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Cancer is one of the leading causes of death worldwide, with currently assessed chances to develop at least one cancer in a lifetime for about 20%. High cases rates and mortality require the development of new anticancer therapies and treatment strategies. Another important concern is toxicity normally associated with conventional therapy methods, such as chemo- and radiotherapy. Among many proposed antitumoral agents, oncolytic viruses are still one of the promising and fast-developing fields of research with almost a hundred studies published data on over 3000 patients since the beginning of the new millennia.
Among all oncolytic viruses, the Vaccinia virus is arguably one of the safest, with an extremely long and prominent history of use, since it was the one and only vaccine used in the Smallpox Eradication Program in the 1970s. Interestingly enough, it was the first oncolytic virus proven to have tumor tropism in vitro and in vivo in laboratory settings, and this year we can celebrate an unofficial 100th anniversary since the publication of the fact. While being highly immunogenic, Vaccinia virus DNA replication takes place in the cytoplasm of the infected cell, and virus genes never integrate into the host genome. Another advantage of using Vaccinia as an oncolytic agent is its high genome capacity, which allows inserting up to 25 kbps of exogenous genes, thus allowing to additionally arm the virus against the tumor.
Oncolytic virus action consists of two major parts: direct oncolysis and immune activation against the tumor, with the latter being the key to successful treatment. To this moment, preclinical research data are mostly generated in immunocompromised xenograft models, which have hurdles to be properly translated for clinical use. In the first part of the current study, fourteen different recombinant Vaccinia virus strains were tested in two different murine tumor cell lines and corresponding immunocompetent animal models. We found, that Copenhagen backbone Vaccinia viruses while being extremely effective in cell culture, do not show significant oncolytic efficacy in animals. In contrast, several of the LIVP backbone viruses tested (specifically, IL-2 expressing ones) have little replication ability when compared to the Copenhagen strain, but are able to significantly delay tumor growth and prolong survival of the treated animals. We have also noted cytokine related toxicity of the animals to be mouse strain specific.
We have also tested the virus with the highest therapeutic benefit in combination with romidepsin and cyclophosphamide. While the combination with histone deacetylase inhibitor romidepsin did not result in therapeutic benefit in our settings, the addition of cyclophosphamide significantly improved the efficacy of the treatment, at the same time reducing cytokine-associated toxicity of the IL-2 expressing virus.
In the second part of the work, we analyzed the ability of adipose-derived mesenchymal stem cells to serve as a carrier for the oncolytic Vaccinia virus. We showed for the first time that the cells can be infected with the virus and can generate virus progeny. They are also able to survive for a substantially long time and, when injected into the bloodstream of tumor-bearing animals, produce the virus that is colonizing the tumor. Analysis of the systemic distribution of the cells after injection revealed that infected and uninfected cells are not distributed in the same manner, possibly suggesting that infected cells are getting recognized and cleared by an impaired immune system of athymic mice faster than non-infected cells. Despite this, injection of virus-loaded adipose-derived mesenchymal stem cells to human A549 tumor-bearing xenograft mice resulted in rapid tumor regression and reduced virus-related side effects of the treatment when compared to injection of the naked virus.
In conclusion, we have tested two different approaches to augmenting oncolytic Vaccinia virus therapy. First, the combination of recombinant Vaccinia virus expressing IL-2 and cyclophosphamide showed promising results in a syngeneic mouse model, despite the low permissivity of murine cells to the virus. Second, we loaded the oncolytic Vaccinia virus into mesenchymal stem cells and have proven that they can potentially serve as a vehicle for the virus.
The original habitat of native European honey bees (\(Apis\) \(mellifera\)) is forest, but currently there is a lack of data about the occurrence of wild honey bee populations in Europe. Prior to being kept by humans in hives, honey bees nested as wild species in hollow trees in temperate forests. However, in the 20th century, intensification of silviculture and agriculture with accompanying losses of nesting sites and depletion of food resources caused population declines in Europe. When the varroa mite (Varroa destructor), an invasive ectoparasite from Asia, was introduced in the late 1970s, wild honey bees were thought to be eradicated in Europe. Nevertheless, sporadic, mostly anecdotal, reports from ornithologists or forest ecologists indicated that honey bee colonies still occupy European forest areas. In my thesis I hypothesize that near-natural deciduous forests may provide sufficient large networks of nesting sites representing refugia for wild-living honey bees. Using two special search techniques, i.e. the tracking of flight routes of honey bee foragers (the “beelining” method) and the inspection of known cavity trees, I collected for the first time data on the occurrence and density of wild-living honey bees in forest areas in Germany (CHAPTER 3). I found wild-living honey bee colonies in the Hainich national park at low densities in two succeeding years. In another forest region, I checked known habitat trees containing black woodpecker cavities for occupation by wild-living honey bee colonies. It turned out that honey bees regularly use these cavities and occur in similar densities in both studied forest regions, independent of the applied detection method. Extrapolating these densities to all German forest areas, I estimate several thousand wild-living colonies in Germany that potentially interact in different ways with the forest environment. I conclude that honey bees regularly colonize forest areas in Germany and that networks of mapped woodpecker cavities offer unique possibilities to study the ecology of wild-living honey bees over several years.
While their population status is ambiguous and the density of colonies low, the fact that honey bees can still be found in forests poses questions about food supply in forest environments. Consequently, I investigated the suitability of woodlands as a honey bee foraging habitat (CHAPTER 4). As their native habitat, forests are assumed to provide important pollen and nectar sources for honey bee colonies. However, resource supply might be spatially and temporally restricted and landscape-scale studies in European forest regions are lacking. Therefore, I set up twelve honey bee colonies in observation hives at locations with varying degree of forest cover. Capitalizing on the unique communication behaviour, the waggle dance, I examined the foraging distances and habitat preferences of honey bees over almost an entire foraging season. Moreover, by connecting this decoded dance information with colony weight recordings, I could draw conclusions about the contribution of the different habitat types to honey yield. Foraging distances generally increased with the amount of forest in the surrounding landscape. Yet, forest cover did not have an effect on colony weight. Compared to expectations based on the proportions of different habitats in the surroundings, colonies foraged more frequently in cropland and grasslands than in deciduous and coniferous forests, especially in late summer when pollen foraging in the forest is most difficult. In contrast, colonies used forests for nectar/honeydew foraging in early summer during times of colony weight gain emphasizing forests as a temporarily significant source of carbohydrates. Importantly, my study shows that the ecological and economic value of managed forest as habitat for honey bees and other wild pollinators can be significantly increased by the continuous provision of floral resources, especially for pollen foraging.
The density of these wild-living honey bee colonies and their survival is driven by several factors that vary locally, making it crucial to compare results in different regions. Therefore, I investigated a wild-living honey bee population in Galicia in north-western Spain, where colonies were observed to reside in hollow electric poles (CHAPTER 5). The observed colony density only in these poles was almost twice as high as in German forest areas, suggesting generally more suitable resource conditions for the bees in Galicia. Based on morphometric analyses of their wing venation patterns, I assigned the colonies to the native evolutionary lineage (M-lineage) where the particularly threatened subspecies \(Apis\) \(mellifera\) \(iberiensis\) also belongs to. Averaged over two consecutive years, almost half of the colonies survived winter (23 out of 52). Interestingly, semi-natural areas both increased abundance and subsequent colony survival. Colonies surrounded by more semi-natural habitat (and therefore less intensive cropland) had an elevated overwintering probability, indicating that colonies need a certain amount of semi-natural habitat in the landscape to survive. Due to their ease of access these power poles in Galicia are, ideally suited to assess the population demography of wild-living Galician honey bee colonies through a long-term monitoring.
