF3 could block the utilization of pPAS, whereas SRSF7 could activate it by directly recruiting polyadenylation factors.
Interestingly, we showed that knockdown of Srsf3 also negatively regulates the expression of Cpsf6 (a subunit of CFIm) through alternative splicing, which subsequently leads to decreased expression of CPSF6 and of CFIm. Reduction of CFIm led to increased expression of transcripts with short 3'UTR, analogous to knockdown of Srsf3. This mirrors the results of previous studies. A direct comparison between SRSF3- and CPSF6-specific transcripts revealed that not all targets were congruent. In addition, we found preliminary evidence for CFIm-related masking of essential cis-pPAS elements by bimodal UGUA motifs at the pPAS. In summary, we present a novel mechanism of indirect 3'UTR-APA regulation through SRSF3-conditional expression of the CFIm subunit CPSF6.
...
Cellular communication is a concept that can be explained as the transfer of signals or material (such as cytokines, ions, small molecules) between cells from the same or different type, across either short or long distances. Once this signal or material is received, it will, as a rule, promote a functional effect. Several routes, involved in this transfer, are well described and are of global importance for organ/tissue communication in an organism.
The brain interacts dynamically with the immune system, and the main route known to mediate this communication, is via the release of cytokines (by peripheral blood cells), which can then activate certain brain cell types, such as microglia, directly, or activate the vagus nerve transferring signals to neuronal populations in the brain. The communication between these two systems plays a key role in the pathophysiology of neurodegenerative diseases, and the mechanisms involved in this interaction are of central importance for understanding disease initiation and progression and search for therapeutic models.
The Momma lab previously addressed the mechanisms of interaction between the peripheral immune system and the brain by investigating cellular fusion of haematopoietic cells with neurons after inflammation. They addressed the question of whether this phenomenon also occurs under non-invasive conditions. To approach this problem, a genetic tracing model that relies on the Cre-Lox recombination system was used. Transgenic mice expressing Cre recombinase specifically in the haematopoietic lineage were crossed into a Cre-reporter background, thus all haematopoietic cells irreversibly express the reporter marker-gene EYFP. Using this model, EYFP was detected in non-haematopoietic tissues, suggesting the existence of a communication mechanism never described before. As cells containing two nuclei were never detected, fusion as a mechanism was excluded, suggesting that Cre reaches non-haematopoietic cells via a different signalling pathway. The Momma lab investigated whether the transfer of material through extracellular vesicles (EVs) could be behind this periphery-to-brain communication. Using the genetic mouse model, they were able to trace the transfer of Cre RNA via EVs between cells in vivo, generating the first in vivo evidence of functional RNA transfer by EVs between blood and brain.
The last decade has witnessed a rapid expansion of the field of EVs. Initially considered as waste disposal material, recent evidence has challenged this view. EVs are currently considered as a widespread intercellular communication system that can transport and transfer all types of biomolecules, from nucleic acids to lipids and proteins. However, several important questions are still under investigation. One of them is whether EVs are involved in brain pathophysiology, as inflammation plays an important role in onset and progression of neurodegenerative diseases and is well described in Parkinson Disease (PD). Based on preliminary data in a mouse, peripherally injected with a low dose of Lipopolysaccharide (LPS, an endotoxin found in the outer-membrane of Gram-negative bacteria, which causes an immune response), neurons and other cell population in the brain take up EVs from the periphery. Particularly, dopaminergic neurons from Substantia Nigra and Ventral Tegmental Area have been shown to receive functional RNA, transported through EVs, which can lead up to 20% of recombination. Furthermore, different neuronal populations from Hippocampus, Cortex and Cerebellum exhibit recombination, indicating a widespread signalling from blood to the brain. Therefore, the goal of my PhD thesis was to investigate the mechanisms of this transfer and the triggers that lead to EV uptake by neural cells in vivo both in pathological and physiological conditions.
In this project, the extent and function of EV-mediated signalling from blood to brain is explored in the context of peripheral inflammation and neurodegenerative diseases. Firstly, EVs isolated from WT mice were further characterized using size-exclusion chromatography (SEC), Western Blot (WB) and electron microscopy in order to extend the knowledge from previous work done in the Momma lab. Secondly, to expand on the biological relevance of the fact that inflammation is correlated with an increase in EV uptake, different approaches using the genetic murine tracing model were used. Recombination events from haematopoietic cells to the brain have been followed after peripheral injection of LPS. Peripheral inflammation caused by LPS injection led to widespread recombination events in the brain, specifically in microglia and neurons, including dopaminergic (DA) neurons. In contrast, astrocytes, oligodendrocytes and endothelial cells were never or very rarely recombined. Additionally, peripheral LPS injection in a murine model, where Cre is expressed only in erythrocytes, led to recombination events only in microglia, suggesting that the type of EV-secreting cell plays a role in the targeting of EVs to a specific cell population.
