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Background
The knowledge of metabolic pathways and fluxes is important to understand the adaptation of organisms to their biotic and abiotic environment. The specific distribution of stable isotope labelled precursors into metabolic products can be taken as fingerprints of the metabolic events and dynamics through the metabolic networks. An open-source software is required that easily and rapidly calculates from mass spectra of labelled metabolites, derivatives and their fragments global isotope excess and isotopomer distribution.
Results
The open-source software “Least Square Mass Isotopomer Analyzer” (LS-MIDA) is presented that processes experimental mass spectrometry (MS) data on the basis of metabolite information such as the number of atoms in the compound, mass to charge ratio (m/e or m/z) values of the compounds and fragments under study, and the experimental relative MS intensities reflecting the enrichments of isotopomers in 13C- or 15 N-labelled compounds, in comparison to the natural abundances in the unlabelled molecules. The software uses Brauman’s least square method of linear regression. As a result, global isotope enrichments of the metabolite or fragment under study and the molar abundances of each isotopomer are obtained and displayed.
Conclusions
The new software provides an open-source platform that easily and rapidly converts experimental MS patterns of labelled metabolites into isotopomer enrichments that are the basis for subsequent observation-driven analysis of pathways and fluxes, as well as for model-driven metabolic flux calculations.
Cosmology often uses intricate formulas and mathematics to derive new theories and concepts. We do something different in this paper: We look at biological processes and derive from these heuristics so that the revised cosmology agrees with astronomical observations but does also agree with standard biological observations. We show that we then have to replace any type of singularity at the start of the universe by a condensation nucleus and that the very early period of the universe usually assumed to be inflation has to be replaced by a period of rapid crystal growth as in Weiss magnetization domains.
Impressively, these minor modifications agree well with astronomical observations including removing the strong inflation perturbations which were never observed in the recent BICEP2 experiments. Furthermore, looking at biological principles suggests that such a new theory with a condensation nucleus at start and a first rapid phase of magnetization-like growth of the ordered, physical laws obeying lattice we live in is in fact the only convincing theory of the early phases of our universe that also is compatible with current observations.
We show in detail in the following that such a process of crystal creation, breaking of new crystal seeds and ultimate evaporation of the present crystal readily leads over several generations to an evolution and selection of better, more stable and more self-organizing crystals. Moreover, this explains the “fine-tuning” question why our universe is fine-tuned to favor life: Our Universe is so self-organizing to have enough offspring and the detailed physics involved is at the same time highly favorable for all self-organizing processes including life.
This biological theory contrasts with current standard inflation cosmologies. The latter do not perform well in explaining any phenomena of sophisticated structure creation or self-organization. As proteins can only thermodynamically fold by increasing the entropy in the solution around them we suggest for cosmology a condensation nucleus for a universe can form only in a “chaotic ocean” of string-soup or quantum foam if the entropy outside of the nucleus rapidly increases. We derive an interaction potential for 1 to n-dimensional strings or quantum-foams and show that they allow only 1D, 2D, 4D or octonion interactions. The latter is the richest structure and agrees to the E8 symmetry fundamental to particle physics and also compatible with the ten dimensional string theory E8 which is part of the M-theory. Interestingly, any other interactions of other dimensionality can be ruled out using Hurwitz compositional theorem. Crystallization explains also extremely well why we have only one macroscopic reality and where the worldlines of alternative trajectories exist: They are in other planes of the crystal and for energy reasons they crystallize mostly at the same time, yielding a beautiful and stable crystal. This explains decoherence and allows to determine the size of Planck´s quantum h (very small as separation of crystal layers by energy is extremely strong).
Ultimate dissolution of real crystals suggests an explanation for dark energy agreeing with estimates for the “big rip”. The halo distribution of dark matter favoring galaxy formation is readily explained by a crystal seed starting with unit cells made of normal and dark matter.
That we have only matter and not antimatter can be explained as there may be right handed mattercrystals and left-handed antimatter crystals. Similarly, real crystals are never perfect and we argue that exactly such irregularities allow formation of galaxies, clusters and superclusters. Finally, heuristics from genetics suggest to look for a systems perspective to derive correct vacuum and Higgs Boson energies.
In a nice assay published in Nature in 1993 the physicist Richard God III started from a human observer and made a number of witty conclusions about our future prospects giving estimates for the existence of the Berlin Wall, the human race and all the rest of the universe. In the same spirit, we derive implications for "the meaning of life, the universe and all the rest" from few principles. Adams´ absurd answer "42" tells the lesson "garbage in / garbage out" - or suggests that the question is non calculable. We show that experience of "meaning" and to decide fundamental questions which can not be decided by formal systems imply central properties of life: Ever higher levels of internal representation of the world and an escalating tendency to become more complex. An observer, "collecting observations" and three measures for complexity are examined. A theory on living systems is derived focussing on their internal representation of information. Living systems are more complex than Kolmogorov complexity ("life is NOT simple") and overcome decision limits (Gödel theorem) for formal systems as illustrated for cell cycle. Only a world with very fine tuned environments allows life. Such a world is itself rather complex and hence excessive large in its space of different states – a living observer has thus a high probability to reside in a complex and fine tuned universe.