In a nutshell, my thesis indicates that honey bees in Europe always existed in the wild. I performed the first survey of wild-living bee density yet done in Germany and Spain. My thesis identifies the landscape as a major factor that compromises winter survival and reports the first data on overwintering rates of wild-living honey bees in Europe. Besides, I established methods to efficiently detect wild-living honey bees in different habitat. While colonies can be found all over Europe, their survival and viability depend on unpolluted, flower rich habitats. The protection of near-natural habitat and of nesting sites is of paramount importance for the conservation of wild-living honey bees in Europe.
Cancer is one of the leading causes of death worldwide. The underlying tumorigenesis is driven by the accumulation of alterations in the genome, eventually disabling tumor suppressors and activating proto-oncogenes.
The MYC family of proto-oncogenes shows a strong deregulation in the majority of tumor entities. However, the exact mechanisms that contribute to MYC-driven oncogenesis remain largely unknown. Over the past decades, the influence of the MYC protein on transcription became increasingly apparent and was thoroughly investigated. Additionally, in recent years several publications provided evidence for so far unreported functions of MYC that are independent of a mere regulation of target genes. These findings suggest an additional role of MYC in the maintenance of genomic stability and this role is strengthened by key findings presented in this thesis.
In the first part, I present data revealing a pathway that allows MYC to couple transcription elongation and DNA double-strand break repair, preventing genomic instability of MYC-driven tumor cells. This pathway is driven by a rapid transfer of the PAF1 complex from MYC onto RNAPII, a process that is mediated by HUWE1. The transfer controls MYC-dependent transcription elongation and, simultaneously, the remodeling of chromatin structure by ubiquitylation of histone H2B. These regions of open chromatin favor not only elongation but also DNA double-strand break repair.
In the second part, I analyze the ability of MYC proteins to form multimeric structures in response to perturbation of transcription and replication. The process of multimerization is also referred to as phase transition. The observed multimeric structures are located proximal to stalled replication forks and recruit factors of the DNA-damage response and transcription termination machinery. Further, I identified the HUWE1-dependent ubiquitylation of MYC as an essential step in this phase transition. Cells lacking the ability to form multimers display genomic instability and ultimately undergo apoptosis in response to replication stress.
Both mechanisms present MYC as a stress resilience factor under conditions that are characterized by a high level of transcriptional and replicational stress. This increased resilience ensures oncogenic proliferation.
Therefore, targeting MYC’s ability to limit genomic instability by uncoupling transcription elongation and DNA repair or disrupting its ability to multimerize presents a therapeutic window in MYC-dependent tumors.
Während der embryonalen Neurogenese spielt die Repression neuraler Gene in nicht neuralen Zellen, sowie in neuralen Vorläuferzellen durch den REST (repressor element silencing transcription factor)-Komplex eine wichtige Rolle. Durch die schrittweise Inaktivierung diese Komplexes im Verlauf der Differenzierung werden neurale Genexpressionsprogramme
gesteuert. Zusätzlich kommt bei der Kontrolle der räumlichen und zeitlichen Regulation der Genexpression während der Neurogenese verschiedenen miRNAs eine wichtige Rolle zu. So konnte in vorangegangenen Arbeiten im Zebrafischen gezeigt werden, dass miR-26b die Transkription eines wichtigen Effektorproteins des REST-Komplexes, CTDSP2 (C-terminal domain small phosphatases), während der Neurogenese negativ reguliert. Da darüber hinaus
die miR-26 Repression zu einer stark verminderten neuronalen Differenzierung führte, kommt diesem regulatorischen Schaltkreis eine zentrale Rolle bei der Neurogenese im Zebrafisch zu.
Die zusammen mit ihren Ctdsp-Wirtsgenen koexprimierte miR-26 Familie liegt in Vertebraten evolutionär hoch konserviert vor. Analog zum Zebrafisch konnte im murinen in vitro ES-Zell Differenzierungssystem gezeigt werden, dass miR-26 die Expression von Ctdsp2 reprimiert.
Weiterhin konnte in diesem System gezeigt werden, dass auch Rest ein miR-26 Zielgen ist und dass der Verlust der miR-26 zu einem Arrest der differenzierenden Zellen im neuronalen Vorläuferstadium führt. Zusammengenommen deuten diese vorangegangenen Arbeiten auf
eine zentrale Rolle der miR-26 während der Neurogenese hin.
Die hier vorgestellte Arbeit zielte zunächst darauf ab die Regulation des REST-Komplexes durch die miR-26 auf molekularer Ebene besser zu verstehen. Der Verlust der miR-26 Bindestelle in der Ctdsp2 mRNA führte zu einer erhöhten Ctdsp2 Expression, beeinflusste aber
nicht die terminale Differenzierung zu Neuronen. Im Gegensatz hierzu führte der Verlust der miR-26 Bindestelle in der Rest mRNA zu einem Arrest der Differenzierung im neuralen Vorläuferzellstadium. Zellen in denen die miR-26 Bindestelle in Rest deletiert war, zeigten zudem, genau wie miR-26 knockout (KO) Zellen, eine erhöhte Expression von REST-Komplex Komponenten, sowie eine verringerte Expression von REST-regulierten miRNAs.
Zusammengenommen weisen diese Daten daraufhin, dass während der Neurogenese im Säugersystem die Inaktivierung von Rest durch miR-26 für die Maturierung von Neuronen eine zentrale Rolle spielt.
Ein weiterer Fokus dieser Arbeit lag auf der Regulation der miR-26 Expression während der Neurogenese. Vorangegangene Arbeiten in nicht-neuronalen Zelltypen identifizierten die lnc (long-non-coding) RNA Malat1 als eine ce (competitive endogenous) RNA der miR-26. Um den Einfluss von Malat1 auf die miR-26 Expression während der Neurogenese zu untersuchen,
wurde zunächst mittels CRISPR/Cas9 der vollständige Malat1-Lokus in ESCs deletiert. Der Verlust von Malat1 führte zu einer erhöhten Expression der miR-26 Familienmitglieder sowie deren Ctdsp-Wirtsgene. Weiterhin war die Proliferation von Malat1 KO neuronalen
Vorläuferzellen stark vermindert, was mit einer Erhöhung der Frequenz seneszenter Zellen einherging. Durch die Inaktivierung von miR-26 in differenzierenden Malat1 KO ESCs konnte dieser proliferative Phänotyp aufgehoben werden. Darüber hinaus konnte eine verstärkte neuronale Differenzierung dieser Zellen beobachtet werden.
Zusammenfassend zeigen diese Daten, dass neben der Regulation des REST-Komplexes durch miR-26 auch die Kontrolle des Zellzyklus über die Malat1-vermittelte Regulation der miR-26
in neuronalen Vorläuferzellen einen kritischen Schritt bei der Differenzierung von neuronalen Vorläuferzellen zu maturen Neuronen darstellt.
Since the advent of high-throughput sequencing technologies in the mid-2010s, RNA se-
quencing (RNA-seq) has been established as the method of choice for studying gene
expression. In comparison to microarray-based methods, which have mainly been used to
study gene expression before the rise of RNA-seq, RNA-seq is able to profile the entire
transcriptome of an organism without the need to predefine genes of interest. Today,
a wide variety of RNA-seq methods and protocols exist, including dual RNA sequenc-
ing (dual RNA-seq) and multi RNA sequencing (multi RNA-seq). Dual RNA-seq and
multi RNA-seq simultaneously investigate the transcriptomes of two or more species, re-
spectively. Therefore, the total RNA of all interacting species is sequenced together and
only separated in silico. Compared to conventional RNA-seq, which can only investi-
gate one species at a time, dual RNA-seq and multi RNA-seq analyses can connect the
transcriptome changes of the species being investigated and thus give a clearer picture of
the interspecies interactions. Dual RNA-seq and multi RNA-seq have been applied to a
variety of host-pathogen, mutualistic and commensal interaction systems.
