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For all animals the cold represents a dreadful danger. In the event of severe heat loss, animals
fall into a chill coma. If this state persists, it is inevitably followed by death. In poikilotherms
(e.g. insects), the optimal temperature range is narrow compared to homeotherms
(e.g. mammals), resulting in a critical core temperature being reached more quickly. As a
consequence, poikilotherms either had to develop survival strategies, migrate or die. Unlike
the majority of insects, the Western honeybee (Apis mellifera) is able to organize itself into
a superorganism. In this process, worker bees warm and cool the colony by coordinated
use of their flight muscles. This enables precise control of the core temperature in the hive,
analogous to the core body temperature in homeothermic animals. However, to survive the
harsh temperatures in the northern hemisphere, the thermogenic mechanism of honeybees
must be in constant readiness. This mechanism is called shivering thermogenesis, in which
honeybees generate heat using their flight muscles.
My thesis presents the molecular and neurochemical background underlying shivering thermogenesis
in worker honeybees. In this context, I investigated biogenic amine signaling.
I found that the depletion of vesicular monoamines impairs thermogenesis, resulting in
a decrease in thoracic temperature. Subsequent investigations involving various biogenic
amines showed that octopamine can reverse this effect. This clearly indicates the involvement
of the octopaminergic system. Proceeding from these results, the next step was to elucidate
the honeybee thoracic octopaminergic system. This required a multidisciplinary approach to
ultimately provide profound insights into the function and action of octopamine at the flight
muscles. This led to the identification of octopaminergic flight muscle controlling neurons,
which presumably transport octopamine to the flight muscle release sites. These neurons
most likely innervate octopamine β receptors and their activation may stimulate intracellular
glycolytic pathways, which ensure sufficient energy supply to the muscles.
Next, I examined the response of the thoracic octopaminergic system to cold stress conditions.
I found that the thoracic octopaminergic system tends towards an equilibrium,
even though the initial stress response leads to fluctuations of octopamine signaling. My
results indicate the importance of the neuro-muscular octopaminergic system and thus the need for its robustness. Moreover, cold sensitivity was observed for the expression of one
transcript of the octopamine receptor gene AmOARβ2. Furthermore, I found that honeybees
without colony context show a physiological disruption within the octopaminergic system.
This disruption has profound effects on the honeybees protection against the cold.
I could show how important the neuro-muscular octopaminergic system is for thermogenesis
in honeybees. In this context, the previously unknown neurochemical modulation of the
honeybee thorax has now been revealed. I also provide a broad basis to conduct further
experiments regarding honeybee thermogenesis and muscle physiology.
T-Zell-aktivierende Formate, wie BiTE (bispecific T-cell engagers) Antikörper und CAR T Zellen haben in den vergangen Jahren die Therapiemöglichkeiten für Tumorpatienten erweitert. Diese Therapeutika verknüpfen T-Zellen mit malignen Zellen über je ein spezifisches Oberflächenmolekül und initiieren, über eine T-Zell-vermittelte Immunantwort, die Lyse der Tumorzelle. Tumorspezifische Antigene sind jedoch selten. Häufig werden Proteine adressiert, die neben den Tumorzellen auch auf gesunden Zellen exprimiert werden. Die Folgen sind toxische Effekte abseits der Tumorzellen auf Antigen-positiven gesunden Zellen (on target/off tumor), welche nicht nur die Dosis des Therapeutikums und dessen Effektivität limitieren, sondern zu geringen bis letalen Begleiterscheinungen führen können. Der Bedarf an effektiven Therapieformen mit geringen Nebenwirkungen ist folglich immer noch sehr hoch. Diese Lücke soll durch ein neues Antikörperformat, sogenannten Hemibodies, geschlossen werden. Hemibodies sind eine neue Klasse von T-Zell-aktivierenden Antikörpern, die sich gegen eine Antigenkombination und nicht einzelne Antigene auf Tumorzellen richten. Sie bestehen aus zwei komplementären Molekülen mit je einer Antigen-bindenden Sequenz, die entweder mit der leichten (VL) oder der schweren (VH) Kette eines T-Zell-aktivierenden anti CD3 Antikörpers fusioniert ist. Nur wenn beide Hemibody-Fragmente gleichzeitig in unmittelbarer Nähe an ihr jeweiliges Antigenepitop auf der Tumorzelle binden, komplementieren die beiden Antikörperkonstrukte über das geteilte anti-CD3 und bilden einen trivalenten T Zell aktivierenden Komplex aus. Diese funktionale Einheit rekrutiert T-Zellen zur Tumorzelle und induzierte die T-Zell-vermittelte Lyse der malignen Zelle.