Our universe may have started by Qubit decoherence:
In quantum computers, qubits have all their states undefined during calculation and become defined as output (“decoherence”). We study the transition from an uncontrolled, chaotic quantum vacuum (“before”) to a clearly interacting “real world”. In such a cosmology, the Big Bang singularity is replaced by a condensation event of interacting strings. This triggers a crystallization process. This avoids inflation, not fitting current observations: increasing long-range interactions limit growth and crystal symmetries ensure the same laws of nature and basic symmetries over the whole crystal. Tiny mis-arrangements provide nuclei of superclusters and galaxies and crystal structure allows arrangement of dark (halo regions) and normal matter (galaxy nuclei) for galaxy formation. Crystals come and go: an evolutionary cosmology is explored: entropic forces from the quantum soup “outside” of the crystal try to dissolve it. This corresponds to dark energy and leads to a “big rip” in 70 Gigayears. Selection for best growth and condensation events over generations of crystals favors multiple self-organizing processes within the crystal including life or even conscious observers in our universe. Philosophically this theory shows harmony with nature and replaces absurd perspectives of current cosmology.
Independent of cosmology, we suggest that a “real world” (so our everyday macroscopic world) happens only inside a crystal. “Outside” there is wild quantum foam and superposition of all possibilities. In our crystallized world the vacuum no longer boils but is cooled down by the crystallization event, space-time exists and general relativity holds. Vacuum energy becomes 10**20 smaller, exactly as observed in our everyday world. We live in a “solid” state, within a crystal, the n quanta which build our world have all their different m states nicely separated. There are only nm states available for this local “multiverse”. The arrow of entropy for each edge of the crystal forms one fate, one world-line or clear development of our world, while layers of the crystal are different system states. Mathematical leads from loop quantum gravity (LQG) point to required interactions and potentials. Interaction potentials for strings or loop quanta of any dimension allow a solid, decoherent state of quanta challenging to calculate. However, if we introduce here the heuristic that any type of physical interaction of strings corresponds just to a type of calculation, there is already since 1898 the Hurwitz theorem showing that then only 1D, 2D, 4D and 8D (octonions) allow complex or hypercomplex number calculations. No other hypercomplex numbers and hence dimensions or symmetries are possible to allow calculations without yielding divisions by zero. However, the richest solution allowed by the Hurwitz theorem, octonions, is actually the observed symmetry of our universe, E8. Standard physics such as condensation, crystallization and magnetization but also solid-state physics and quantum computing allow us to show an initial mathematical treatment of our new theory by LQG to describe the cosmological state transformations by equations, and, most importantly, point out routes to parametrization of free parameters looking at testable phenomena, experiments and formulas that describe processes of crystallization, protein folding, magnetization, solid-state physics and quantum computing. This is presented here for LQG, for string theory it would be more elegant but was too demanding to be shown here.
Note: While my previous Opus server preprint “A new cosmology of a crystallization process (decoherence) from the surrounding quantum soup provides heuristics to unify general relativity and quantum physics by solid state physics” (https://doi.org/10.25972/OPUS-23076) deals with the same topics and basic formulas, this new version is improved: clearer in title, better introduction, more stringent in its mathematics and improved discussion of the implications including quantum computing, hints for parametrization and connections to LQG and other current cosmological efforts.
This 5th of June 2021 version is again an OPUS preprint, but this will next be edited for Archives https://arxiv.org.
Why is our universe so fine-tuned? In this preprint we discuss that this is not a strange accident but that fine-tuned universes can be considered to be exceedingly large if one counts the number of observable different states (i.e. one aspect of the more general preprint http://www.opus-bayern.de/uni-wuerzburg/volltexte/2009/3353/). Looking at parameter variation for the same set of physical laws simple and complex processes (including life) and worlds in a multiverse are compared in simple examples. Next the anthropocentric principle is extended as many conditions which are generally interpreted anthropocentric only ensure a large space of different system states. In particular, the observed over-tuning beyond the level for our existence is explainable by these system considerations. More formally, the state space for different systems becomes measurable and comparable looking at their output behaviour. We show that highly interacting processes are more complex then Chaitin complexity, the latter denotes processes not compressible by shorter descriptions (Kolomogorov complexity). The complexity considerations help to better study and compare different processes (programs, living cells, environments and worlds) including dynamic behaviour and can be used for model selection in theoretical physics. Moreover, the large size (in terms of different states) of a world allowing complex processes including life can in a model calculation be determined applying discrete histories from quantum spin-loop theory. Nevertheless there remains a lot to be done - hopefully the preprint stimulates further efforts in this area.
In a modified inflation scenario we replace the “big bang” by a condensation event in an eternal all-compassing big ocean of free qubits in our modified cosmology. Interactions of qubits in the qubit ocean are rare. If they happen, they provide a nucleus for a new universe as the qubits become decoherent and freeze-out into defined bit ensembles. Second, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth) – the crystal unit cell guarantees same symmetries everywhere. Hence, the textbook inflation scenario to explain the same laws of nature in our domain is replaced by the crystal unit cell of the crystal formed. We give here only the perspective or outline of this modified inflation theory, as the detailed mathematical physics behind this has still to be formulated and described.
Interacting qubits solidify, quantum entropy decreases (but increases in the ocean around). The interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After that very early events, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements, but more importantly can explain well by such a type of cosmological crystallization instead of inflation the early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: no annihilation of antimatter necessary, rather the unit cell of our crystal universe has a matter handedness avoiding anti-matter.
We prove a triggering of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, this means that in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below h dash liquidity left). However, the E8 symmetry of heterotic string theory has six rolled-up, small dimensions which help to keep the qubit crystal together and will never expand.
Finally, we give first energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction and gravity derive from the permeating qubit-interaction field in the crystal. Hence, vacuum energy gets low inside the qubit crystal. Condensed mathematics may advantageously help to model free (many states denote the same qubit) and bound qubits in phase space.