We applied dual RNA-seq to a host-pathogen system of human mast cells and Staphylo-
coccus aureus (S. aureus). S. aureus, a commensal gram-positive bacterium, can become
an opportunistic pathogen and infect skin lesions of atopic dermatitis (AD) patients.
Among the first immune cells S. aureus encounters are mast cells, which have previously
been shown to be able to kill the bacteria by discharging antimicrobial products and re-
leasing extracellular traps made of protein and deoxyribonucleic acid (DNA). However,
S. aureus is known to evade the host’s immune response by internalizing within mast
cells. Our dual RNA-seq analysis of different infection settings revealed that mast cells
and S. aureus need physical contact to influence each other’s gene expression. We could
show that S. aureus cells internalizing within mast cells undergo profound transcriptome
changes to adjust their metabolism to survive in the intracellular niche. On the host side,
we found out that infected mast cells elicit a type-I interferon (IFN-I) response in an
autocrine manner and in a paracrine manner to non-infected bystander-cells. Our study
provides the first evidence that mast cells are capable to produce IFN-I upon infection
with a bacterial pathogen.
Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes.
The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration.
Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.
Mammalian embryonic development is subject to complex biological relationships that need to be understood. However, before the whole structure of development can be put together, the individual building blocks must first be understood in more detail. One of these building blocks is the second cell fate decision and describes the differentiation of cells of the inner cell mass of the embryo into epiblast and primitive endoderm cells. These cells then spatially segregate and form the subsequent bases for the embryo and yolk sac, respectively. In organoids of the inner cell mass, these two types of progenitor cells are also observed to form, and to some extent to spatially separate. This work has been devoted to these phenomena over the past three years. Plenty of studies already provide some insights into the basic mechanics of this cell differentiation, such that the first signs of epiblast and primitive endoderm differentiation, are the expression levels of transcription factors NANOG and GATA6. Here, cells with low expression of GATA6 and high expression of NANOG adopt the epiblast fate. If the expressions are reversed, a primitive endoderm cell is formed. Regarding the spatial segregation of the two cell types, it is not yet clear what mechanism leads to this. A common hypothesis suggests the differential adhesion of cell as the cause for the spatial rearrangement of cells. In this thesis however, the possibility of a global cell-cell communication is investigated. The approach chosen to study these phenomena follows the motto "mathematics is biology's next microscope". Mathematical modeling is used to transform the central gene regulatory network at the heart of this work into a system of equations that allows us to describe the temporal evolution of NANOG and GATA6 under the influence of an external signal. Special attention is paid to the derivation of new models using methods of statistical mechanics, as well as the comparison with existing models. After a detailed stability analysis the advantages of the derived model become clear by the fact that an exact relationship of the model parameters and the formation of heterogeneous mixtures of two cell types was found. Thus, the model can be easily controlled and the proportions of the resulting cell types can be estimated in advance. This mathematical model is also combined with a mechanism for global cell-cell communication, as well as a model for the growth of an organoid. It is shown that the global cell-cell communication is able to unify the formation of checkerboard patterns as well as engulfing patterns based on differently propagating signals. In addition, the influence of cell division and thus organoid growth on pattern formation is studied in detail. It is shown that this is able to contribute to the formation of clusters and, as a consequence, to breathe some randomness into otherwise perfectly sorted patterns.
Einflüsse der Photophysik und Photochemie von Cyaninfarbstoffen auf die Lokalisationsmikroskopie
(2023)
In den letzten Jahren haben sich hochauflösende Fluoreszenzmikroskopiemethoden, basierend auf der Lokalisation einzelner Fluorophore, zu einem leistungsstarken Werkzeug etabliert, um Fluoreszenzbilder weit unterhalb der Auflösungsgrenze zu generieren. Hiermit können räumliche Auflösungen von ~ 20 nm erzielt werden, was weit unterhalb der Beugungsgrenze liegt. Dabei haben zahlreiche Optimierungen und Entwicklungen neuer Methoden in der Einzelmolekül-Lokalisationsmikroskopie die Genauigkeit der orstspezifischen Bestimmung einzelner Fluorophore auf bis zu ~ 1 – 3 nm erhöht. Eine Auflösung im molekularen Bereich, weit unterhalb von ~ 10 nm bleibt allerdings herausfordernd, da die Lokalisationsgenauigkeit nur ein Kriterium hierfür ist. Allerdings wurde sich in den letzten Jahren überwiegend auf die Verbesserung dieses Parameters konzentriert. Weitere Kriterien für die fluoreszenzmikroskopische Auflösung sind dabei unter anderem die Markierungsdichte und die Kopplungseffizienz der Zielstruktur, sowie der Kopplungsfehler (Abstand zur Zielstruktur nach Farbstoffkopplung), die sich herausfordernd für eine molekulare Auflösung darstellen. Auch wenn die Kopplungseffizienz und -dichte hoch und der Kopplungsfehler gering ist, steigt bei Interfluorophordistanzen < 5nm, abhängig von den Farbstoffen, die Wahrscheinlichkeit von starken und schwachen Farbstoffwechselwirkungen und damit von Energieübertragungsprozessen zwischen den Farbstoffen, stark an. Daneben sollten Farbstoffe, abhänging von der Lokalisationsmikroskopiemethode, spezifische Kriterien, wie beispielsweise die Photoschaltbarkeit bei dSTORM, erfüllen, was dazu führt, dass diese Methoden häufig nur auf einzelne Farbstoffe beschränkt sind. In dieser Arbeit konnte mithilfe von definierten DNA-Origami Konstrukten gezeigt werden, dass das Blinkverhalten von Cyaninfarbstoffen unter dSTORM-Bedingungen einer Abstandsabhängigkeit aufgrund von spezifischen Energieübertragungsprozessen folgt, womit Farbstoffabstände im sub-10 nm Bereich charakterisiert werden konnten. Darüber hinaus konnte diese Abstandsabhängigkeit an biologischen Proben gezeigt werden. Hierbei konnten verschiedene zelluläre Rezeptoren effizient und mit geringem Abstandsfehler zur Zielstruktur mit Cyaninfarbstoffen gekoppelt werden. Diese abstandsabhänigen Prozesse und damit Charakterisierungen könnten dabei nicht nur spezifisch für die häufig unter dSTORM-Bedingungen verwendeten Cyaninfarbstoffen gültig sein, sondern auch auf andere Farbstoffklassen, die einen Auszustand zeigen, übertragbar sein. Darüber hinaus konnte gezeigt werden, dass hochauflösende dSTORM Aufnahmen unabhängig vom Farbstoffkopplungsgrad der Antikörpern sind, welche häufig für Standardfärbungen von zellulären Strukturen verwendet werden. Dabei konnte durch Photonenkoinzidenzmessungen dargelegt werden, dass aufgrund komplexer Farbstoffwechselwirkungen im Mittel nur ein Farbstoff aktiv ist, wobei höhere Kopplungsgrade ein komplexes Blinkverhalten zu Beginn der Messung zeigen. Durch die undefinierten Farbstoffabstände an Antikörpern konnte hier kein eindeutiger Energieübertragungsmechanismus entschlüsselt werden. Dennoch konnte gezeigt werden, dass Farbstoffaggregate bzw. H-Dimere unter dSTORM-Bedingungen destabilisiert werden. Durch die zuvor erwähnten DNA-Origami Konstrukte definierter Interfluorophordistanzen konnten Energieübertragungsmechanismen entschlüsselt werden, die auch für die Antikörper diverser Kopplungsgrade gültig sind. Des Weiteren konnten, ausgelöst durch komplexe Energieübertragungsprozesse höherer Kopplungsgrade am Antikörper, Mehrfarbenaufnahmen zellulärer Strukturen generiert werden, die über die spezifische Fluoreszenzlebenszeit separiert werden konnten. Dies stellt hier eine weitere Möglichkeit dar, unter einfachen Bedingungen, schnelle Mehrfarbenaufnahmen zellulärer Strukturen zu generieren. Durch die Verwendung des selben Farbstoffes unterschiedlicher Kopplungsgrade kann hier nur mit einer Anregungswellenlänge und frei von chromatischer Aberration gearbeitet werden. Neben den photophysikalischen Untersuchungen der Cyaninfarbstoffe Cy5 und Alexa Fluor 647 wurden diese ebenso photochemisch näher betrachtet. Dabei konnte ein neuartiger chemischer Mechanismus entschlüsselt werden. Dieser Mechanismus führt, ausgelöst durch Singulett-Sauerstoff (1O2), zu einer Photozerschneidung des konjugierten Doppelbindungssystems um zwei Kohlenstoffatome, was zu strukturellen und spektroskopischen Veränderungen dieser Farbstoffe führt. Auf Grundlage dieses Mechanismus konnte eine neue DNA-PAINT Methode entwickelt werden, die zu einer Beschleunigung der Aufnahmezeit führt.