Im Rahmen der vorliegenden Arbeit wurden geeignete Antigenkombinationen identifiziert und die erste effektive und spezifische Hemibody-basierte Immuntherapie gegen das Multiple Myelom (MM), ohne Nebenwirkungen auf Antigen-einfach-positiven gesunden Zellen, entwickelt. Basierend auf einer umfangreichen Analyse von Kandidaten-Antigenen wurden Kombinationen aus bekannten MM Zielmolekülen, wie BCMA, CD38, CD138, CD229 und SLAMF7, und für das MM unbekannte Oberflächenmolekülen, wie CHRM5 und LAX1, untersucht. Gegen die vielversprechendsten Antigene wurden Hemibodies entwickelt und produziert. Im Zusammenhang mit Analysen zur Produzierbarkeit sowie biochemischen und funktionalen Charakterisierungen, konnte aus 75 initialen Hemibody-Kombinationen drei Kombinationen mit geeigneten Eigenschaften identifiziert werden. Die Bindung von zwei Hemibody-Partnern auf der Oberfläche der MM Zelle führte zur Ausbildung eines trivalenten T-Zell-rekrutierenden Komplexes. Dieser initiierte nachfolgend über eine T-Zell-vermittelte Immunantwort die spezifische Lyse der malignen Zellen, ohne die Viabilität von Antigen-einfach-positiven gesunden Körper- oder Effektor-Zellen zu beeinflussen. Zusätzlich führte eine Hemibody-Therapie in vivo in einem NOD SCID MM-Mausmodel innerhalb von 7 Tagen zur kompletten Remission der MM Zellen. Diese Daten zeigten Hemibodies als ein neues, sehr vielversprechendes Antikörperformat für eine effektive und tumorspezifische Immuntherapie mit potentiell geringen Nebenwirkungen.
Cellular growth and proliferation are among the most important processes for cells and
organisms. One of the major determinants of these processes is the amount of proteins
and consequently also the amount of ribosomes. Their synthesis involves several hundred
proteins and four different ribosomal RNA species, is highly coordinated and very
energy-demanding. However, the molecular mechanims of transcriptional regulation of
the protein-coding genes involved, is only poorly understood in mammals.
In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming
to identify previously unknown transcriptional regulators of ribosome biogenesis
factors (RiBis), which are important for the assembly and maturation of ribosomes,
and ribosomal proteins (RPs), which are ribosomal components themself. With that
approach and follow-up (validation) experiments, ALDOA and RBM8A among others,
could be identified as regulators of ribosome biogenesis.
Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter
driven reporters on protein and transcript level, as well as to a downregulation
of ribosome biogenesis gene transcripts and of mRNAs of other genes important for
proliferation.
Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent
downregulation of one of the fluorescent reporters, but this regulation was independent
of the promoter driving the expression of the reporter. However, acute protein
depletion experiments in combination with nascent RNA sequencing (4sU-Seq)
revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon
acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of
RP genes, but also to other chromatin regions. Total POL II or elongating and initiating
POL II levels were not altered upon acute RBM8A depletion.
These data provide a starting point for further research on the mechanisms of transcriptional
regulation of RP and RiBi genes in mammals.
Honeybees are among the few animals that rely on eusociality to survive. While the
task of queen and drones is only reproduction, all other tasks are accomplished by sterile
female worker bees. Different tasks are mostly divided by worker bees of different ages
(temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and
nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding
the hive entrance. The oldest honeybees eventually leave the hive to forage for resources
until they die. However, uncontrollable circumstances might force the colony to adapt or
perish. For example, the introduced Varroa destructor mite or the deformed wing virus
might erase a lot of in-hive bees. On the other hand, environmental events might kill a
lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of
task allocation must be a priority for a honeybee colony.
In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of
the honeybee division of labor, especially in conjunction with honeybee malnourishment.
The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to
be important for the transition from in-hive tasks to foraging. I have studied in detail
expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus
lay on gene expression in the honeybee brain and fat body. I found an increase in the
AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to
foraging and a decrease in expression of the other genes in both tissues. Interestingly,
I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the
honeybee fat body during orientation flights. Furthermore, I closely observed juvenile
hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers
increased with the transition from in-hive tasks to foraging and triglyceride levels decreased.
Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level.
For example, foragers are more responsive towards light and sucrose. I proposed that
modulation via biogenic amines, especially via octopamine and tyramine, can increase
or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified
1
using electroretinography. In addition, I studied the behavioral response of the bees to
light using a phototaxis assay. Injecting octopamine increased the receptor response and
tyramine decreased it. Also, both groups of honeybees showed an increased phototactic
response when injected with octopamine and a decreased response when injected with
tyramine, independent of locomotion.
Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to
cope with the missing resources. Furthermore, larval undernourishment has also been
implied to speed up the transition from in-hive bees to foragers, due to increasing levels
of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared
honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to
bees reared under the standard in-vitro rearing diet. However, first I had to investigate
whether the in-vitro rearing method affects adult honeybees.
I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging
earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was
unaffected.
Afterwards, I investigated the effects of different larval diets on adult honeybee workers.
I found no effects of malnourishment on behavioral or physiological factors besides a
difference in weight. Honeybee weight increased with increasing amounts of larval food,
but the effect seemed to vanish after a week.
These results show the complexity and adaptability of the honeybee division of labor.