Proteins fold in water and achieve a clear structure despite a huge parameter space. Inside a (protein) crystal you have everywhere the same symmetries as there is everywhere the same unit cell. We apply this to qubit interactions to do fundamental physics:
We modify cosmological inflation: we replace the big bang by a condensation event in an eternal all-encompassing ocean of free qubits. Rare interactions of qubits in the ocean provide a nucleus or seed for a new universe (domain), as the qubits become decoherent and freeze-out into defined bit ensembles. Next, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth). The crystal unit cell guarantees same symmetries (and laws of nature) everywhere inside the crystal, no inflation scenario is needed.
Interacting qubits solidify, quantum entropy decreases in the crystal, but increases outside in the ocean. The interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After this very early modified steps, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements.
Applying the Hurwitz theorem to qubits we prove that initiation of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. We describe a six-bit-ensemble toy model of qubit interaction and the repulsive forces of qubits for ultra-short distances. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below Planck´s quantum is liquidity left). The E8 symmetry of heterotic string theory has six curled-up, small dimensions. These keep the qubit crystal together and never expand. We give energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit crystal formation.
Implications are fundamental answers, e.g. why there is fine-tuning for life-friendliness, why there is string theory with rolled-up dimension and so many free parameters. We explain by cosmological crystallization instead of inflation the early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: the unit cell of our crystal universe has a matter handedness avoiding anti-matter. Importantly, crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness. Vacuum energy gets appropriate low inside the crystal by its qubit binding energy, outside it is 10**20 higher. Scalar fields for color interaction/confinement and gravity could be derived from the qubit-interaction field.
Protein folding achieves a clear solution structure in a huge parameter space (the so-called protein folding problem). Proteins fold in water, and get by this a highly ordered structure. Finally, inside a protein crystal for structure resolution, you have everywhere the same symmetries as there is everywhere the same unit cell. We apply this to qubit interactions to do fundamental physics:
in a modified cosmology, we replace the big bang by a condensation event in an eternal all-encompassing ocean of free qubits. Interactions of qubits in the qubit ocean are quite rare but provide a nucleus or seed for a new universe (domain) as the qubits become decoherent and freeze-out into defined bit ensembles. Second, we replace inflation by a crystallization event triggered by the nucleus of interacting qubits to which rapidly more and more qubits attach (like in everyday crystal growth). The crystal unit cell guarantees same symmetries everywhere inside the crystal. The textbook inflation scenario to explain the same laws of nature in our domain is replaced by the unit cell of the crystal formed.
Interacting qubits solidify, quantum entropy decreases (but increases in the ocean around). In a modified inflation scenario, the interacting qubits form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. Then standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements. We explain by cosmological crystallization instead of inflation: early creation of large-scale structure of voids and filaments, supercluster formation, galaxy formation, and the dominance of matter: the unit cell of our crystal universe has a matter handedness avoiding anti-matter.
We prove initiation of qubit interactions can only be 1,2,4 or 8-dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
The phase space of the crystal agrees with the standard model of the basic four forces for n quanta. It includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients. However, in our four dimensions there is only one bit overlap to neighbor states left (almost solid, only below Planck quantum there is liquidity left). The E8 symmetry of heterotic string theory has six curled-up, small dimensions which help to keep the qubit crystal together and will never expand.
Mathematics focusses on the Hurwitz proof applied to qubit interaction, a toy model of qubit interaction and repulsive forces of qubits. Vacuum energy gets appropriate low inside the crystal. We give first energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction/confinement and gravity are derived from the qubit-interaction field.
The phase space for the standard model of the basic four forces for n quanta includes all possible ensemble combinations of their quantum states m, a total of n**m states. Neighbor states reach according to transition possibilities (S-matrix) with emergent time from entropic ensemble gradients.
We replace the “big bang” by a condensation event (interacting qubits become decoherent) and inflation by a crystallization event – the crystal unit cell guarantees same symmetries everywhere. Interacting qubits solidify and form a rapidly growing domain where the n**m states become separated ensemble states, rising long-range forces stop ultimately further growth. After that very early events, standard cosmology with the hot fireball model takes over. Our theory agrees well with lack of inflation traces in cosmic background measurements, large-scale structure of voids and filaments, supercluster formation, galaxy formation, dominance of matter and life-friendliness.
We prove qubit interactions to be 1,2,4 or 8 dimensional (agrees with E8 symmetry of our universe). Repulsive forces at ultrashort distances result from quantization, long-range forces limit crystal growth. Crystals come and go in the qubit ocean. This selects for the ability to lay seeds for new crystals, for self-organization and life-friendliness.
We give energy estimates for free qubits vs bound qubits, misplacements in the qubit crystal and entropy increase during qubit decoherence / crystal formation. Scalar fields for color interaction and gravity derive from the permeating qubit-interaction field. Hence, vacuum energy gets low only inside the qubit crystal. Condensed mathematics may advantageously model free / bound qubits in phase space.