Der Verzehr von radioaktiv belasteten Pilzfruchtkörpern stellt ein Gesundheitsrisiko für den Menschen dar und auch fast 35 Jahre nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986 sind Pilze aus Waldökosystemen zum Teil noch stark durch das ausgetretene radioaktive 137Cs belastet. Die Einschätzung der Belastung und somit des Gesundheitsrisikos ist aufgrund einer Vielzahl von Einflussfaktoren, wie z. B. der Pilzart, der Tiefe des Myzels, der Bodenkontamination und der Feuchtigkeit des Bodens, schwierig. Ziel dieser Arbeit war es die Variabilität, den Einfluss verschiedener Faktoren sowie die effektive Halbwertszeit der 137Cs-Aktivität in Pilzfruchtkörpern zu ermitteln. Des Weiteren wurde überprüft, ob die Bodenkontamination für eine Abschätzung der 137Cs-Aktivität von Pilzfruchtkörpern herangezogen werden kann. Für die Untersuchungen wurden über mehrere Jahre Proben von Maronenröhrlingen (Imleria badia) und Steinpilzen (Boletus edulis) aus vier Waldgebieten in Mittel- und Süddeutschland mit unterschiedlichem Aktivitätseintrag nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986 analysiert. Die Gebiete waren Eichenzell, Wülfersreuth, Oberschönenfeld und der Nationalpark Bayerischer Wald. Als Ergänzung dienten zugesendete Proben derselben Pilzarten von Mitgliedern aus Pilzvereinen aus ganz Deutschland. Zusätzlich zu den Pilzproben wurden Bodenproben gemessen, um zum einen die aktuelle Bodenkontamination zu bestimmen und zum anderen zu überprüfen, ob der Großteil des 137Cs weiterhin im Bereich des Pilzmyzels zu finden ist.
Für die Untersuchung der örtlichen Variabilität der 137Cs-Aktivität wurden Maronenröhrlinge (Imleria badia) aus dem Waldgebiet Eichenzell in den Jahren 2017 bis 2019 analysiert. Innerhalb eines Sammeltages variierten die Messwerte verschiedener Proben innerhalb des Waldgebietes teilweise um den Faktor sechs. Dabei ist die Variabilität innerhalb eines Teilgebietes größer als zwischen beiden Teilgebieten des Waldgebietes Eichenzell. Für ein repräsentatives Ergebnis eines Gebietes ist es aufgrund der Variabilität erforderlich, eine ausreichende Menge an Fruchtkörpern zu analysieren.
Um die effektive Halbwertszeit der 137Cs-Aktivität in Maronenröhrlingen (Imleria badia) zu ermitteln, wurden Proben aus drei Waldgebieten über fünf bis neun Jahre analysiert. Die Wahl der drei Waldgebiete erfolgte anhand des 137Cs-Aktivitätseintrags nach der Reaktorkatastrophe von Tschernobyl im Jahr 1986. Die Bodenkontaminationswerte variieren von 3.000 Bq/m² in Eichenzell über 12.500 Bq/m² in Wülfersreuth bis 35.000 Bq/m² in Oberschönenfeld. Die effektiven Halbwerts-zeiten liegen in einem engen Bereich von 5,2 bis 5,8 Jahre mit einem Mittelwert von 5,4 ± 0,3 Jahren. Damit reduziert sich die radioaktive Belastung der Pilzfruchtkörper in etwa fünfmal schneller als durch die rein physikalische Halbwertszeit des 137Cs von 30,08 Jahren. Durch die Hinzunahme von bereits im Jahr 1990 veröffentlichten Daten ergab sich eine längere effektive Halbwertszeit von 7,7 ± 0,6 Jahren.
Für die Untersuchung der zwei Einflussfaktoren Exposition des Sammelgebiets (Hangausrichtung nach Ost oder West) und Höhenlage wurden sowohl Maronenröhrlinge (Imleria badia) als auch Steinpilze (Boletus edulis) hinsichtlich der 137Cs-Aktivität gemessen, um die Auswirkung auf Pilzarten mit unterschiedlichem Akkumulationsvermögen zu analysieren. Als Untersuchungsgebiet diente der Nationalpark Bayerischer Wald, da dieser ein großes Gebiet umfasst und verschiedene Ausprägungen der beiden Faktoren abbildet. Zudem wurde das Gebiet in Folge der Reaktorkatastrophe von Tschernobyl stark kontaminiert und der Park ist ein beliebtes Pilzsammelgebiet. Anhand der 137Cs-Aktivität von Bodenproben konnte das Gebiet in zwei Regionen (Cluster) eingeteilt werden: eine Region mit hohem und eine mit niedrigem Aktivitätseintrag. Im Vergleich wiesen Maronenröhrlinge (Imleria badia) durchschnittlich eine um den Faktor fünf höhere 137Cs-Aktivität als Steinpilze (Boletus edulis) auf. Der Faktor Höhenlage zeigte im Gegensatz zur Exposition einen Einfluss auf die Kontamination der Pilzfruchtkörper. In Bezug auf die Höhenlage war der Einfluss nur im Falle eines hohen Aktivitätseintrags signifikant, wobei die Pilzproben aus der niedrigsten Höhenlage am höchsten belastet waren.