The synaptic cleft is of central importance for synaptic transmission, neuronal plasticity and memory and thus well studied in neurobiology. To target proteins of interest with high specificity and strong signal to noise conventional immunohistochemistry relies on the use of fluorescently labeled antibodies. However, investigations on synaptic receptors remain challenging due to the defined size of the synaptic cleft of ~20 nm between opposing pre- and postsynaptic membranes. At this limited space, antibodies bear unwanted side effects such as crosslinking, accessibility issues and a considerable linkage error between fluorophore and target of ~10 nm. With recent single molecule localization microscopy (SMLM) methods enabling localization precisions of a few nanometers, the demand for labeling approaches with minimal linkage error and reliable recognition of the target molecules rises.
Within the scope of this work, different labeling techniques for super-resolution fluorescence microscopy were utilized allowing site-specific labeling of a single amino acid in synaptic proteins like kainate receptors (KARs), transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory proteins (TARPs), γ-aminobutyric acid type A receptors (GABA-ARs) and neuroligin 2 (NL2). The method exploits the incorporation of unnatural amino acids (uAAs) in the protein of interest using genetic code expansion (GCE) via amber suppression technology and subsequent labeling with tetrazine functionalized fluorophores. Implementing this technique, hard-to-target proteins such as KARs, TARPs and GABA-ARs could be labeled successfully, which could only be imaged insufficiently with conventional labeling approaches. Furthermore, functional studies involving electrophysiological characterization, as well as FRAP and FRET experiments validated that incorporation of uAAs maintains the native character of the targeted proteins. Next, the method was transferred into primary hippocampal neurons and in combination with super-resolution microscopy it was possible to resolve the nanoscale organization of γ2 and γ8 TARPs. Cluster analysis of dSTORM localization data verified synaptic accumulation of γ2, while γ8 was homogenously distributed along the neuron. Additionally, GCE and bioorthogonal labeling allowed visualization of clickable GABA-A receptors located at postsynaptic compartments in dissociated hippocampal neurons. Moreover, saturation experiments and FRET imaging of clickable multimeric receptors revealed successful binding of multiple tetrazine functionalized fluorophores to uAA-modified dimeric GABA-AR α2 subunits in close proximity (~5 nm). Further utilization of tetrazine-dyes via super-resolution microscopy methods such as dSTORM and click-ExM will provide insights to subunit arrangement in receptors in the future.
This work investigated the nanoscale organization of synaptic proteins with minimal linkage error enabling new insights into receptor assembly, trafficking and recycling, as well as protein-protein interactions at synapses. Ultimately, bioorthogonal labeling can help to understand pathologies such as the limbic encephalitis associated with GABA-AR autoantibodies and is already in application for cancer therapies.
Die Fluoreszenzmikroskopie ist eine vielseitig einsetzbare Untersuchungsmethode für biologische Proben, bei der Biomoleküle selektiv mit Fluoreszenzfarbstoffen markiert werden, um sie dann mit sehr gutem Kontrast abzubilden. Dies ist auch mit mehreren verschiedenartigen Zielmolekülen gleichzeitig möglich, wobei üblicherweise verschiedene Farbstoffe eingesetzt werden, die über ihre Spektren unterschieden werden können.
Um die Anzahl gleichzeitig verwendbarer Färbungen zu maximieren, wird in dieser Arbeit zusätzlich zur spektralen Information auch das zeitliche Abklingverhalten der Fluoreszenzfarbstoffe mittels spektral aufgelöster Fluoreszenzlebensdauer-Mikroskopie (spectrally resolved fluorescence lifetime imaging microscopy, sFLIM) vermessen. Dazu wird die Probe in einem Konfokalmikroskop von drei abwechselnd gepulsten Lasern mit Wellenlängen von 485 nm, 532nm und 640nm angeregt. Die Detektion des Fluoreszenzlichtes erfolgt mit einer hohen spektralen Auflösung von 32 Kanälen und gleichzeitig mit sehr hoher zeitlicher Auflösung von einigen Picosekunden. Damit wird zu jedem detektierten Fluoreszenzphoton der Anregungslaser, der spektrale Kanal und die Ankunftszeit registriert. Diese detaillierte multidimensionale Information wird von einem Pattern-Matching-Algorithmus ausgewertet, der das Fluoreszenzsignal mit zuvor erstellten Referenzpattern der einzelnen Farbstoffe vergleicht. Der Algorithmus bestimmt so für jedes Pixel die Beiträge der einzelnen Farbstoffe.
Mit dieser Technik konnten pro Anregungslaser fünf verschiedene Färbungen gleichzeitig dargestellt werden, also theoretisch insgesamt 15 Färbungen. In der Praxis konnten mit allen drei Lasern zusammen insgesamt neun Färbungen abgebildet werden, wobei die Anzahl der Farben vor allem durch die anspruchsvolle Probenvorbereitung limitiert war. In anderen Versuchen konnte die sehr hohe Sensitivität des sFLIM-Systems genutzt werden, um verschiedene Zielmoleküle voneinander zu unterscheiden, obwohl sie alle mit demselben Farbstoff markiert waren. Dies war möglich, weil sich die Fluoreszenzeigenschaften eines Farbstoffmoleküls geringfügig in Abhängigkeit von seiner Umgebung ändern. Weiterhin konnte die sFLIM-Technik mit der hochauflösenden STED-Mikroskopie (STED: stimulated emission depletion) kombiniert werden, um so hochaufgelöste zweifarbige Bilder zu erzeugen, wobei nur ein einziger gemeinsamer STED-Laser benötigt wurde.
Die gleichzeitige Erfassung von mehreren photophysikalischen Messgrößen sowie deren Auswertung durch den Pattern-Matching-Algorithmus ermöglichten somit die Entwicklung von neuen Methoden der Fluoreszenzmikroskopie für Mehrfachfärbungen.
The Role of Acid Sphingomyelinase in \(Staphylococcus\) \(aureus\) Infection of Endothelial Cells
(2022)
Staphylococcus aureus is a human bacterial pathogen responsible for a variety of diseases including bacterial pneumonia and sepsis. Recent studies provided an explanation, how S. aureus and its exotoxins contribute to the degradation of endothelial junction proteins and damage lung tissue [4]. Previous findings were indicating an involvement of acid sphingomyelinase (ASM) activity in cell barrier degradation [5]. In the presented study the impact of singular virulence factors, such as staphylococcal α-toxin, on in vitro cell barrier integrity as well as their ability to elicit an activation of ASM were investigated.