They show the importance of the biogenic amines octopamine and tyramine and of the
corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on
nursing behavior, but that it speeds up the transition from nursing to foraging, showing
strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems
that larval malnourishment can be easily compensated during the early lifetime of adult
honeybees.
The western honeybee (Apis mellifera) is widely known as the honey producer and pollinator managed by beekeepers but neglected as a wild bee species. Central European honeybee populations have been anthropogenically disturbed since about 1850 through introgression and moderate artificial selection but have never been truly domesticated due to a lack of mating control. While their decline in the wild was historically attributed to the scarcity of nesting cavities, a contemporary view considers the invasion of the parasitic mite Varroa destructor in the 1970s as the major driver. However, there are no longitudinal population data available that could substantiate either claim. Based on the insight that introduced European honeybees form viable wild populations in eastern North America and reports on the occurrence of wild-living colonies from various European countries, we systematically studied the ecology of wild-living honeybees in Germany. First, we investigated whether wild-living honeybees colonising German forests form a self-sustaining population. Second, we asked how the parasite burden of wild-living colonies relates to that of managed colonies. And third, we explored whether the winter mortality of wild-living colonies is associated with parasite burden, nest depredation, or the lack of resources on the landscape scale.
Between 2017 and 2021, we monitored listed trees with black woodpecker cavities for honeybees in the managed forests of three study regions (Swabian Alb, counties Coburg and Lichtenfels, county Weilheim-Schongau). Continuity of occupation was determined using microsatellite genetic markers. Wild-living colonies predictably colonised forests in summer, when about 10% of all cavities were occupied. The annual colony survival rate and colony lifespan (based on N=112 colonies) were 10.6% and 0.6 years, with 90% of colonies surviving summer (July–September), 16% surviving winter (September–April), and 72% surviving spring (April–July). The average maximum and minimum colony densities were 0.23 (July) and 0.02 (April) colonies per km^2. During the (re-)colonisation of forests in spring, swarms preferred cavities that had already been occupied by other honeybee colonies. We estimate the net reproductive rate of the population to be R0= 0.318, meaning that it is currently not self-sustaining but maintained by the annual immigration of swarms from managed hives. The wild-living colonies are feral in a behavioural sense.
We compared the occurrence of 18 microparasites among feral colonies (N=64) and managed colonies (N=74) using qPCR. Samples were collected in four regions (the three regions mentioned above and the city of Munich) in July 2020; they consisted of 20 workers per colony captured at flight entrances. We distinguished five colony types representing differences in colony age and management histories. Besides strong regional variation, feral colonies consistently hosted fewer microparasite taxa (median: 5, range 1–8) than managed colonies (median: 6, range 4–9) and had different parasite communities. Microparasites that were notably less prevalent among feral colonies were Trypanosomatidae, Chronic bee paralysis virus, and Deformed wing viruses A and B. In the comparison of five colony types, parasite burden was lowest in newly founded feral colonies, intermediate in overwintered feral colonies and managed nucleus colonies, and highest in overwintered managed colonies and hived swarms. This suggests that the natural mode of colony reproduction by swarming, which creates pauses in brood production, and well-dispersed nests, which reduce horizontal transmission, explain the reduced parasite burden in feral compared to managed colonies.
To explore the roles of three potential drivers of feral colony winter mortality, we combined colony observations gathered during the monitoring study with data on colony-level parasite burden, observations and experiments on nest depredation, and landscape analyses. There was no evidence for an effect of summertime parasite burden on subsequent winter mortality: colonies that died (N=57) did not have a higher parasite burden than colonies that survived (N=10). Camera traps (N=15) installed on cavity trees revealed that honeybee nests are visited by a range of vertebrate species throughout the winter at rates of up to 10 visits per week. Four woodpecker species, great tits, and pine martens acted as true nest depredators. The winter survival rate of colonies whose nest entrances were protected by screens of wire mesh (N=32) was 50% higher than that of colonies with unmanipulated entrances (N=40). Analyses of land cover maps revealed that the landscapes surrounding surviving colonies (N=19) contained on average 6.4 percentage points more resource-rich cropland than landscapes surrounding dying colonies (N=94).
We estimate that tens of thousands of swarms escape from apiaries each year to occupy black woodpecker cavities and other hollow spaces in Germany and that feral colonies make up about 5% of the regional honeybee populations. They are unlikely to contribute disproportionately to the spread of bee diseases. Instead, by spatially complementing managed colonies, they contribute to the pollination of wild plants in forests. Honeybees occupying tree cavities likely have various effects on forest communities by acting as nest site competitors or prey, and by accumulating biomass in tree holes. Nest depredation (a consequence of a lack of well-protected nest sites) and food resource limitation seem to be more important than parasites in hampering feral colony survival. The outstanding question is how environmental and intrinsic factors interact in preventing population establishment. Nest boxes with movable frames could be used to better study the environmental drivers of feral colonies’ mortality. Pairs of wild (self-sustaining) and managed populations known to exist outside Europe could provide answers to whether modern apiculture creates honeybee populations maladapted to life in the wild. In Europe, large continuous forests might represent evolutionary refuges for wild honeybees.