Zur Ermittlung der vertikalen Verteilung des 137Cs im Boden wurden in den Waldgebieten Eichenzell und Nationalpark Bayerischer Wald Proben bis zu einer Tiefe von 24 cm entnommen und anschließend in 2 cm Schichten analysiert. Alle Verteilungen konnten mit einem Gauß-Fit oder einem multiplen Gauß-Fit mit 2 bis 3 Maxima abgebildet werden. Das erste Maximum lag in allen Fällen in den organischen Horizonten oder im Übergangsbereich zum Ah-Horizont. Folglich befindet sich der Großteil des 137Cs fast 35 Jahre nach der Reaktorkatastrophe von Tschernobyl immer noch im Bereich des Pilzmyzels und kann somit von den Pilzen aufgenommen und in den Fruchtkörpern angereichert werden.
Der Vergleich der 137Cs-Aktivität der Pilz- und Bodenproben aus dem Nationalpark Bayerischer Wald ergab sowohl für Maronenröhrlinge (Imleria badia) als auch für Steinpilze (Boletus edulis) eine positive Korrelation. Nach Unterteilung der Proben anhand der Höhenlage zeigte sich eine noch stärkere Korrelation. Dies zeigt, dass neben der Bodenkontamination auch die Höhenlage einen Einfluss auf die 137Cs-Aktivität der Fruchtkörper hat.
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Despite constant progress in basic and translational research, cancer is still one of the leading cause of death. In particular, tumors of the central nervous system (CNS) are usually associated with dismal prognosis. Although about 100 distinct subtypes of primary CNS tumors have been classified molecularly, metastases derived from primaries outside the CNS (= brain metastases, BrM) are more frequently observed across brain tumor patients. It is estimated that approximately 20 - 40 % of all cancer patients will develop BrM during their course of disease, and basically every tumor type is able to metastasize to the brain. Nevertheless, BrM are most frequently derived from primaries of the lung, breast, and skin (melanoma). Treatment options for patients with BrM are very limited, and standard of care therapies include surgery, ionizing radiation (e.g. whole brain radio-therapy, WBRT), and some systemic and immuno-therapeutic approaches.
The brain represents a unique organ, which in part is due to the presence of the blood-brain barrier, a unit of the neuro-vascular interface ensuring tightly regulated exchange of nutrients, molecules, and cells. Furthermore, apart from microglia the brain parenchyma does not harbor other immune cells. Those cells however can be found at the borders of the CNS residing in the meninges, for instance. Based on recent insight on the immune landscape in the CNS, a paradigm shift occurred after which the brain is no longer regarded as immune-privileged but rather immune distinct. The phenomenon of immune cell infiltration has been described before in the context of neurological disorders including Multiple Sclerosis, as well as in brain tumors.
Since the development of immune-therapeutic approaches for tumors outside the CNS that aim to evoke sustainable anti-tumor effects, it became increasingly interesting to understand and harness the immune landscape (= tumor microenvironment, TME) of brain tumors, as well. Interestingly, most of the knowledge about the TME is based on studies of primary brain tumors. However, it is known that BrM compared to primary brain tumors induce a different TME like e.g. the recruitment of much more lymphocytes, which is one of the reasons primary brain tumors are considered immunologically “cold” and poorly respond to immuno-therapies. Previous insight into the functional contribution of tumor-associated cells in BrM progression revealed for example that brain-resident cell types (e.g. astrocytes or microglia) promote BrM development and outgrowth. However, until recently a comprehensive view on the cellular composition and functional role of the brain metastases-associated TME was missing and little was known how it changes during tumor progression or standard therapy.