Experiments with bacterial supernatants performed on human endothelial cells demonstrated a rapid dissociation after treatment, whereas murine endothelial cells were rather resistant against cell barrier degradation. Furthermore, amongst all tested staphylococcal toxins it was found that only α-toxin had a significant impact on endothelial junction proteins and ASM activity. Ablation of this single toxin was sufficient to protect endothelial cells from cell barrier degradation and activation of ASM was absent.
In this process it was verified, that α-toxin induces a recruitment of intracellular ASM, which is accompanied by rapid and oscillating changes in cytoplasmic Ca2+ concentration and an increased exposure of Lysosomal associated membrane protein 1 (LAMP1) on the cell surface. Recruitment of lysosomal ASM is associated, among other aspects, to plasma membrane repair and was previously described to be involved with distinct pathogens as well as other pore forming toxins (PFT). However, with these findings a novel feature for α-toxin has been revealed, indicating that the staphylococcal PFT is able to elicit a similar process to previously described plasma membrane repair mechanisms.
Increased exposure and intake of surface membrane markers questioned the involvement of ASM activity in S. aureus internalization by non-professional phagocytes such as endothelial cells. By modifying ASM expression pattern as well as application of inhibitors it was possible to reduce the intracellular bacterial count. Thus, a direct connection between ASM activity and S. aureus infection mechanisms was observed, therefore this study exemplifies how S. aureus is able to exploit the host cell sphingolipid metabolism as well as benefit of it for invasion into non-professional phagocytic cells
Humans and animals alike use the sun, the moon, and the stars to guide their ways.
However, the position of celestial cues changes depending on daytime, season, and
place on earth. To use these celestial cues for reliable navigation, the rotation of the
sky has to be compensated. While humans invented complicated mechanisms like the
Antikythera mechanism to keep track of celestial movements, animals can only rely on
their brains. The desert ant Cataglyphis is a prime example of an animal using celestial
cues for navigation. Using the sun and the related skylight polarization pattern as a
compass, and a step integrator for distance measurements, it can determine a vector
always pointing homewards. This mechanism is called path integration. Since the sun’s
position and, therefore, also the polarization pattern changes throughout the day,
Cataglyphis have to correct this movement. If they did not compensate for time, the
ants’ compass would direct them in different directions in the morning and the evening.
Thus, the ants have to learn the solar ephemeris before their far-reaching foraging
trips.
To do so, Cataglyphis ants perform a well-structured learning-walk behavior during the
transition phase from indoor worker to outdoor forager. While walking in small loops
around the nest entrance, the ants repeatedly stop their forward movements to perform
turns. These can be small walked circles (voltes) or tight turns about the ants’ body
axes (pirouettes). During pirouettes, the ants gaze back to their nest entrance during
stopping phases. These look backs provide a behavioral read-out for the state of the
path integrator. The ants “tell” the observer where they think their nest is, by looking
back to it. Pirouettes are only performed by Cataglyphis ants inhabiting an environment
with a prominent visual panorama. This indicates, that pirouettes are performed to
learn the visual panorama. Voltes, on the other hand, might be used for calibrating the
celestial compass of the ants.
In my doctoral thesis, I employed a wide range of state-of-the-art techniques from
different disciplines in biology to gain a deeper understanding of how navigational
information is acquired, memorized, used, and calibrated during the transition phase
from interior worker to outdoor forager. I could show, that celestial orientation cues that
provide the main compass during foraging, do not guide the ants during the look-backbehavior
of initial learning walks. Instead Cataglyphis nodus relies on the earth’s
magnetic field as a compass during this early learning phase. While not guiding the
ants during their first walks outside of the nest, excluding the ants from perceiving the
natural polarization pattern of the skylight has significant consequences on learning-related
plasticity in the ants’ brain. Only if the ants are able to perform their learning-walk
behavior under a skylight polarization pattern that changes throughout the day,
plastic neuronal changes in high-order integration centers are induced. Especially the
mushroom bogy collar, a center for learning and memory, and the central complex, a
center for orientation and motor control, showed an increase in volume after learning
walks. This underlines the importance of learning walks for calibrating the celestial
compass. The magnetic compass might provide the necessary stable reference
system for the ants to calibrate their celestial compass and learn the position of
landmark information. In the ant brain, visual information from the polarization-sensitive
ocelli converge in tight apposition with neuronal afferents of the mechanosensitive
Johnston’s organ in the ant’s antennae. This makes the ants’ antennae an interesting
candidate for studying the sensory bases of compass calibration in Cataglyphis ants.
The brain of the desert navigators is well adapted to successfully accomplish their
navigational needs. Females (gynes and workers) have voluminous mushroom bodies,
and the synaptic complexity to store large amount of view-based navigational
information, which they acquire during initial learning walks. The male Cataglyphis
brain is better suited for innate behaviors that support finding a mate.
The results of my thesis show that the well adapted brain of C. nodus ants undergoes
massive structural changes during leaning walks, dependent on a changing celestial
polarization pattern. This underlies the essential role of learning walks in the calibration
of orientation systems in desert ants.
Biodiversity is in rapid decline worldwide. These declines are more pronounced in areas that are currently biodiversity rich, but economically poor – essentially describing many tropical regions in the Global South where landscapes are dominated by smallholder agriculture. Agriculture is an important driver of biodiversity decline, through habitat destruction and unsustainable practices. Ironically, agriculture itself is dependent on a range of ecosystem services, such as pollination and pest control, provided by biodiversity. Biodiversity on fields and the delivery of ecosystem services to crops is often closely tied to the composition of the surrounding landscape – complex landscapes with a higher proportion of (semi-)natural habitats tend to support a high abundances and biodiversity of pollinators and natural enemies that are beneficial to crop production. However, past landscape scale studies have focused primarily on industrialized agricultural landscapes in the Global North, and context dependent differences between regions and agricultural systems are understudied. Smallholder agriculture supports 2 billion people worldwide and contributes to over half the world’s food supply. Yet smallholders, particularly in sub-Saharan Africa, are underrepresented in research investigating the consequences of landscape change and agricultural practices. Where research in smallholder agriculture is conducted, the focus is often on commodity crops, such as cacao, and less on crops that are directly consumed by smallholder households, though the loss of services to these crops could potentially impact the most vulnerable farmers the hardest. Agroecology – a holistic and nature-based approach to agriculture, provides an alternative to unsustainable input-intensive agriculture. Agroecology has been found to benefit smallholders through improved agronomical and food-security outcomes. Co-benefits of agroecological practices with biodiversity and ecosystem services are assumed, but not often empirically tested. In addition, the local and landscape effects on biodiversity and ecosystem services are more commonly studied in isolation, but their potentially interactive effects are so far little explored. Our study region in northern Malawi exemplifies many challenges experienced by smallholder farmers throughout sub-Saharan Africa and more generally in the Global South. Malawi is located in a global biodiversity hotspot, but biodiversity is threatened by rapid habitat loss and a push for input-intensive agriculture by government and other stakeholders. In contrast, agroecology has been effectively promoted and implemented in the study region. We investigated how land-use differences and the agroecological practices affects biodiversity and ecosystem services of multiple taxa in a maize-bean intercropping system (Chapter 2), and pollination of pumpkin (Chapter 3) and pigeon pea (Chapter 4). Additionally, the effects of local and landscape scale shrub- to farmland habitat conversion was investigated on butterfly communities, as well as the potential for agroecology to mitigate these effects (Chapter 5).
Understanding the causal relationship between genotype and phenotype is a major objective in biology. The main interest is in understanding trait architecture and identifying loci contributing to the respective traits. Genome-wide association mapping (GWAS) is one tool to elucidate these relationships and has been successfully used in many different species. However, most studies concentrate on marginal marker effects and ignore epistatic and gene-environment interactions. These interactions are problematic to account for, but are likely to make major contributions to many phenotypes that are not regulated by independent genetic effects, but by more sophisticated gene-regulatory networks. Further complication arises from the fact that these networks vary in different natural accessions. However, understanding the differences of gene regulatory networks and gene-gene interactions is crucial to conceive trait architecture and predict phenotypes.