In this view point we do not change cosmology after the hot fireball starts (hence agrees well with observation), but the changed start suggested and resulting later implications lead to an even better fit with current observations (voids, supercluster and galaxy formation; matter and no antimatter) than the standard model with big bang and inflation: In an eternal ocean of qubits, a cluster of qubits crystallizes to defined bits. The universe does not jump into existence (“big bang”) but rather you have an eternal ocean of qubits in free super-position of all their quantum states (of any dimension, force field and particle type) as permanent basis. The undefined, boiling vacuum is the real “outside”, once you leave our everyday universe. A set of n Qubits in the ocean are “liquid”, in very undefined state, they have all their m possibilities for quantum states in free superposition. However, under certain conditions the qubits interact, become defined, and freeze out, crystals form and give rise to a defined, real world with all possible time series and world lines. GR holds only within the crystal. In our universe all n**m quantum possibilities are nicely separated and crystallized out to defined bit states: A toy example with 6 qubits each having 2 states illustrates, this is completely sufficient to encode space using 3 bits for x,y and z, 1 bit for particle type and 2 bits for its state. Just by crystallization, space, particles and their properties emerge from the ocean of qubits, and following the arrow of entropy, time emerges, following an arrow of time and expansion from one corner of the toy universe to everywhere else. This perspective provides time as emergent feature considering entropy: crystallization of each world line leads to defined world lines over their whole existence, while entropy ensures direction of time and higher representation of high entropy states considering the whole crystal and all slices of world lines. The crystal perspective is also economic compared to the Everett-type multiverse, each qubit has its m quantum states and n qubits interacting forming a crystal and hence turning into defined bit states has only n**m states and not more states. There is no Everett-type world splitting with every decision but rather individual world trajectories reside in individual world layers of the crystal. Finally, bit-separated crystals come and go in the qubit ocean, selecting for the ability to lay seeds for new crystals. This self-organizing reproduction selects over generations also for life-friendliness. Mathematical treatment introduces quantum action theory as a framework for a general lattice field theory extending quantum chromo dynamics where scalar fields for color interaction and gravity have to be derived from the permeating qubit-interaction field. Vacuum energy should get appropriately low by the binding properties of the qubit crystal. Connections to loop quantum gravity, string theory and emergent gravity are discussed. Standard physics (quantum computing; crystallization, solid state physics) allow validation tests of this perspective and will extend current results.
We explore a cosmology where the Big Bang singularity is replaced by a condensation event of interacting strings. We study the transition from an uncontrolled, chaotic soup (“before”) to a clearly interacting “real world”. Cosmological inflation scenarios do not fit current observations and are avoided. Instead, long-range interactions inside this crystallization event limit growth and crystal symmetries ensure the same laws of nature and basic symmetries over our domain. Tiny mis-arrangements present nuclei of superclusters and galaxies and crystal structure leads to the arrangement of dark (halo regions) and normal matter (galaxy nuclei) so convenient for galaxy formation. Crystals come and go, allowing an evolutionary cosmology where entropic forces from the quantum soup “outside” of the crystal try to dissolve it. These would correspond to dark energy and leads to a big rip scenario in 70 Gy. Preference of crystals with optimal growth and most condensation nuclei for the next generation of crystals may select for multiple self-organizing processes within the crystal, explaining “fine-tuning” of the local “laws of nature” (the symmetry relations formed within the crystal, its “unit cell”) to be particular favorable for self-organizing processes including life or even conscious observers in our universe.
Independent of cosmology, a crystallization event may explain quantum-decoherence in general: The fact, that in our macroscopic everyday world we only see one reality. This contrasts strongly with the quantum world where you have coherence, a superposition of all quantum states. We suggest that a “real world” (so our everyday macroscopic world) happens only in our domain, i.e. inside a crystal. “Outside” of our domain and our observable universe there is the quantum soup of boiling quantum foam and superposition of all possibilities. In our crystallized world the vacuum no longer boils but is cooled down by the crystallization event and hence is 10**20 smaller, exactly as observed in our everyday world. As we live in a “solid” state, within a crystal, the different quanta which build our world have all their different states nicely separated. This theory postulates there are only n quanta and m states available for them (there is no Everett-like ever splitting multiverse after each decision). In the solid state we live in, there is decoherence, the states are nicely separated. The arrow of entropy for each edge of the crystal forms one fate, one worldline or clear development of a world, while the layers of the crystal are different system states.
Some mathematical leads from loop quantum gravity point to required interactions and potentials. A complete mathematical treatment of this unified theory is far too demanding currently. Interaction potentials for strings or membranes of any dimension allow a solid state of quanta, so allowing decoherence in our observed world are challenging to calculate. However, if we introduce here the heuristic that any type of physical interaction of strings corresponds just to a type of calculation, there is already since 1898 the Hurwitz theorem showing that then only 1D, 2D, 4D and 8D (octonions) allow complex or hypercomplex number calculations. No other hypercomplex numbers and hence dimensions or symmetries are possible to allow calculations without yielding divisions by zero. However, the richest solution allowed by the Hurwitz theorem, octonions, is actually the observed symmetry of our universe, E8.
MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.
Natural enemies have been shown to be effective agents for controlling insect pests in crops. However, it remains unclear how different natural enemy guilds contribute to the regulation of pests and how this might be modulated by landscape context. In a field exclusion experiment in oilseed rape (OSR), we found that parasitoids and ground-dwelling predators acted in a complementary way to suppress pollen beetles, suggesting that pest control by multiple enemies attacking a pest during different periods of its occurrence in the field improves biological control efficacy. The density of pollen beetle significantly decreased with an increased proportion of non-crop habitats in the landscape. Parasitism had a strong effect on pollen beetle numbers in landscapes with a low or intermediate proportion of non-crop habitats, but not in complex landscapes. Our results underline the importance of different natural enemy guilds to pest regulation in crops, and demonstrate how biological control can be strengthened by complementarity among natural enemies. The optimization of natural pest control by adoption of specific management practices at local and landscape scales, such as establishing non-crop areas, low-impact tillage, and temporal crop rotation, could significantly reduce dependence on pesticides and foster yield stability through ecological intensification in agriculture.
Background
Spermatogenesis is a complex differentiation process that involves the successive and simultaneous execution of three different gene expression programs: mitotic proliferation of spermatogonia, meiosis, and spermiogenesis. Testicular cell heterogeneity has hindered its molecular analyses. Moreover, the characterization of short, poorly represented cell stages such as initial meiotic prophase ones (leptotene and zygotene) has remained elusive, despite their crucial importance for understanding the fundamentals of meiosis.