Hence, within this thesis it was sought to describe novel aspects of the TME of preclinical BrM models, which include two xenograft and one syngeneic mouse model. BrM was induced via intra-cardiac injection of tumor cells with a high brain tropism. Both xenograft models were based on immuno-compromised nude mice (Balb/c nude) and included the melanoma-to-brain (M2B) model H1_DL2, and the lung-to-brain (L2B) model H2030. In addition the breast-to-brain model 99LN-BrM was used in wild-type mice (BL6), and therefore represented an immuno-competent, syngeneic model. First BrMs could be detected in the xenograft models at 3 weeks after injection, whereas first 99LN BrMs were detected at 5 weeks. BrM development and progression were monitored by bioluminescence imaging once per week in the xenograft models. Tumor progression in the 99LN model was examined by magnetic resonance imaging. Based on the measurement methods, and for further histologic and cytometric experiments, mice were stratified into groups with small or large BrMs, respectively. Some initial immuno-stainings confirmed previous findings, showing that brain-resident cells like astrocytes and microglia become activated in the presence of tumor cells, whereas neurons for example rather give the impression of passive bystanders. Importantly, an accumulation of IBA1+ cells was observed during BrM progression. IBA1 is a pan-macrophage marker that stains all tumor-associated macrophages (TAMs). However previous work suggested that the TAM population consists of at least two main subpopulations in BrM as well: the resident-infiltrating microglia (MG, TAM-MG), as well as the peripheral and monocytic-derived macrophages (TAM-MDM). Since both cell types within the tumor share morphological traits, and due to the lack of markers to distinguish them, an exact discrimination of both cell types was complicated in the past. Recently, an integrative lineage-tracing-based study identified the integrin CD49d as MDM-specific in the context of brain tumor-associated myeloid cells, hence enabling a reliable dissection of both TAM populations in e.g. flow cytometric experiments.
One of the main aims of this thesis was to dissect the myeloid TME in the three different BrM models during tumor progression. Using a 5-marker flow cytometry (FCM) (CD45/CD11b/Ly6C/Ly6G/CD49d) approach, the following cell populations were examined in more detail: granulocytes, inflammatory monocytes, MDM, and MG.
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Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
(2021)
Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation.
The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation.
In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities.
The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD.
The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A).
The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted.
The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend.
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Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets.
First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery.
In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes.
In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.
Die Physiologie des Schmerzes umfasst komplexe immunologische, sensorische und inflammatorische Prozesse im Rückenmark, im Gehirn und in der Peripherie. Wiederholte nozizeptive Stimulation induziert pathophysiologische Veränderungen bei der Schmerzweiterleitung, aus denen eine periphere oder zentrale Sensibilisierung resultiert. Diese kann bei dafür anfälligen Patienten zu der Ausbildung von chronischen Schmerzzuständen führen. Obwohl das Wissen über die genauen molekularen Vorgänge der Schmerz-Chronifizierung noch immer unvollständig ist, sind die Identifizierung von Risikofaktoren vernünftige Schritte, um die individuelle Anfälligkeit für die Entwicklung chronischer Schmerzen zu bestimmen. Das Hauptziel dieser Doktorarbeit bestand daher in der Identifikation humaner genetischer Biomarker für chronische Schmerzzustände.
Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative.
Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain.
The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated.
A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts.
In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts.
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The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change.
Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid.
Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong
inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In
addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants.
The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However,
combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared.
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Exploring the in vivo subthreshold membrane activity of phasic firing in midbrain dopamine neurons
(2021)
Dopamine is a key neurotransmitter that serves several essential functions in daily behaviors such as locomotion, motivation, stimulus coding, and learning. Disrupted dopamine circuits can result in altered functions of these behaviors which can lead to motor and psychiatric symptoms and diseases. In the central nervous system, dopamine is primarily released by dopamine neurons located in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) within the midbrain, where they signal behaviorally-relevant information to downstream structures by altering their firing patterns. Their “pacemaker” firing maintains baseline dopamine levels at projection sites, whereas phasic “burst” firing transiently elevates dopamine concentrations. Firing activity of dopamine neurons projecting to different brain regions controls the activation of distinct dopamine pathways and circuits. Therefore, characterization of how distinct firing patterns are generated in dopamine neuron populations will be necessary to further advance our understanding of dopamine circuits that encode environmental information and facilitate a behavior.
However, there is currently a large gap in the knowledge of biophysical mechanisms of phasic firing in dopamine neurons, as spontaneous burst firing is only observed in the intact brain, where access to intrinsic neuronal activity remains a challenge. So far, a series of highly-influential studies published in the 1980s by Grace and Bunney is the only available source of information on the intrinsic activity of midbrain dopamine neurons in vivo, in which sharp electrodes were used to penetrate dopamine neurons to record their intracellular activity. A novel approach is thus needed to fill in the gap. In vivo whole-cell patch-clamp method is a tool that enables access to a neuron’s intrinsic activity and subthreshold membrane potential dynamics in the intact brain. It has been used to record from neurons in superficial brain regions such as the cortex and hippocampus, and more recently in deeper regions such as the amygdala and brainstem, but has not yet been performed on midbrain dopamine neurons. Thus, the deep brain in vivo patch-clamp recording method was established in the lab in an attempt to investigate the subthreshold membrane potential dynamics of tonic and phasic firing in dopamine neurons in vivo.