Most of the studies in cell biology primarily focus on models from the opisthokont group of eukaryotes. However, opisthokonts do not encompass the full diversity of eukaryotes. Thus, it is necessary to broaden the research focus to other organisms to gain a comprehensive understanding of basic cellular processes shared across the tree of life. In this sense, Trypanosoma brucei, a unicellular eukaryote, emerges as a viable alternative. The collaborative efforts in genome sequencing and protein tagging over the past two decades have significantly expanded our knowledge on this organism and have provided valuable tools to facilitate a more detailed analysis of this parasite. Nevertheless, numerous questions still remain.
The survival of T. brucei within the mammalian host is intricately linked to the endo-lysosomal system, which plays a critical role in surface glycoprotein recycling, antibody clearance, and plasma membrane homeostasis. However, the dynamics of the duplication of the endo-lysosomal system during T. brucei proliferation and its potential relationship with plasma membrane growth remain poorly understood. Thus, as the primary objective, this thesis explores the endo-lysosomal system of T. brucei in the context of the cell cycle, providing insights on cell surface growth, endosome duplication, and clathrin recruitment. In addition, the study revisits ferritin endocytosis to provide quantitative data on the involvement of TbRab proteins (TbRab5A, TbRab7, and TbRab11) and the different endosomal subpopulations (early, late, and recycling endosomes, respectively) in the transport of this fluid-phase marker. Notably, while these subpopulations function as distinct compartments, different TbRabs can be found within the same region or structure, suggesting a potential physical connection between the endosomal subpopulations. The potential physical connection of endosomes is further explored within the context of the cell cycle and, finally, the duplication and morphological plasticity of the lysosome are also investigated. Overall, these findings provide insights into the dynamics of plasma membrane growth and the coordinated duplication of the endo-lysosomal system during T. brucei proliferation. The early duplication of endosomes suggests their potential involvement in plasma membrane growth, while the late duplication of the lysosome indicates a reduced role in this process. The recruitment of clathrin and TbRab GTPases to the site of endosome formation supports the assumption that the newly formed endosomal system is active during cell division and, consequently, indicates its potential role in plasma membrane homeostasis.
Furthermore, considering the vast diversity within the Trypanosoma genus, which includes ~500 described species, the macroevolution of the group was investigated using the combined information of the 18S rRNA gene sequence and structure. The sequence-structure analysis of T. brucei and other 42 trypanosome species was conducted in the context of the diversity of Trypanosomatida, the order in which trypanosomes are placed. An additional analysis focused on Trypanosoma highlighted key aspects of the group’s macroevolution. To explore these aspects further, additional trypanosome species were included, and the changes in the Trypanosoma tree topology were analyzed. The sequence-structure phylogeny confirmed the independent evolutionary history of the human pathogens T. brucei and Trypanosoma cruzi, while also providing insights into the evolution of the Aquatic clade, paraphyly of groups, and species classification into subgenera.
Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus
(2023)
Der Name der transposablen Elemente beruht auf ihrer Fähigkeit, ihre genomische Position verändern zu können. Durch Chromosomenaberrationen, Insertionen oder Deletionen können ihre genomischen Transpositionen genetische Instabilität verursachen. Inwieweit sie darüber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterführung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der ältesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des bösartigen schwarzen Hautkrebses, welcher nach wie vor die tödlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit beschäftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.
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.
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.
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.
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.
Coxiella burnetii, a Gram negative obligate intracellular bacterium, is the causative
agent of Q fever. It has a world wide distribution and has been documented to
be capable of causing infections in several domestic animals, livestock species,
and human beings. Outbreaks of Q fever are still being observed in livestock
across animal farms in Europe, and primary transmission to humans still oc-
curs especially in animal handlers. Public health authorities in some countries
like Germany are required by law to report human acute cases denoting the
significance of the challenge posed by C. burnetii to public health.
In this thesis, I have developed a platform alongside methods to address the
challenges of genomic analyses of C. burnetii for typing purposes. Identification
of C. burnetii isolates is an important task in the laboratory as well as in the
clinics and genotyping is a reliable method to identify and characterize known
and novel isolates. Therefore, I designed and implemented several methods
to facilitate the genotyping analyses of C. burnetii genomes in silico via a web
platform. As genotyping is a data intensive process, I also included additional
features such as visualization methods and databases for interpretation and
storage of obtained results. I also developed a method to profile the resistome
of C. burnetii isolates using a machine learning approach. Data about antibiotic
resistance in C. burnetii are scarce majorly due to its lifestyle and the difficulty
of cultivation in laboratory media. Alternative methods that rely on homology
identification of resistance genes are also inefficient in C. burnetii, hence, I
opted for a novel approach that has been shown to be promising in other
bacteria species. The applied method relied on an artificial neural network as
well as amino acid composition of position specific scoring matrix profile for
feature extraction. The resulting model achieved an accuracy of ≈ 0.96 on test
data and the overall performance was significantly higher in comparison to
existing models. Finally, I analyzed two new C. burnetii isolates obtained from
an outbreak in Germany, I compared the genome to the RSA 493 reference
isolate and found extensive deletions across the genome landscape.