The basic subject of this study – using data from the Arabidopsis 1001 Genomes Project – is the analysis of pre-mature stop codons. These have been incurred in nearly one-third of the ~ 30k genes. A gene-gene interaction network of the co-occurrence of stop codons has been built and the over and under representation of different pairs has been statistically analyzed. To further classify the significant over and under- represented gene-gene interactions in terms of molecular function of the encoded proteins, gene ontology terms (GO-SLIM) have been applied. Furthermore, co- expression analysis specifies gene clusters that co-occur over different genetic and phenotypic backgrounds. To link these patterns to evolutionary constrains, spatial location of the respective alleles have been analyzed as well. The latter shows clear patterns for certain gene pairs that indicate differential selection.
Over the past centuries, anthropogenic utilization has fundamentally changed the appearance of European forest ecosystems. Constantly growing and changing demands have led to an enormous decline in ecological key elements and a structural homogenization of most forests. These changes have been accompanied by widespread declines of many forest-dwelling and especially saproxylic, i.e. species depending on deadwood. In order to counteract this development, various conservation strategies have been developed, but they primarily focus on a quantitative deadwood enrichment. However, the diversity of saproxylic species is furthermore driven by a variety of abiotic and biotic determinants as well as interactions between organisms. A detailed understanding of these processes has so far been largely lacking. The aim of the present thesis was therefore to improve the existing ecological knowledge of determinants influencing saproxylic species and species communities in order to provide the basis for evidence-based and adapted conservation measures.
In chapter II of this thesis, I first investigated the impact of sun exposure, tree species, and their combination on saproxylic beetles, wood-inhabiting fungi, and spiders. Therefore, logs and branches of six tree species were set up under different sun exposures in an experimental approach. The impact of sun exposure and tree species strongly differed among single saproxylic taxa as well as diameters of deadwood. All investigated taxa were affected by sun exposure, whereby sun exposure resulted in a higher alpha-diversity of taxa recorded in logs and a lower alpha-diversity of saproxylic beetles reared from branches compared to shading by canopy. Saproxylic beetles and wood-inhabiting fungi as obligate saproxylic species were additionally affected by tree species. In logs, the respective impact of both determinants also resulted in divergent community compositions. Finally, a rarefaction/extrapolation method was used to evaluate the effectiveness of different combinations of tree species and sun exposure for the conservation of saproxylic species diversity. Based on this procedure, a combination of broadleaved and coniferous as well as hard- and softwood tree species was identified to support preferably high levels of saproxylic species diversity.
The aim of chapter III was to evaluate the individual conservational importance of tree species for the protection of saproxylic beetles. For this, the list of tree species sampled for saproxylic beetles was increased to 42 different tree species. The considered tree species represented large parts of taxonomic and phylogenetic diversity native to Central Europe as well as the most important non-native tree species of silvicultural interest. Freshly cut branches were set up for one year and saproxylic beetles were reared afterwards for two subsequent years.
The study revealed that some tree species, in particular Quercus sp., host a particular high diversity of saproxylic beetles, but tree species with a comparatively medium or low overall diversity were likewise important for red-listed saproxylic beetle species. Compared to native tree species, non-native tree species hosted a similar overall species diversity of saproxylic beetles but differed in community composition.
In chapter IV, I finally analysed the interactions of host beetle diversity and the diversity of associated parasitoids by using experimentally manipulated communities of saproxylic beetles and parasitoid Hymenoptera as a model system. Classical approaches of species identification for saproxylic beetles were combined with DNA-barcoding for parasitoid Hymenoptera. The diversity of the host communities was inferred from their phylogenetic composition as well as differences in seven functional traits. Abundance, species richness, and Shannon-diversity of parasitoid Hymenoptera increased with increasing host abundance. However, the phylogenetic and functional dissimilarity of host communities showed no influence on the species communities of parasitoid Hymenoptera. The results clearly indicate an abundance-driven system in which the general availability, not necessarily the diversity of potential hosts, is decisive.
In summary, the present thesis corroborates the general importance of deadwood heterogeneity for the diversity of saproxylic species by combining different experimental approaches. In order to increase their efficiency, conservation strategies for saproxylic species should generally promote deadwood from different tree species under different conditions of sun exposure on landscape-level in addition to the present enrichment of a certain deadwood amount. The most effective combinations of tree species should consider broadleaved and coniferous as well as hard- and softwood tree species. Furthermore, in addition to dominant tree species, special attention should be given to native, subdominant, silviculturally unimportant, and rare tree species.
Despite belonging to the best described patterns in ecology, the mechanisms driving biodiversity along broad-scale climatic gradients, like the latitudinal gradient in diversity, remain poorly understood. Because of their high biodiversity, restricted spatial ranges, the continuous change in abiotic factors with altitude and their worldwide occurrence, mountains constitute ideal study systems to elucidate the predictors of global biodiversity patterns. However, mountain ecosystems are increasingly threatened by human land use and climate change. Since the consequences of such alterations on mountainous biodiversity and related ecosystem services are hardly known, research along elevational gradients is also of utmost importance from a conservation point of view. In addition to classical biodiversity research focusing on taxonomy, the significance of studying functional traits and their prominence in biodiversity ecosystem functioning (BEF) relationships is increasingly acknowledged. In this dissertation, I explore the patterns and drivers of mammal and dung beetle diversity along elevational and land use gradients on Mt. Kilimanjaro, Tanzania. Furthermore, I investigate the predictors of dung decomposition by dung beetles under different extinction scenarios.
Mammals are not only charismatic, they also fulfil important roles in ecosystems. They provide important ecosystem services such as seed dispersal and nutrient cycling by turning over high amounts of biomass. In chapter II, I show that mammal diversity and community biomass both exhibited a unimodal distribution with elevation on Mt.Kilimanjaro and were mainly impacted by primary productivity, a measure of the total food abundance, and the protection status of study plots. Due to their large size and endothermy, mammals, in contrast to most arthopods, are theoretically predicted to be limited by food availability. My results are in concordance with this prediction. The significantly higher diversity and biomass in the Kilimanjaro National Park and in other conservation areas underscore the important role of habitat protection is vital for the conservation of large mammal biodiversity on tropical mountains.
Dung beetles are dependent on mammals since they rely upon mammalian dung as a food and nesting resource. Dung beetles are also important ecosystem service providers: they play an important role in nutrient cycling, bioturbation, secondary seed dispersal and parasite suppression. In chapter III, I show that dung beetle diversity declined with elevation while dung beetle abundance followed a hump-shaped pattern along the elevational gradient. In contrast to mammals, dung beetle diversity was primarily predicted by temperature. Despite my attempt to accurately quantifiy mammalian dung resources by calculating mammalian defecation rates, I did not find an influence of dung resource availability on dung beetle richness. Instead, higher temperature translated into higher dung beetle diversity.
Apart from being important ecosystem service providers, dung beetles are also model organisms for BEF studies since they rely on a resource which can be quantified easily. In chapter IV, I explore dung decomposition by dung beetles along the elevational gradient by means of an exclosure experiment in the presence of the whole dung beetle community, in the absence of large dung beetles and without any dung beetles. I show that dung decomposition was the highest when the dung could be decomposed by the whole dung beetle community, while dung decomposition was significantly reduced in the sole presence of small dung beetles and the lowest in the absence of dung beetles. Furthermore, I demonstrate that the drivers of dung decomposition were depend on the intactness of the dung beetle community. While body size was the most important driver in the presence of the whole dung beetle community, species richness gained in importance when large dung beetles were excluded. In the most perturbed state of the system with no dung beetles present, temperature was the sole driver of dung decomposition. In conclusion, abiotic drivers become more important predictors of ecosystem services the more the study system is disturbed.