The use of this method allowed the first in-depth examination of burst firing and its subthreshold membrane potential activity of in vivo midbrain dopamine neurons, which illuminated that firing activity and subthreshold membrane activity of dopamine neurons are very closely related. Furthermore, systematic characterization of subthreshold membrane patterns revealed that tonic and phasic firing patterns of in vivo dopamine neurons can be classified based on three distinct subthreshold membrane signatures: 1) tonic firing, characterized by stable, non-fluctuating subthreshold membrane potentials; 2) rebound bursting, characterized by prominent hyperpolarizations that initiate bursting; and 3) plateau bursting, characterized by transient, depolarized plateaus on which bursting terminates. The results thus demonstrated that different types of phasic firing are driven by distinct patterns of subthreshold membrane activity, which may potentially signal distinct types of information. Taken together, the deep brain in vivo patch-clamp technique can be used for the investigation of firing mechanisms of dopamine neurons in the intact brain and will help address open questions in the dopamine field, particularly regarding the biophysical mechanisms of burst firing in dopamine neurons that control behavior.
Plastics contain a complex mixture of chemicals including polymers, additives, starting substances and side-products of processing. These plastic chemicals are prone to leach into the packaged goods, in the case of food contact materials (FCMs), or into the natural environment, in the case of plastic debris. Thus, plastics represent an exposure source of chemicals for humans and wildlife alike. While it is widely known that individual plastic chemicals, such as bisphenol A and phthalates, are hazardous, little is known on the overall chemical composition and toxicity of plastics. When fragmented into smaller particles, referred to as microplastics (< 5 mm), the plastic itself can be ingested by many species. It is well established that microplastic ingestion can have negative consequences for a wide range of organisms including invertebrates, but the contribution of plastic chemicals to the toxicity of microplastics is unclear.
Given the above, the present thesis aimed at a comprehensive toxicological, ecotoxicological and chemical characterization of everyday plastics. For a comparative evaluation, 77 plastic products were selected covering 16 material types (e.g., polyethylene) made from petroleum or renewable feedstocks. These products included biodegradable products, FCMs and non-FCMs, as well as raw materials and final products, respectively. In the first two studies, the chemical mixtures contained in the 77 products were extracted with methanol and extracts were analyzed in a set of four in vitro bioassays and by non-target high-resolution gas or liquid chromatography mass spectrometry. Since an exposure only occurs if chemicals actually leach under realistic conditions, in a third study migration experiments with water were conducted for 24 out of the 77 products. The aqueous migrates were assessed in the same way as the methanolic extracts. In addition, the freshwater invertebrate Daphnia magna was exposed chronically to microplastics made of polyvinylchloride (PVC), polyurethane (PUR) and polylactic acid (PLA) to investigate the contribution of chemicals in microplastic toxicity, in a fourth study.
The experimental findings demonstrate that a wide variety of chemicals is present in plastics. A single plastic product can contain up to several thousand chemical features, most of which unique to that product and at the same time unknown. The results also indicate that the majority of these chemical mixtures are toxic in vitro. Accordingly, 65% of the plastic extracts induced baseline toxicity and 42% an oxidative stress response, while 25% had an antiandrogenic and 6% an estrogenic activity. This implies that chemicals causing unspecific toxicity are more prevalent in plastics than such with endocrine effects. These chemicals can also leach from plastics under realistic conditions. Between 17 and 8936 chemical features were detected in a single migrate sample and all 24 tested migrates induced in vitro toxicity. This means that humans and wildlife can actually be exposed to toxic plastic chemicals under realistic conditions. Generally, each product has its individual toxicological and chemical fingerprint. Thus, neither material type, feedstock, biodegradability nor the food contact suitability of a product can serve as a predictor for the toxicity, the chemical composition or complexity of a product. Likewise, this means that bio-based and biodegradable materials are not superior to their petroleum-based counterparts from a toxicological perspective despite being promoted as sustainable alternatives to conventional plastics.
Moreover, the present thesis demonstrates that plastic chemicals can be the main driver for microplastic toxicity. Irregular microplastics made of PVC, PUR and PLA adversely affected life-history traits of D. magna in a polymer type- and endpoint-dependent manner at concentrations between 100 and 500 mg L-1 and with a higher efficiency than natural kaolin particles. While the toxicity of PVC was triggered by the chemicals used in the material, the effects of PUR and PLA were induced by the physical properties of the particle.
In addition, in the fifth study, results and observations made during this thesis were integrated inter- and transdisciplinarily with the perspectives of a social scientist and a product manufacturer. This elucidated that knowledge on plastic ingredients is often concealed, is lacking or not applicable in practice. These intransparencies hinder the safety evaluation of plastic products as well as the choice and sale of the least toxic packaging material.