This work has provided a new digital infrastructure to analyze and character-
ize C. burnetii genomes that was not in existence before and it has also made a
significant contribution to the existing information about antibiotic resistance
genes in C. burnetii.
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.
The monarch butterfly (Danaus plexippus) performs one of the most astonishing behaviors in the animal kingdom: every fall millions of these butterflies leave their breeding grounds in North Amerika and migrate more than 4.000 km southwards until they reach their overwintering habitat in Central Mexico. To maintain their migratory direction over this enormous distance, the butterflies use a time-compensated sun compass. Beside this, skylight polarization, the Earth’s magnetic field and specific mountain ranges seem to guide the butterflies as well the south. In contrast to this fascinating orientation ability, the behavior of the butterflies in their non-migratory state received less attention. Although they do not travel long distances, they still need to orient themselves to find food, mating partners or get away from competitors. The aim of the present doctoral thesis was to investigate use of visual cues for orientation in migrating as well as non-migrating monarch butterflies. For this, field experiments investigating the migration of the butterflies in Texas (USA) were combined with experiments testing the orientation performance of non-migratory butterflies in Germany.
In the first project, I recorded the heading directions of tethered butterflies during their annual fall migration. In an outdoor flight simulator, the butterflies maintained a southwards direction as long as they had a view of the sun’s position. Relocating the position of the sun by 180° using a mirror, revealed that the sun is the animals’ main orientation reference. Furthermore, I demonstrated that when the sun is blocked and a green light stimulus (simulated sun) is introduced, the animals interpreted this stimulus as the ‘real’ sun. However, this cue was not sufficient to set the migratory direction when simulated as the only visual cue in indoor experiments. When I presented the butterflies a linear polarization pattern additionally to the simulated sun, the animals headed in the correct southerly direction showing that multiple skylight cues are required to guide the butterflies during their migration.
In the second project, I, furthermore, demonstrated that non-migrating butterflies are able to maintain a constant direction with respect to a simulated sun. Interestingly, they ignored the spectral component of the stimulus and relied on the intensity instead. When a panoramic skyline was presented as the only orientation reference, the butterflies maintained their direction only for short time windows probably trying to stabilize their flight based on optic-flow information. Next, I investigated whether the butterflies combine celestial with local cues by simulating a sun stimulus together with a panoramic skyline. Under this conditions, the animals’ directedness was increased demonstrating that they combine multiple visual cues for spatial orientation.
Following up on the observation that a sun stimulus resulted in a different behavior than the panoramic skyline, I investigated in my third project which orientation strategies the butterflies use by presenting different simulated cues to them. While a bright stripe on a dark background elicited a strong attraction of the butterflies steering in the direction of the stimulus, the inverted version of the stimulus was used for flight stabilization. In contrast to this, the butterflies maintained arbitrary directions with a high directedness with respect to a simulated sun. In an ambiguous scenery with two identical stimuli (two bright stripes, two dark stripes, or two sun stimuli) set 180° apart, a constant flight course was only achieved when two sun stimuli were displayed suggesting an involvement of the animals’ internal compass. In contrast, the butterflies used two dark stripes for flight stabilization and were alternatingly attracted by two bright stripes. This shows that monarch butterflies use stimulus-dependent orientation strategies and gives the first evidence for different neuronal pathways controlling the output behavior.
The fusion of methods from several disciplines is a crucial component of scientific development. Artificial Neural Networks, based on the principle of biological neuronal networks, demonstrate how nature provides the best templates for technological advancement. These innovations can then be employed to solve the remaining mysteries of biology, including, in particular, processes that take place on microscopic scales and can only be studied with sophisticated techniques. For instance, direct Stochastic Optical Reconstruction Microscopy combines tools from chemistry, physics, and computer science to visualize biological processes at the molecular level. One of the key components is the computer-aided reconstruction of super-resolved images. Improving the corresponding algorithms increases the quality of the generated data, providing further insights into our biology. It is important, however, to ensure that the heavily processed images are still a reflection of reality and do not originate in random artefacts.
Expansion microscopy is expanding the sample by embedding it in a swellable hydrogel. The method can be combined with other super-resolution techniques to gain additional resolution. We tested this approach on microtubules, a well-known filamentous reference structure, to evaluate the performance of different protocols and labelling techniques.
We developed LineProfiler an objective tool for data collection. Instead of collecting perpendicular profiles in small areas, the software gathers line profiles from filamentous structures of the entire image. This improves data quantity, quality and prevents a biased choice of the evaluated regions. On the basis of the collected data, we deployed theoretical models of the expected intensity distribution across the filaments. This led to the conclusion that post-expansion labelling significantly reduces the labelling error and thus, improves the data quality. The software was further used to determine the expansion factor and arrangement of synaptonemal complex data.
Automated Simple Elastix uses state-of-the-art image alignment to compare pre- and post-expansion images. It corrects linear distortions occurring under isotropic expansion, calculates a structural expansion factor and highlights structural mismatches in a distortion map. We used the software to evaluate expanded fungi and NK cells. We found that the expansion factor differs for the two structures and is lower than the overall expansion of the hydrogel.