Results
We have developed a flow cytometry-based approach for obtaining highly pure stage-specific spermatogenic cell populations, including early meiotic prophase. Here we combined this methodology with next generation sequencing, which enabled the analysis of meiotic and postmeiotic gene expression signatures in mouse with unprecedented reliability. Interestingly, we found that a considerable number of genes involved in early as well as late meiotic processes are already on at early meiotic prophase, with a high proportion of them being expressed only for the short time lapse of lepto-zygotene stages. Besides, we observed a massive change in gene expression patterns during medium meiotic prophase (pachytene) when mostly genes related to spermiogenesis and sperm function are already turned on. This indicates that the transcriptional switch from meiosis to post-meiosis takes place very early, during meiotic prophase, thus disclosing a higher incidence of post-transcriptional regulation in spermatogenesis than previously reported. Moreover, we found that a good proportion of the differential gene expression in spermiogenesis corresponds to up-regulation of genes whose expression starts earlier, at pachytene stage; this includes transition protein-and protamine-coding genes, which have long been claimed to switch on during spermiogenesis. In addition, our results afford new insights concerning X chromosome meiotic inactivation and reactivation.
Conclusions
This work provides for the first time an overview of the time course for the massive onset and turning off of the meiotic and spermiogenic genetic programs. Importantly, our data represent a highly reliable information set about gene expression in pure testicular cell populations including early meiotic prophase, for further data mining towards the elucidation of the molecular bases of male reproduction in mammals.
One of the fascinating features of meiotic prophase I, is the highly conserved
vigorous movements of homologous chromosomes. These movements are
critical for the success of essential events as homologs alignment, synapsis and
recombination. Several organisms studied so far, including mammals, worms,
yeast and plants achieve these movements by anchoring the chromosome ends
to specialized sites in the nuclear envelope (NE). This attachment requires
telomere adaptor proteins which have to date been identified in fission yeast
and mice.
The mouse meiosis-specific telomere adaptor proteins TERB1, TERB2, and
MAJIN are involved in the attachment of ubiquitous shelterin telomere to the
LINC complex, in an analogous mechanism as those described in fission yeast.
Despite the essential role of meiosis-specific telomere adaptor proteins, the
precise mechanism of anchorage of telomeres to the nuclear envelope, as well
as their evolutionary history, are still not well understood. Therefore, the main
aim of this thesis is to investigate the organization of the mouse meiosis-specific
telomere adaptor complex TERB1-TERB2-MAJIN and its evolutionary history.
In the first part of this thesis high-resolution Structured Illumination Microscopy
(SIM), indirect immunofluorescence and Telo-FISH on mouse spermatocytes
were used to determine precisely how the telomere complex proteins are
localized with relation to the shelterin telomeric TRF1 protein and telomeric
DNA. During zygotene and pachytene stages staining patterns revealed
extensively overlapping of meiotic telomere complex proteins distributions in
which TERB2 organization is more heterogeneous than TERB1 and MAJIN at
the chromosome ends. Further, TRF1 localization was shown at the side of
lateral elements (LEs) ends with grasp-like distribution surrounding the TERB1
and MAJIN signals in zygotene and pachytene stages. Interestingly, telomeric
DNA was shown to be laterally distributed and partially overlapping with the
more central distribution displayed by meiotic telomere complex proteins of LEs
ends. The combination of these results allowed to describe an alternative model
of the telomere attachment to the NE during meiotic prophase I. The second part of this thesis, analyses mouse TERB1, TERB2, and MAJIN
evolutionary history. The lack of similarity between mouse and fission yeast
meiotic-specific telomere adaptor proteins has raised the question about the
origin of this specific complex through evolution. To identify mouse TERB1,
TERB2, and MAJIN putative orthologues, computational approaches and
phylogenetic analyses were performed. Besides, to test their potential function
during meiosis, expression studies were conducted. From these analyses, it was
revealed that mouse meiosis-specific telomere complex is ancient, as it
originated as early as eumetazoans pointing to a single origin. The absence of
any homologs in Nematoda and only a few candidates detected in Arthropoda
for meiosis-specific telomere complex, seemed, that these proteins have been
lost/replaced or highly diversified in these lineages. Remarkably, TERB1, TERB2,
and MAJIN protein domains involved in the formation of the complex as well as
those required for the interaction with the telomere shelterin protein and the
LINC complexes revealed high sequence similarity across all clades. Finally,
gene expression in the cnidarian Hydra Vulgaris provided evidence that the
TERB1-TERB2-MAJIN complex is selectively expressed in the germline
suggesting conservation of meiotic functions across metazoan evolution.
In summary, this thesis provides significant insights into the meiosis-specific
telomere complex mechanism to engage telomeres to the nuclear envelope and
the elucidation of its origin in metazoans.
The mode of action of phloretin and its analogs on the permeability of natural membranes for neutral and charged molecules, such as urea, glucose and chloride has been characterized 25 years ago. In contrast to signal molecules with primary effects on transport systems of natural membranes, phloretin also affects model membranes, i.e., artificial membranes, which do not contain proteins. Since the dipole potential reducing effect of phloretin on mono- and bilayers has been found, it became clear that its primary effect must be a biophysical one: phloretin adsorbs to lipid layers and changes biophysical parameters of these layers. The aim of this work was the characterization of the interaction between the surface-active molecule phloretin and artificial lipid layers. We were able to describe structural and functional parameters of the model systems mono- and bilayer as functions of one or few variables. One of these parameters, the dipole potential, measured as a function of the aqueous phloretin concentration, allowed a critical examination of the Langmuir adsorption model that has been postulated for the interaction between phloretin and lipid layers. Surface pressure versus area per lipid molecule isotherms and surface (dipole) potential change versus area per lipid molecule isotherms, measured at lipid monolayers, allowed a structural description of the phloretin-lipid interaction: phloretin integrates into monolayers dependent on the surface pressure and the phase state of the lipid. Calorimetric measurements confirmed the integration of phloretin into membranes because of the strong decrease of the phase transition temperature, but they also showed that the cooperativity of phase transition is hardly affected, even at very high amounts of phloretin in the membrane. Obviously the interaction between phloretin and lipids is restricted to the head groups, an integration into the hydrocarbon layer is unlikely. 2H NMR measurements with spherical unilamellar vesicles of headgroup-deuterated lipid showed changed quadrupolar splittings indicating the interaction between phloretin and headgroups of the lipids.