Overall, the present thesis highlights that the chemical safety of plastics and their bio-based and biodegradable alternatives is currently not ensured. Thus, chemicals require more consideration in the toxicity and risk assessment of plastics and microplastics. Product-specific and complex chemical compositions, including unknown compounds, pose a challenge here. Two essential steps towards non-toxic products are to increase transparency along the product life cycle and to reduce the chemical complexity of plastics by communication and regulation. The results of the present thesis indicate that products exist which do not contain toxic chemicals. These can serve to direct the design of safer plastics. Since toxicity and chemical complexity seem to increase with processing, the integration of toxicity testing during the production steps would further support the safe and sustainable production and use of plastic products.
Sympathetic influences on articular cartilage regeneration capacity and osteoarthritis manifestation
(2021)
The pathogenesis of osteoarthritis (OA) involves articular cartilage, synovial tissue and subchondral bone and is therefore a disease of the whole joint. OA is characterized by progressive degradation of cartilage, synovial inflammation, osteophyte formation and subchondral bone sclerosis. Cartilage-surrounding tissues are innervated by tyrosine hydroxylase (TH)-positive sympathetic nerve fibers with the most important sympathetic neurotransmitter norepinephrine (NE) detected in the synovial fluid of OA patients. Furthermore, adrenergic receptors are expressed in different knee joint tissues. Most in vitro studies indicate a potential role of the β2-adrenergic receptor, which has been not investigated during OA pathogenesis in vivo. The role of the sympathetic nervous system (SNS) in OA progression has not yet been studied. Therefore, the objective of this study was to analyze how the SNS and NE influence the MSC dependent cartilage regeneration in vitro and the OA pathogenesis and manifestation in vivo.
In the first part of this study, the effect of NE on the chondrogenesis of sASC, which are known to play an important role in cartilage regeneration was analyzed in vitro. In the second part of this study, the role of the SNS was studied in vivo in mice that were sympathectomized chemically followed by surgically induced OA. The specific focus was on the β2-adrenergic receptor effects on OA pathogenesis, which were analyzed in β2-adrenergic receptor-deficient mice.
The in vitro experiments have shown that NE reduced the chondrogenic potential of sASCs by decreasing the expression of type II collagen and sGAG. NE mediated these effects mainly by the α2-AR signalling. Furthermore, NE treatment led to activation of the ERK1/2 signal pathway. These findings suggested that the sympathetic neurotransmitter NE might suppress the chondrogenic capacity of MSC and their dependent cartilage regeneration and may also play a role in OA progression and manifestation.
The in vivo study has shown that sympathectomy reduced synovial TH-positive nerve fibers in the synovium and the NE concentration in the spleen significantly. In WT mice, DMM leads to increased NE concentrations in the spleen compared to sham mice indicating an increased SNS activity after mechanical stress or inflammation due to DMM. Sympathectomy leads to less pronounced cartilage degeneration (OARSI score) after DMM compared to DMM in WT mice. Furthermore, the release of the type II collagen degradation fragment CTX-II was abolished in Syx DMM mice compared to WT DMM mice, suggesting that less SNS activity due to sympathectomy reduced the cartilage degeneration during OA pathogenesis. Similarly, sympathectomy decreased the synovitis score significantly after DMM compared to DMM in
WT mice. Synovitis in WT mice was accompanied by increased MMP-13 expression in the synovium after DMM, compared to Syx mice. Cartilage degeneration seemed to be driven mainly by the increased synovial inflammation accompanied by an increased MMP13 expression in synoviocytes and not in chondrocytes. The pathological changes in synovium and cartilage might also be linked to each other, as indicated by the moderate correlation between the synovial inflammation (synovitis score) and cartilage degeneration (OARSI score). Subchondral bone volume as well the thickness of the subchondral bone plate (SCBP) and calcified cartilage (CC) were increased in Syx mice compared to WT after DMM. The data on DMM induction in β2-AR deficient mice revealed that the β2-AR signaling is involved in cartilage degeneration and the aggravated subchondral bone changes as these mice had less pronounced cartilage degeneration compared to WT mice. While the cartilage degeneration was similar, the subchondral bone changes were more pronounced in β2-AR deficient mice compared to the Syx mice.
Overall, the SNS had differential effects in cartilage, synovium and subchondral bone. A reduced SNS activity by sympathectomy attenuated cartilage degeneration and synovitis but aggravated the OA specific subchondral bone changes. These findings provide new insights into the development of novel therapeutic strategies for OA by targeting the SNS in a tissue- specific manner.