In this dissertation, I exemplify that the drivers of diversity along broad-scale climatic gradients on Mt. Kilimanjaro depend on the thermoregulatory strategy of organisms. While mammal diversity was mainly impacted by food/energy resources, dung beetle diversity was mainly limited by temperature. I also demonstrate the importance of protected areas for the preservation of large mammal biodiversity. Furthermore, I show that large dung beetles were disproportionately important for dung decomposition as dung decomposition significantly decreased when large dung beetles were excluded. As regards land use, I did not detect an overall effect on dung beetle and mammal diversity nor on dung beetle-mediated dung decomposition. However, for the most specialised mammal trophic guilds and dung beetle functional groups, negative land use effects were already visible. Even though the current moderate levels of land use on Mt. Kilimanjaro can sustain high levels of biodiversity, the pressure of the human population on Mt. Kilimanjaro is increasing and further land use intensification poses a great threat to biodiversity. In synergy wih land use, climate change is jeopardizing current patterns and levels of biodiversity with the potential to displace communities, which may have unpredictable consequences for ecosystem service provisioning in the future.
Chapter 1 – General introduction
Anthropogenic land-use and climate change are the major drivers of the global biodiversity loss. Yet, biodiversity is essential for human well-being, as we depend on the availability of potable water, sufficient food and further benefits obtained from nature. Each species makes a somewhat unique contribution to these ecosystem services. Furthermore, species tolerate environmental stressors, such as climate change, differently. Thus, biodiversity is both the "engine" and the "insurance" for human well-being in a changing climate. Here, I investigate the effects of temperature and land use on herbivory (Chapter 2), predation (Chapter 3) and pest control (Chapter 4), and at the same time identify features of habitats (e.g. plant richness, proximity to different habitat types) and landscapes (e.g. landscape diversity, proportion of oilseed rape area) as potential management targets in an adaptation strategy to climate change. Finally, I discuss the similarities and differences between factors influencing herbivory, predation and pest control, while placing the observations in the context of climate change as a multifaceted phenomenon, and highlighting starting points for sustainable insect pest management (Chapter 5).
Chapter 2 – Plant richness, land use and temperature differently shape invertebrate leaf-chewing herbivory on major plant functional groups
Invertebrate herbivores are temperature-sensitive. Rising temperatures increase their metabolic rates and thus their demand for carbon-rich relative to protein-rich resources, which can lead to changes in the diets of generalist herbivores. Here, we quantified leaf-area loss to chewing invertebrates among three plant functional groups (legumes, non-leguminous forbs and grasses), which largely differ in C:N (carbon:nitrogen) ratio. This reseach was conducted along spatial temperature and land-use gradients in open herbaceous vegetation adjacent to different habitat types (forest, grassland, arable field, settlement). Herbivory largely differed among plant functional groups and was higher on legumes than forbs and grasses, except in open areas in forests. There, herbivory was similar among plant functional groups and on legumes lower than in grasslands. Also the presence of many plant families lowered herbivory on legumes. This suggests that open areas in forests and diverse vegetation provide certain protection against leaf damage to some plant families (e.g. legumes). This could be used as part of a conservation strategy for protected species. Overall, the effects of the dominant habitat type in the vicinity and diverse vegetation outweighed those of temperature and large-scale land use (e.g. grassland proportion, landscape diversity) on herbivory of legumes, forbs and grasses at the present time.
Chapter 3 – Landscape diversity and local temperature, but not climate, affect arthropod predation among habitat types
Herbivorous insects underlie top-down regulation by arthropod predators. Thereby, predation rates depend on predator community composition and behaviour, which is shaped by temperature, plant richness and land use. How the interaction of these factors affects the regulatory performance of predators was unknown. Therefore, we assessed arthropod predation rates on artificial caterpillars along temperature, and land-use gradients. On plots with low local mean temperature (≤ 7°C) often not a single caterpillar was attacked, which may be due to the temperature-dependent inactivity of arthropods. However, multi-annual mean temperature, plant richness and the dominant habitat type in the vicinity did not substantially affect arthropod predation rates. Highest arthropod predation rates were observed in diverse landscapes (2-km scale) independently of the locally dominanting habitat type. As landscape diversity, but not multi-annual mean temperature, affected arthropod predation rates, the diversification of landscapes may also support top-down regulation of herbivores independent of moderate increases of multi-annual mean temperature in the near future.
Chapter 4 – Pest control and yield of winter oilseed rape depend on spatiotemporal crop-cover dynamics and flowering onset: implications for global warming
Winter oilseed rape is an important oilseed crop in Europe, yet its seed yield is diminished through pests such as the pollen beetle and stem weevils. Damage from pollen beetles depends on pest abundances, but also on the timing of infestation relative to crop development as the bud stage is particularly vulnerable. The development of both oilseed rape and pollen beetles is temperature-dependent, while temperature effects on pest abundances are yet unknown, which brings opportunities and dangers to oilseed rape cropping under increased temperatures. We obtained measures of winter oilseed rape (flowering time, seed yield) and two of its major pests (pollen beetle, stem weevils) for the first time along both land-use and temperature gradients. Infestation with stem weevils was not influenced by any temperature or land-use aspect considered, and natural pest regulation of pollen beetles in terms of parasitism rates of pollen beetle larvae was low (< 30%), except on three out of 29 plots. Nonetheless, we could identify conditions favouring low pollen beetle abundances per plant and high seed yields. Low pollen beetle densities were favoured by a constant oilseed rape area relative to the preceding year (5-km scale), whereas a strong reduction in area (> 40%) caused high pest densities (concentration effect). This occurred more frequently in warmer regions, due to drought around sowing, which contributed to increased pollen beetle numbers in those regions. Yet, in warmer regions, oilseed rape flowered early, which possibly led to partial escape from pollen beetle infestation in the most vulnerable bud stage. This is also suggested by higher seed yields of early flowering oilseed rape fields, but not per se at higher temperatures. Thus, early flowering (e.g. cultivar selection) and the interannual coordination of oilseed rape area offer opportunities for environmental-friendly pollen beetle management.
Chapter 5 – General discussion
Anthropogenic land-use and climate change are major threats to biodiversity, and consequently to ecosystem functions, although I could show that ecosystem functions such as herbivory and predation barely responded to temperature along a spatial gradient at present time. Yet, it is important to keep several points in mind: (i) The high rate of climate warming likely reduces the time that species will have to adapt to temperature in the future; (ii) Beyond mean temperatures, many aspects of climate will change; (iii) The compensation of biodiversity loss through functional redundancy in arthropod communities may be depleted at some point; (iv) Measures of ecosystem functions are limited by methodological filters, so that changes may be captured incompletely. Although much uncertainty of the effects of climate and land-use change on ecosystem functions remains, actions to halt biodiversity loss and to interfere with natural processes in an environmentally friendly way, e.g. reduction of herbivory on crops, are urgently needed. With this thesis, I contribute options to the environment-friendly regulation of herbivory, which are at least to some extent climate resilient, and at the same time make a contribution to halt biodiversity loss. Yet, more research and a transformation process is needed to make human action more sustainable. In terms of crop protection, this means that the most common method of treating pests with fast-acting pesticides is not necessarily the most sustainable. To realize sustainable strategies, collective efforts will be needed targeted at crop damage prevention through reducing pest populations and densities in the medium to long term. The sooner we transform human action from environmentally damaging to biodiversity promoting, the higher is our insurance asset that secures human well-being under a changing climate.