L-type voltage-gated calcium channels (LTCC) are heteromultimeric membrane proteins that allow Ca2+ entry into the cell upon plasma membrane depolarization. The β subunit of voltage-dependent calcium channels (Cavβ) binds to the α-interaction domain in the pore-forming α1 subunit and regulates the trafficking and biophysical properties of these channels. Of the four Cavβ isoforms, Cavβ2 is predominantly expressed in cardiomyocytes. This subunit associates with diverse proteins besides LTCC, but the molecular composition of the Cavβ2 nanoenvironments in cardiomyocytes is yet unresolved. Here, we used a protein-labeling technique in living cells based on an engineered ascorbate peroxidase 2 (APEX2). In this strategy, Cavβ2b was fused to APEX2 and expressed in adult rat cardiomyocytes using an adenovirus system. Nearby proteins covalently labeled with biotin-phenol were purified using streptavidin-coated beads and identified by mass spectrometry (MS). Analysis of the in situ APEX2-based biotin labeling by MS revealed 61 proteins located in the nanoenvironments of Cavβ2b, with a high specificity and consistency in all the replicates. These proteins are involved in diverse cellular functions such as cellular trafficking, sarcomere organization and excitation-contraction coupling. Among these proteins, we demonstrated an interaction between the ryanodine receptor 2 (RyR2) and Cavβ2b, probably coupling LTCC and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src homology 3 (SH3) domain of Cavβ2b and is necessary for an effective pacing frequency‐dependent increase in Ca2+-induced Ca2+ release in cardiomyocytes.
In this thesis two genes involved in causing neurodegenerative phenotypes in Drosophila are described. olk (omb-like), a futsch allele, is a micotubule associated protein (MAP) which is homologous to MAP1B and sws (swiss cheese) a serine esterase of yet unknown function within the nervous system. The lack of either one of these genes causes progressive neurodegeneration in two different ways. The sws mutant is characterized by general degeneration of the adult nervous system, glial hyperwrapping and neuronal apoptosis. Deletion of NTE (neuropathy target esterase), the SWS homolog in vertebrates, has been shown to cause a similar pattern of progressive neural degeneration in mice. NTE reacts with organophosphates causing axonal degeneration in humans. Inhibition of vertebrate NTE is insufficient to induce paralyzing axonal degeneration, a reaction called "aging reaction" is necessary for the disease to set in. It is hypothesized that a second "non-esterase" function of NTE is responsible for this phenomenon. The biological function of SWS within the nervous system is still unknown. To characterize the function of this protein several transgenic fly lines expressing different mutated forms of SWS were established. The controlled expression of altered SWS protein with the GAL4/UAS system allowed the analysis of isolated parts of the protein that were altered in the respective constructs. The characterization of a possible non-esterase function was of particular interest in these experiments. One previously described aberrant SWS construct lacking the first 80 amino acids (SWSΔ1-80) showed a deleterious, dominant effect when overexpressed and was used as a model for organophosphate (OP) intoxication. This construct retains part of its detrimental effect even without catalytically active serine esterase function. This strongly suggests that there is another characteristic to SWS that is not defined solely by its serine esterase activity. Experiments analyzing the lipid contents of sws mutant, wildtype (wt) and SWS overexpressing flies gave valuable insights into a possible biological function of SWS. Phosphatidylcholine, a major component of cell membranes, accumulates in sws mutants whereas it is depleted in SWS overexpressing flies. This suggests that SWS is involved in phosphatidylcholine regulation. The produced α-SWS antibody made it possible to study the intracellular localization of SWS. Images of double stainings with ER (endoplasmic reticulum) markers show that SWS is in great part localized to the ER. This is consistent with findings of SWS/ NTE localization in yeast and mouse cells. The olk mutant also shows progressive neurodegeneration but it is more localized to the olfactory system and mushroom bodies. Regarding specific cell types it seemed that specifically the projection neurons (PNs) are affected. A behavioral phenotype consisting of poor olfactory memory compared to wt is also observed even before histologically visible neurodegeneration sets in. Considering that the projection neurons connect the antennal lobes to the mushroom bodies, widely regarded as the "learning center", this impairment was expected. Three mutants where identified (olk1-3) by complementation analysis with the previously known futschN94 allele and sequencing of the coding sequence of olk1 revealed a nonsense mutation early in the protein. Consistent with the predicted function of Futsch as a microtubule associated protein (MAP), abnormalities are most likely due to a defective microtubule network and defects in axonal transport. In histological sections a modified cytoskeletal network is observed and western blots confirm a difference in the amount of tubulin present in the olk1 mutant versus the wt. The elaboration of neuronal axons and dendrites is dependent on a functional cytoskeleton. Observation of transport processes in primary neural cultures derived from olk1 mutant flies also showed a reduction of mitochondrial transport. Interaction with the fragile X mental retardation gene (dfmr1) was observed with the olk mutant. A dfmr1/ olk1 double mutant shows an ameliorated phenotype compared to the olk1 single mutant. tau, another MAP gene, was also shown to be able to partially rescue the olk1 mutant.