A large number of chemicals are constantly introduced to surface water from anthropogenic and natural sources. Although substantial efforts have been made to identify these chemicals (e.g potentially anthropogenic contaminants) in surface waters using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), a large number of LC-HRMS chemical signals often with high peak intensity are left unidentified. In addition to synthetic chemicals and transformation products, these signals may also represent plant secondary metabolites (PSMs) released from vegetation through various pathways such as leaching, surface run-off and rain sewers or input of litter from vegetation. While this may be considered as a confounding factor in screening of water contaminants, it could also contribute to the cumulative toxic risk of water contamination. However, it is hardly known to what extent these metabolites contribute to the chemical mixture of surface waters. Thus, reducing the number of unknowns in water samples by identifying also PSMs in significant concentrations in surface waters will help to improve monitoring and assessment of water quality potentially impacted by complex mixtures of natural and synthetic compounds. Therefore, the main focus of the present study was to identify the occurrence of PSMs in river waters and explore the link between the presence of vegetation along rivers and detection of their corresponding PSMs in river
water.
In order to achieve the goals of the present thesis, two chemical screening approaches, namely, non-target and target screening using LC-HRMS were implemented. (1) Non-target analysis involving a novel approach has been applied to associate unknown peaks of high intensity in LC-HRMS to PSMs from surrounding vegetation by focusing on peaks overlapping between river water and aqueous plant extracts (Annex A1). (2) LC–HRMS target screening in river waters were performed for about 160 PSMs, which were selected from a large phytotoxin database (Annex A2 and A3) considering their expected abundance in the vegetation, their potential mobility, persistence and toxicity in the water cycle and commercial availability of standards.
In non-target screening (Annex A1), a high number of overlapping peaks has been found in between aqueous plant extracts and water from adjacent location, suggesting a significant impact of vegetation on chemical mixtures detectable in river waters. The chemical structures were assigned for 12 pairs of peaks while several pairs of peaks
whose MS/MS spectra matched but no structure suggestion were made by the implemented software tools for retrieving possible chemical structure. Nevertheless, the pairs of peaks with matching spectra represented the same chemical structure. The identified compound belonged to different compound classes such as coumarins, flavonoids besides others. For the identified PSMs individual concentration up to 5 µg/L were measured. The concentration and the number of detected PSMs per sample were correlated with the rain event and vegetation coverage.
Target screening unraveled the occurrence of 33 out of 160 target compounds in river waters (Annex A2 and A3). The identified compounds belonged to different classes such as alkaloids, coumarins, flavonoids, and other compounds. Individual compound concentrations were up to several thousand ng/L with the toxic alkaloids narciclasine and
lycorine recording highest maximum concentrations. The neurotoxic alkaloid coniine from poison hemlock was detected at concentrations up to 0.4 µg/L while simple coumarins
esculetin and fraxidin occurred at concentrations above 1 µg/L. The occurrence of some PSMs in river water were correlated to the specific vegetation growing along the rivers while the others were linked to a wide range of vegetation. As an example, narciclasine and lycorine was emitted by the dominant plant species from Amaryllidaceae family (e.g. Galanthus nivalis (snow drop), Leucojum vernum and Anemone nemorosa) while intermedine and echimidine were from Symphytum officinale. The ubiquitous occurrence of simple coumarins fraxidin, scopoletin and aesculetin could be linked to their presence in a wide range of vegetation.
Due to lack of aquatic toxicity data for the identified PSMs (in both target and non-target) and extremely scarce exposure data, no reliable risk assessment was possible.
Alternatively, risk estimation was performed using the threshold for toxicological concern (TTC) concept developed for drinking water contaminants. Many of the identified PSMs
exceeded the TTC value (0.1 µg/L) thus caution should be taken when using such surface waters for drinking water abstraction or recreational use.
This thesis provides an overview of the occurrence of PSMs in river water impacted by the massive presence of vegetation. Concentration for many of the identified PSMs are well within the range of those of synthetic environmental contaminants. Thus, this study adds to a series of recent results suggesting that possibly toxic PSMs occur in relevant concentrations in European surface waters and should be considered in monitoring and risk assessment of water resources. Aquatic toxicity data for PSMs are extensively lacking but are required to include these compounds in the assessment of risks to aquatic organisms and for eliminating risks to human health during drinking water production.
In der vorliegenden Arbeit wurde das Zinkfinger-µ-Protein HVO_2753 des halophilen Archaeons Haloferax volcanii hinsichtlich seiner biologischen Funktion und seiner Struktur charakterisiert.