Assessing the fluorescence lifetime of emitters used for direct Stochastic Optical Reconstruction Microscopy can reveal additional information about the molecular environment or distinguish dyes emitting with a similar wavelength. The corresponding measurements require a confocal scanning of the sample in combination with the fluorescent switching of the underlying emitters. This leads to non-linear, interrupted Point Spread Functions. The software ReCSAI targets this problem by combining the classical algorithm of compressed sensing with modern methods of artificial intelligence. We evaluated several different approaches to combine these components and found, that unrolling compressed sensing into the network architecture yields the best performance in terms of reconstruction speed and accuracy.
In addition to a deep insight into the functioning and learning of artificial intelligence in combination with classical algorithms, we were able to reconstruct the described non-linearities with significantly improved resolution, in comparison to other state-of-the-art architectures.
Die Fanconi-Anämie (FA) ist eine seltene, heterogene Erbkrankheit. Sie weist ein sehr variables klinisches Erscheinungsbild auf, das sich aus angeborenen Fehlbildungen, hämatologischen Funktionsstörungen, einem erhöhten Risiko für Tumorentwicklung und endokrinen Pathologien zusammensetzt. Die Erkrankung zählt zu den genomischen Instabilitätssyndromen, welche durch eine fehlerhafte DNA-Schadensreparatur gekennzeichnet sind. Bei der FA zeigt sich dies vor allem in einer charakteristischen Hypersensitivität gegenüber DNA-quervernetzenden Substanzen (z. B. Mitomycin C, Cisplatin). Der zelluläre FA-Phänotyp zeichnet sich durch eine erhöhte Chromosomenbrüchigkeit und einen Zellzyklusarrest in der G2-Phase aus. Diese Charakteristika sind bereits spontan vorhanden und werden durch Induktion mit DNA-quervernetzenden Substanzen verstärkt. Der Gendefekt ist dabei in einem der 22 bekannten FA-Gene (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U, -V, -W) oder in noch unbekannten FA-Genen zu finden. Die FA-Gendefekte werden mit Ausnahme von FANCR (dominant-negative de novo Mutationen) und FANCB (X-chromosomal) autosomal rezessiv vererbt. Die FA-Genprodukte bilden zusammen mit weiteren Proteinen den FA/BRCA-Signalweg. Das Schlüsselereignis dieses Signalwegs stellt die Monoubiquitinierung von FANCD2 und FANCI (ID2-Komplex) dar. Ausgehend davon lässt sich zwischen upstream- und downstream-gelegenen FA-Proteinen unterscheiden. Letztere sind direkt an der DNA-Schadensreparatur beteiligt. Zu den upstream-gelegenen Proteinen zählt der FA-Kernkomplex, der sich aus bekannten FA-Proteinen und aus FA-assoziierten-Proteinen (FAAPs) zusammensetzt und für die Monoubiquitinierung des ID2-Komplexes verantwortlich ist. Für FAAPs wurden bisher keine pathogenen humanen Mutationen beschrieben. Zu diesen Proteinen gehört auch FAAP100, das mit FANCB und FANCL innerhalb des FA-Kernkomplexes den Subkomplex LBP100 bildet.
Durch die vorliegende Arbeit wurde eine nähere Charakterisierung dieses Proteins erreicht. In einer Amnion-Zelllinie konnte eine homozygote Missense-Mutation identifiziert werden. Der Fetus zeigte einen typischen FA-Phänotyp und auch seine Zellen wiesen charakteristische FA-Merkmale auf. Der zelluläre Phänotyp ließ sich durch FAAP100WT komplementieren, sodass die Pathogenität der Mutation bewiesen war. Unterstützend dazu wurden mithilfe des CRISPR/Cas9-Systems weitere FAAP100-defiziente Zelllinien generiert. Diese zeigten ebenfalls einen typischen FA-Phänotyp, welcher sich durch FAAP100WT komplementieren ließ. Die in vitro-Modelle dienten als Grundlage dafür, die Funktion des FA-Kernkomplexes im Allgemeinen und die des Subkomplexes LBP100 im Besonderen besser zu verstehen. Dabei kann nur durch intaktes FAAP100 das LBP100-Modul gebildet und dieses an die DNA-Schadensstelle transportiert werden. Dort leistet FAAP100 einen essentiellen Beitrag für den FANCD2-Monoubiquitinierungsprozess und somit für die Aktivierung der FA-abhängigen DNA-Schadensreparatur. Um die Funktion von FAAP100 auch in vivo zu untersuchen, wurde ein Faap100-/--Mausmodell generiert, das einen mit anderen FA-Mausmodellen vergleichbaren, relativ schweren FA-Phänotyp aufwies. Aufgrund der Ergebnisse lässt sich FAAP100 als neues FA-Gen klassifizieren. Zudem wurde die Rolle des Subkomplexes LBP100 innerhalb des FA-Kernkomplexes weiter aufgeklärt. Beides trägt zu einem besseren Verständnis des FA/BRCA-Signalweges bei. Ein weiterer Teil der vorliegenden Arbeit beschäftigt sich mit der Charakterisierung von FAAP100138, einer bisher nicht validierten Isoform von FAAP100. Durch dieses Protein konnte der zelluläre FA-Phänotyp von FAAP100-defizienten Zelllinien nicht komplementiert werden, jedoch wurden Hinweise auf einen dominant-negativen Effekt von FAAP100138 auf den FA/BRCA-Signalweg gefunden. Dies könnte zu der Erklärung beitragen, warum und wie der Signalweg, beispielsweise in bestimmtem Gewebearten, herunterreguliert wird. Zudem wäre eine Verwendung in der Krebstherapie denkbar.