In many animals the ability to navigate over long distances is an important prerequisite for foraging. For example, it is widely accepted that desert ants and honey bees, but also mammals, use path integration for finding the way back to their home site. It is however a matter of a long standing debate whether animals in addition are able to acquire and use so called cognitive maps. Such a 'map', a global spatial representation of the foraging area, is generally assumed to allow the animal to find shortcuts between two sites although the direct connection has never been travelled before. Using the artificial neural network approach, here we develop an artificial memory system which is based on path integration and various landmark guidance mechanisms ( a bank of individual and independent landmark-defined memory elements). Activation of the individual memory elements depends on a separate motivation network and an, in part, asymmetrical lateral inhibition network. The information concerning the absolute position of the agent is present, but resides in a separate memory that can only be used by the path integration subsystem to control the behaviour, but cannot be used for computational purposes with other memory elements of the system. Thus, in this simulation there is no neural basis of a cognitive map. Nevertheless, an agent controlled by this network is able to accomplish various navigational tasks known from ants and bees and often discussed as being dependent on a cognitive map. For example, map-like behaviour as observed in honey bees arises as an emergent property from a decentralized system. This behaviour thus can be explained without referring to the assumption that a cognitive map, a coherent representation of foraging space, must exist. We hypothesize that the proposed network essentially resides in the mushroom bodies of the insect brain.
The microbial communities that live inside the human gastrointestinal tract -the human gut
microbiome- are important for host health and wellbeing. Characterizing this new “organ”,
made up of as many cells as the human body itself, has recently become possible through
technological advances. Metagenomics, the high-throughput sequencing of DNA directly from
microbial communities, enables us to take genomic snapshots of thousands of microbes living
together in this complex ecosystem, without the need for isolating and growing them.
Quantifying the composition of the human gut microbiome allows us to investigate its
properties and connect it to host physiology and disease. The wealth of such connections was
unexpected and is probably still underestimated. Due to the fact that most of our dietary as well
as medicinal intake affects the microbiome and that the microbiome itself interacts with our
immune system through a multitude of pathways, many mechanisms have been proposed to
explain the observed correlations, though most have yet to be understood in depth.
An obvious prerequisite to characterizing the microbiome and its interactions with the host is
the accurate quantification of its composition, i.e. determining which microbes are present and
in what numbers they occur. Historically, standard practices have existed for sample handling,
DNA extraction and data analysis for many years. However, these were generally developed for
single microbe cultures and it is not always feasible to implement them in large scale
metagenomic studies. Partly because of this and partly because of the excitement that new
technology brings about, the first metagenomic studies each took the liberty to define their own
approach and protocols. From early meta-analysis of these studies it became clear that the
differences in sample handling, as well as differences in computational approaches, made
comparisons across studies very difficult. This restricts our ability to cross-validate findings of
individual studies and to pool samples from larger cohorts. To address the pressing need for
standardization, we undertook an extensive comparison of 21 different DNA extraction methods
as well as a series of other sample manipulations that affect quantification. We developed a
number of criteria for determining the measurement quality in the absence of a mock
community and used these to propose best practices for sampling, DNA extraction and library
preparation. If these were to be accepted as standards in the field, it would greatly improve
comparability across studies, which would dramatically increase the power of our inferences
and our ability to draw general conclusions about the microbiome.
Most metagenomics studies involve comparisons between microbial communities, for example
between fecal samples from cases and controls. A multitude of approaches have been proposed
to calculate community dissimilarities (beta diversity) and they are often combined with
various preprocessing techniques. Direct metagenomics quantification usually counts
sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to
technology-inherent differences in sampling depth, normalizing counts is necessary, for
instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by
subsampling. To derive a single value for community (dis-)similarity, multiple distance
measures have been proposed. Although it is theoretically difficult to benchmark these
approaches, we developed a biologically motivated framework in which distance measures can
be evaluated. This highlights the importance of data transformations and their impact on the
measured distances.
Building on our experience with accurate abundance estimation and data preprocessing
techniques, we can now try and understand some of the basic properties of microbial
communities. In 2011, it was proposed that the space of genus level variation of the human gut
microbial community is structured into three basic types, termed enterotypes. These were
described in a multi-country cohort, so as to be independent of geography, age and other host
properties. Operationally defined through a clustering approach, they are “densely populated
areas in a multidimensional space of community composition”(source) and were proposed as a
general stratifier for the human population. Later studies that applied this concept to other
datasets raised concerns about the optimum number of clusters and robustness of the
clustering approach. This heralded a long standing debate about the existence of structure and
the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in
the context of the vastly increased amounts of available data. We propose a refined framework
in which the different types should be thought of as weak attractors in compositional space and
we try to implement an approach to determining which attractor a sample is closest to. To this
end, we train a classifier on a reference dataset to assign membership to new samples. This way,
enterotypes assignment is no longer dataset dependent and effects due to biased sampling are
minimized. Using a model in which we assume the existence of three enterotypes characterized
by the same driver genera, as originally postulated, we show the relevance of this stratification
and propose it to be used in a clinical setting as a potential marker for disease development.
Moreover, we believe that these attractors underline different rules of community assembly and
we recommend they be accounted for when analyzing gut microbiome samples.
While enterotypes describe structure in the community at genus level, metagenomic sequencing
can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide
polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis
methodology for this level of resolution has only recently been developed and little exploration
has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the
landscape of genomic variation seems highly structured even beyond species resolution,
indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several
cases, these subspecies exhibit geo-stratification, with some subspecies only found in the
Chinese population. Generally however, they present only minor dispersion limitations and are
seen across most of our study populations. Within one individual, one subspecies is commonly
found to dominate and only rarely are several subspecies observed to co-occur in the same
ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable
over periods of more than three years. When interrogating their functional properties we find
many differences, with specific ones appearing relevant to the host. For example, we identify a
subspecies of E. rectale that is lacking the flagellum operon and find its presence to be
significantly associated with lower body mass index and lower insulin resistance of their hosts;
it also correlates with higher microbial community diversity. These associations could not be
seen at the species level (where multiple subspecies are convoluted), which illustrates the
importance of this increased resolution for a more comprehensive understanding of microbial
interactions within the microbiome and with the host.