Zinkfinger-µ-Proteine wurden bisher nur sehr wenig untersucht, während ihnen jedoch in den letzten Jahren steigendes Interesse entgegengebracht wird. Im Genom von H. volcanii sind mehr als 40 solcher Zinkfinger-µ-Proteine codiert. Von diesen besitzt mit HVO_2753 lediglich eines nicht nur zwei, sondern vier der charakteristischen C(P)XCG-Muster, was für die Anwesenheit von zwei Zinkfinger-Motiven spricht. Während Homologe von HVO_2753 in vielen Euryachaeota vorkommen und manche davon als Zink-Ribbon RNA-Bindeproteine annotiert sind, ist über ihre Funktion jedoch nichts bekannt. Zur Charakterisierung des Proteins wurde zunächst eine in frame-Deletionsmutante seines Gens erstellt und diese einer phänotypischen Charakterisierung unterzogen. Die Mutante wies, verglichen mit dem Wildtyp, keine Unterschiede im Wachstum in Komplexmedium oder in synthetischem Medium mit Glukose als Kohlenstoffquelle auf. Ein schweres Defizit konnte jedoch sowohl bei der Adhäsion und Biofilmbildung als auch der Schwärmfähigkeit der Deletionsmutante festgestellt werden. Während die Schwärmfähigkeit des Wildtyps durch plasmidische Expression von HVO_2753 in der Deletionsmutante teilweise wiederhergestellt werden konnte, war eine solche Komplementation bei der Biofilmbildung nicht möglich. Die Analyse der Relevanz ausgewählter Aminosäuren, wie beispielsweise das jeweils erste Cystein in jedem C(P)XCG-Muster zeigte, dass die Substitution jeder einzelnen der getesteten Aminosäuren einen Funktionsverlust des Proteins nach sich zieht. Die Untersuchung des HVO_2753-Transkripts mittels Northern Blot-Analyse bestätigte erste Hinweise aus vorangegangenen dRNA- und RNA-Seq-Studien, die eine Co-Transkription von HVO_2753 mit dem Nachbargen HVO_2752, das für den Translations-Elongationsfaktor aEF-1 beta codiert, aufzeigten. Daraufhin erfolgte eine Untersuchung des Ribosomenprofils, bei der keine Unterschiede zwischen der Deletionsmutante und der Überexpressionsmutante von HVO_2753 festgestellt werden konnten.
Eine Variante von HVO_2753 mit N-terminalem Hexahistidin-Tag wurde homolog überproduziert und aufgereinigt. Die Überproduktion und Aufreinigung wurden im Zuge dieser Arbeit weiter, speziell für HVO_2753, optimiert. So konnten große Mengen von HVO_2753n überproduziert und bei nativen Salzbedingungen mittels Nickel-Affinitätschromatographie und anschließender Größenausschlusschromatographie aufgereinigt werden. Eine massenspektrometrische Analyse bestätigte sowohl das Molekulargewicht als auch die Abwesenheit posttranslationaler Modifikationen. Die Untersuchung der Menge an gebundenem Zink im Protein erfolgte beim Zink-Assay mit Hilfe des hochsensitiven und hochspezifischen Fluorophors ZnAF-2F. Dabei konnte gezeigt werden, dass überraschenderweise lediglich ein Zink-Ion in HVO_2753 gebunden vorliegt.
Zur weiteren Funktionsaufklärung erfolgte eine Interaktionspartnersuche. Hierfür wurde HVO_2753 überproduziert, ein in vivo-Crosslink und anschließend eine native Aufreinung durchgeführt. Die massenspektrometrische Analyse ausgewählter Fraktionen nach der Größenausschlusschromatographie ergaben eine Vielzahl an möglichen Bindepartnern. Besonders häufig wurde hier die GalE family Epimerase/Dehydratase gefunden. Eine weitere Methode zur Suche nach Interaktionspartnern richtete sich auf RNAs. Hier konnten mittels eines eigens entwickelten Protokolls neben RNAs des Translationsapparates auch mehrfach die tRNA(Glu) gefunden werden.
Zusätzlich sollte die Transkriptomanalyse mittels RNA-Sequenzierung Unterschiede zwischen Wildtyp, Deletionsmutante und Komplementationsmutante aufzeigen. Hier wurden weitreichende Auswirkungen der Deletion von HVO_2753 gefunden. Zahlreiche Gene in mehreren Operons zur Motilität und Chemotaxis lagen in der Deletionsmutante stark herunterreguliert vor, während die Gene einiger Metallionen-Transporter und der Eisen(III)-Siderophor-Biosynthese hochreguliert vorlagen. In der Komplementationsmutante konnten nur von den letzteren Genen Transkriptlevel vergleichbar mit denen des Wildtyps wiedergefunden werden.
In dieser Arbeit konnte gezeigt werden, dass das kleine Zinkfinger-Protein HVO_2753 eine essenzielle Rolle in der positiven Regulation der Motilität, Chemotaxis und der Adhäsion bzw. Biofilmbildung spielt. Gleichzeitig übt HVO_2753 eine negative Regulation auf den Metallionen-Transport und die Biosynthese des Eisen(III)-Siderophors aus.