Forests are multi-functional system, which have to fulfil different objectives at the same time. The main functions include the production of wood, storage of carbon, the promotion of biological diversity and the provision of recreational space. Yet, global forests are affected by large and intense natural disturbances, like bark beetle infestations. While natural disturbances threaten wood production and are perceived as ‘catastrophe’ diminishing recreational value, biodiversity can benefit from the disturbance-induced changes in forest structures. This trade-off poses a dilemma to managers of bark beetle affected stands, particularly in protected areas designated to both nature conservation and recreation. Forest landscapes need a sustainable management concept aligning these different objectives. In order to support this goal with scientific knowledge, the aim of this work is to analyse ecological and social effects along a gradient of different disturbance severities. In this context, I studied the effects of a disturbance severity gradient on the diversity of different taxonomic groups including vascular plants, mosses, lichens, fungi, arthropods and birds in five national parks in Central Europe. To analyse the recreational value of the landscape I conducted visitor surveys in the same study areas in which the biodiversity surveys were performed. To analyse possible psychological or demographic effects on preferences for certain disturbance intensities, an additional online survey was carried out.
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.
Summary
Chapters I & II: General Introduction & General Methods
Agriculture is confronted with a rampant loss of biodiversity potentially eroding ecosystem service potentials and adding up to other stressors like climate change or the consequences of land-use change and intensive management. To counter this ‘biodiversity crisis’, agri-environment schemes (AES) have been introduced as part of ecological intensification efforts. These AES combine special management regimes with the establishment of tailored habitats to create refuges for biodiversity in agricultural landscapes and thus ensure biodiversity mediated ecosystem services such as pest control. However, little is known about how well different AES habitats fulfil this purpose and whether they benefit ecosystem services in adjacent crop fields. Here I investigated how effective different AES habitats are for restoring biodiversity in different agricultural landscapes (Chapter V) and whether they benefit natural pest control in adjacent oilseed rape (Chapter VI) and winter cereal fields (Chapter VII). I recorded biodiversity and pest control potentials using a variety of different methods (Chapters II, V, VI & VII). Moreover, I validated the methodology I used to assess predator assemblages and predation rates (Chapters III & IV).
Chapter III: How to record ground dwelling predators?
Testing methodology is critical as it ensures scientific standards and trustworthy results. Pitfall traps are widely used to record ground dwelling predators, but little is known about how different trap types affect catches. I compared different types of pitfall traps that had been used in previous studies in respect to resulting carabid beetle assemblages. While barrier traps collected more species and deliver more complete species inventories, conventional simple pitfall traps provide reliable results with comparatively little handling effort. Placing several simple pitfall traps in the field can compensate the difference while still saving handling effort.
Chapter IV: How to record predation rates?
A plethora of methods has been proposed and used for recording predation rates, but these have rarely been validated before use. I assessed whether a novel approach to record predation, the use of sentinel prey cards with glued on aphids, delivers realistic results. I compared different sampling efforts and showed that obtained predation rates were similar and could be linked to predator (carabid beetle) densities and body-sizes (a proxy often used for food intake rates). Thus, the method delivers reliable and meaningful predation rates.
Chapter V: Do AES habitats benefit multi-taxa biodiversity?
The main goal of AES is the conservation of biodiversity in agricultural landscapes. I investigated how effectively AES habitats with different temporal continuity fulfil this goal in differently structured landscapes. The different AES habitats investigated had variable effects on local biodiversity. Temporal continuity of AES habitats was the most important predictor with older, more temporally continuous habitats harbouring higher overall biodiversity and different species assemblages in most taxonomic groups than younger AES habitats. Results however varied among taxonomic groups and natural enemies were equally supported by younger habitats. Semi-natural habitats in the surrounding landscape and AES habitat size were of minor importance for local biodiversity and had limited effects. This stresses that newly established AES habitats alone cannot restore farmland biodiversity. Both AES habitats as well as more continuous semi-natural habitats synergistically increase overall biodiversity in agricultural landscapes.