Im ersten Teil dieser Doktorarbeit wurde die kurz nach Elektroporation eintretende hämolytische Zellbewegung von humanen Erythrozyten erstmals quantitativ untersucht, um den zu Grunde liegenden Mechanismus aufzuklären. Die Ergebnisse legen nahe, dass die Bewegung aus dem Ausstoß von unter Druck stehendem Zytosol resultierte. Durch weitere Experimente wurde die Beteiligung des Nicht-Muskel-Myosins NMIIA am Aufbau des zytosolischen Überdrucks nachgewiesen. Ausgehend von diesen Ergebnissen wurde ein molekular-mechanischer bisher unbekannter NMII-basierter Mechanismus der rapiden Ghostbildung beschrieben. Diese Erkenntnis könnte biomedizinische Relevanz besitzen, da der Abbau von Erythrozyten in der Milz die Transformation zu Hb-armen Ghosts voraussetzt.
Der zweite Teil dieser Arbeit befasste sich mit dem Hirntumor Glioblastoma multiforme (GBM), dessen Rezidiv hauptsächlich auf Strahlenresistenz und Zellinvasion zurückzuführen ist. Deshalb wurde mittels hochauflösender Fluoreszenzmikroskopie (dSTORM) die Nanostruktur des DSB-Markers Histon γH2AX und des DNA-Reparaturfaktors DNA-PKcs in bestrahlten GBM-Zellen analysiert. Anhand von dSTORM-Rekonstruktionen wurde erstmals gezeigt, dass die beiden Proteine kaum Kolokalisation im Nanometerbereich aufweisen.
Zunehmend wird die anomale Expression von Membrantransportern aus der SLC-Familie mit der Migration von Krebszellen in Verbindung gebracht. Der finale Abschnitt befasste sich daher mit der subzellulären Lokalisierung der Transporterproteine SLC5A1 und SLC5A3 in GBM-Zellen, um ihre Beteiligung an der Zellmigration nachzuweisen. Dabei wurde erstmals gezeigt, dass der Leitsaum der untersuchten GBM-Zellen deutliches SLC5A1- und SLC5A3-Signal aufwies. Basierend auf diesen Befunden wurden den Transportern unterschiedliche Aufgaben bei der zellmigrativen lokalen Volumenregulation zugeschrieben. Somit ergänzen SLC5A1 und SLC5A3 das migrationsassoziierte Krebszell-Transportom.
Anthropogenic activities are causing air pollution. Amongst air pollutants, tropospheric ozone is a major threat to human health and ecosystem functioning. In this dissertation, I present three studies that aimed at increasing our knowledge on how plant exposure to ozone affects its reproduction and its interactions with insect herbivores and pollinators.
For this purpose, a new fumigation system was built and placed in a greenhouse. The annual plant Sinapis arvensis (wild mustard) was used as the model plant.
Plants were exposed to either 0 ppb (control) or 120 ppb of ozone, for variable amounts of time and at different points of their life cycle. After fumigation, plants were exposed to herbivores or pollinators in the greenhouse, or to both groups of insects in the field.
My research shows that ozone affected reproductive performance differently, depending on the timing of exposure: plants exposed at earlier ages had their reproductive fitness increased, while plants exposed later in their life cycle showed a tendency for reduced reproductive fitness. Plant phenology was a key factor influencing reproductive fitness: ozone accelerated flowering and increased the number of flowers produced by plants exposed at early ages, while plants exposed to ozone at later ages tended to have fewer flowers. On the other hand, the ozone-mediated changes in plant-insect interactions had little impact on plant reproductive success.
The strongest effect of ozone on plant-pollinator interactions was the change in the number of flower visits received per plant, which was strongly linked to the number of open flowers. This means that, as a rule, exposure of plants to ozone early in the life cycle resulted in a higher number of pollinator visits, while exposure later in the life cycle resulted in fewer flower visits by potential pollinators. An exception was observed: the higher number of visits performed by large syrphid flies to young ozone-exposed plants than to the respective control plants went beyond the increase in the number of open flowers in those plants. Also, honeybees spent more time per flower in plants exposed to ozone than on control plants, while other pollinators spent similar amounts of time in control and ozone-exposed plants. This guild-dependent preference for ozone-exposed plants may be due to species-specific preferences related to changes in the quality and quantity of floral rewards.
In the field, ozone-exposed plants showed only a tendency for increased colonization by sucking herbivores and slightly more damage by chewing herbivores than control plants. On the other hand, in the greenhouse experiment, Pieris brassicae butterflies preferred control plants over ozone-exposed plants as oviposition sites. Eggs laid on ozone-exposed plants took longer to hatch, but the chances of survival were higher. Caterpillars performed better in control plants than in ozone-exposed plants, particularly when the temperature was high.
Most of the described effects were dependent on the duration and timing of the ozone exposure and the observed temperature, with the strongest effects being observed for longer exposures and higher temperatures. Furthermore, the timing of exposure altered the direction of the effects.
The expected climate change provides ideal conditions for further increases in tropospheric ozone concentrations, therefore for stronger effects on plants and plant-insect interactions. Acceleration of flowering caused by plant exposure to ozone may put plant-pollinator interactions at risk by promoting desynchronization between plant and pollinator activities. Reduced performance of caterpillars feeding on ozone-exposed plants may weaken herbivore populations. On the other hand, the increased plant reproduction that results from exposing young plants to ozone may be a source of good news in the field of horticulture, when similar results would be achieved in high-value crops. However, plant response to ozone is highly species-specific. In fact, Sinapis arvensis is considered a weed and the advantage conferred by ozone exposure may increase its competitiveness, with negative consequences for crops or plant communities in general. Overall, plant exposure to ozone might constitute a threat for the balance of natural and agro-ecosystems.
Zinkoxid-Nanopartikel (ZnO-NP) finden in vielen Produkten des täglichen Verbrauchs Verwendung. Daten über die toxikologischen Eigenschaften von ZnO-NP werden kontrovers diskutiert. Die menschliche Haut ist in Bezug auf die ZnO-NP Exposition das wichtigste Kontakt-Organ. Intakte Haut stellt eine suffiziente Barriere gegenüber NP dar. Bei defekter Haut ist ein Kontakt zu den proliferierenden Stammzellen möglich, sodass diese als wichtiges toxikologische Ziel für NP darstellen. Das Ziel dieser Dissertation war die Bewertung der genotoxischen und zytotoxischen Effekte an humanen mesenchymalen Stammzellen (hMSC) durch niedrig dosierte ZnO-NP nach 24 stündiger Exposition, repetitiven Expositionen und im Langzeitversuch bis zu 6 Wochen. Zytotoxische Wirkungen von ZnO-NP wurden mit 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid-Test (MTT) gemessen. Darüber hinaus wurde die Genotoxizität durch den Comet-Assay bewertet. Zur Langzeitbeobachtung bis zu 6 Wochen wurde die Transmissionselektronenmikroskopie (TEM) verwendet. Zytotoxizität nach 24-stündiger ZnO-NP-Exposition war ab einer Konzentration von 50 µg/ml nachweisbar. Genotoxizität konnten bereits bei Konzentrationen von 1 und 10 µg/ml ZnO-NP beschrieben werden. Wiederholte Exposition verstärkte die Zyto-, aber nicht die Genotoxizität. Eine intrazelluläre NP-Akkumulation mit Penetration der Zellorganelle wurde bei einer Exposition bis zu 6 Wochen beobachtet. Die Ergebnisse deuten auf zytotoxische und genotoxisches Effekte von ZnO-NP hin. Bereits geringe Dosen von ZnO-NP können bei wiederholter Exposition toxische Wirkungen hervorrufen sowie eine langfristige Zellakkumulation. Diese Daten sollten bei der Verwendung von ZnO-NP an geschädigter Haut berücksichtigt werden.