Chapter VI: The effects of AES habitats on predators in adjacent oilseed rape fields
Apart from biodiversity conservation, ensuring ecosystem service delivery in agricultural landscapes is a crucial goal of AES. I therefore investigated the effects of adjacent AES habitats on ground dwelling predator assemblages in oilseed rape fields. I found clear distance decay effects from the field edges into the field centres on both richness and densities of ground dwelling predators. Direct effects of adjacent AES habitats on assemblages in oilseed rape fields however were limited and only visible in functional traits of carabid beetle assemblages. Adjacent AES habitats doubled the proportion of predatory carabid beetles indicating a beneficial role for pest control. My results show that pest control potentials are largest close to the field edges and beneficial effects are comparably short ranged.
Chapter VII: The effects of AES habitats on pest control in adjacent cereal fields
Whether distance functions and potential effects of AES habitats are universal across crops is unknown. Therefore, I assessed distance functions of predators, pests, predation rates and yields after crop rotation in winter cereals using the same study design as in the previous year. Resulting distance functions were not uniform and differed from those found in oilseed rape in the previous year, indicating that the interactions between certain adjacent habitats vary with habitat and crop types. Distance functions of cereal-leaf beetles (important cereal pests) and parasitoid wasps were moreover modulated by semi-natural habitat proportion in the surrounding landscapes. Field edges buffered assemblage changes in carabid beetle assemblages over crop rotation confirming their important function as refuges for natural enemies. My results emphasize the beneficial role of field edges for pest control potentials. These findings back the calls for smaller field sizes and more diverse, more heterogeneously structured agricultural landscapes.
Chapter VIII: General Discussion
Countering biodiversity loss and ensuring ecosystem service provision in agricultural landscapes is intricate and requires strategic planning and restructuring of these landscapes. I showed that agricultural landscapes could benefit maximally from (i) a mixture of AES habitats and semi-natural habitats to support high levels of overall biodiversity and from (ii) smaller continuously managed agricultural areas (i.e. smaller field sizes or the insertion of AES elements within large fields) to maximize natural pest control potentials in crop fields. I propose a mosaic of younger AES habitats and semi-natural habitats to support ecosystem service providers and increase edge density for ecosystem service spillover into adjacent crops. The optimal extent and density of this network as well as the location in which AES and semi-natural habitats interact most beneficially with adjacent crops need further investigation. My results provide a further step towards more sustainable agricultural landscapes that simultaneously allow biodiversity to persist and maintain agricultural production under the framework of ecological intensification.
New insights into the histone variant H2A.Z incorporation pathway in \(Trypanosoma\) \(brucei\)
(2022)
The histone variant H2A.Z is a key player in transcription regulation in eukaryotes. Histone acetylations by the NuA4/TIP60 complex are required to enable proper incorporation of the histone variant and to promote the recruitment of other complexes and proteins required for transcription initiation. The second key player in H2A.Z-mediated transcription is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant. By the time this project started little was known about H2A.Z in the unicellular parasite Trypanosoma brucei. Like in other eukaryotes H2A.Z was exclusively found in the transcription start sites of the polycistronic transcription units where it keeps the chromatin in an open conformation to enable RNA-polymerase II-mediated transcription. Previous studies showed the variant colocalizing with an acetylation of lysine on histone H4 and a methylation of lysine 4 on histone H3. Data indicated that HAT2 is linked to H2A.Z since it is required for acetylation of lyinse 10 on histone H4. A SWR1-like complex and a complex homologous to the NuA4/TIP60 could not be identified yet. This study aimed at identifying a SWR1-like remodelling complex in T. brucei and at identifying a protein complex orthologous to NuA4/TIP60 as well as at answering the question whether HAT2 is part of this complex or not. To this end, I performed multiple mass spectrometry-coupled co-Immunoprecipitation assays with potential subunits of a SWR1 complex, HAT2 and a putative homolog of a NuA4/TIP60 subunit. In the course of these experiments, I was able to identify the TbSWR1 complex. Subsequent cell fractionation and chromatin immunoprecipitation-coupled sequencing analysis experiments confirmed, that this complex is responsible for the incorporation of the histone variant H2A.Z in T. brucei. In addition to this chromatin remodelling complex, I was also able to identify two histone acetyltransferase complexes assembled around HAT1 and HAT2. In the course of my study data were published by the research group of Nicolai Siegel that identified the histone acetyltransferase HAT2 as being responsible for histone H4 acetylation, in preparation to promote H2A.Z incorporation. The data also indicated that HAT1 is responsible for acetylation of H2A.Z. According to the literature, this acetylation is required for proper transcription initiation. Experimental data generated in this study indicated, that H2A.Z and therefore TbSWR1 is involved in the DNA double strand break response of T. brucei. The identification of the specific complex composition of all three complexes provided some hints about how they could interact with each other in the course of transcription regulation and the DNA double strand break response. A proximity labelling approach performed with one of the subunits of the TbSWR1 complex identified multiple transcription factors, PTM writers and proteins potentially involved in chromatin maintenance. Overall, this work will provide some interesting insights about the composition of the complexes involved in H2A.Z incorporation in T. brucei. Furthermore, it is providing valuable information to set up experiments that could shed some light on RNA-polymerase II-mediated transcription and chromatin remodelling in T. brucei in particular and Kinetoplastids in general.