Taken together, our results provide a rigorous basis for performing comparative metagenomics
of the human gut, encompassing recommendations for both experimental sample processing
and computational analysis. We furthermore refine the concept of community stratification into
enterotypes, develop a reference-based approach for enterotype assignment and provide
compelling evidence for their relevance. Lastly, by harnessing the full resolution of
metagenomics, we discover a highly structured genomic variation landscape below the
microbial species level and identify common subspecies of the human gut microbiome. By
developing these high-precision metagenomics analysis tools, we thus hope to contribute to a
greatly improved understanding of the properties and dynamics of the human gut microbiome.
Population genomics of prokaryotes has been studied in depth in only a small number of primarily pathogenic bacteria, as genome sequences of isolates of diverse origin are lacking for most species. Here, we conducted a large‐scale survey of population structure in prevalent human gut microbial species, sampled from their natural environment, with a culture‐independent metagenomic approach. We examined the variation landscape of 71 species in 2,144 human fecal metagenomes and found that in 44 of these, accounting for 72% of the total assigned microbial abundance, single‐nucleotide variation clearly indicates the existence of sub‐populations (here termed subspecies). A single subspecies (per species) usually dominates within each host, as expected from ecological theory. At the global scale, geographic distributions of subspecies differ between phyla, with Firmicutes subspecies being significantly more geographically restricted. To investigate the functional significance of the delineated subspecies, we identified genes that consistently distinguish them in a manner that is independent of reference genomes. We further associated these subspecies‐specific genes with properties of the microbial community and the host. For example, two of the three Eubacterium rectale subspecies consistently harbor an accessory pro‐inflammatory flagellum operon that is associated with lower gut community diversity, higher host BMI, and higher blood fasting insulin levels. Using an additional 676 human oral samples, we further demonstrate the existence of niche specialized subspecies in the different parts of the oral cavity. Taken together, we provide evidence for subspecies in the majority of abundant gut prokaryotes, leading to a better functional and ecological understanding of the human gut microbiome in conjunction with its host.
Acromyrmex fracticornis grass-cutting ants construct conspicuous chimney-shaped nest turrets made of intermeshed grass fragments. We asked whether turrets are constructed by merely piling up nearby materials around the entrance, or whether ants incorporate different materials as the turret develops. By removing the original nest turrets and following their rebuilding process over three consecutive days, age-dependent changes in wall morphology and inner lining fabrics were characterized. Micromorphological descriptions based on thin sections of turret walls revealed the building behaviors involved. Ants started by collecting nearby twigs and dry grass fragments that are piled up around the nest entrance. Several large fragments held the structure like beams. As a net-like structure grew, soil pellets were placed in between the intermeshed plant fragments from the turret base to the top, reinforcing the structure. Concomitantly, the turret inner wall was lined with soil pellets, starting from the base. Therefore, the consolidation of the turret occurred both over time and from its base upwards. It is argued that nest turrets do not simply arise by the arbitrary deposition of nearby materials, and that workers selectively incorporate large materials at the beginning, and respond to the developing structure by reinforcing the intermeshed plant fragments over time.
Plant communities in the European Alps are assumed to be highly affected by climate change, as the temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010, an experiment simulating advanced and delayed snowmelt as well as a drought event was conducted along an altitudinal transect approximately every 250 m (600–2000 m above sea level) in the Berchtesgaden National Park, Germany. The study showed that flower phenology was strongly affected by altitude; however, there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes may be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes, as changes may be more pronounced at higher altitudes. These data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
Plant communities in the European Alps are assumed to be highly affected by climate change since temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010 an experiment simulating advanced and delayed snowmelt as well as drought event was conducted along an altitudinal transect ca. every 250m (600-2000 m a.s.l.) in the Berchtesgaden National Park, Germany. The study showed that flower phenology is strongly affected by altitude; however there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes might be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes since changes may be more pronounced at higher altitudes. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
Protection of healthy tissues from infection with systemically administered vaccinia virus strains
(2012)
Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host’s own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell’s own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3’end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.
In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
Sex determination (SD) is a complex and diverse developmental process that leads to the decision whether the bipotential gonad anlage will become a testis or an ovary. This mechanism is regulated by gene cascades, networks and/or chromosomal systems, and can be influenced by fluctuations of extrinsic factors like temperature, exposure to hormones and pollution. Within vertebrates, the group of fish show the widest variety of sex determination mechanism. This whole diversity of processes and mechanisms converges to the formation of two different gametes, the eggs and the sperm, the first bigger and static, and the second smaller and motile. Meiosis is crucial for the formation of both types of gametes, and the timing of meiosis entry is one of the first recognizable differences between male and female in vertebrates. The germ cells go into meiosis first in female than in male, and in mammals, this event has been shown to be regulated by retinoic acid (RA). This small polar molecule induces in the germ cells the expression of the pre-meiotic marker Stra8 (stimulated by retinoic acid gene 8), which is necessary for meiosis initiation. Interestingly, genome analyzes have shown that the majority of fish (including medaka) lack the stra8 gene, adding a question mark to the role of RA in meiosis induction in this group. Since a role of RA in entry of meiosis and sexual development of fish is still far from being understood, I investigated in medaka (Oryzias latipes) a possible signaling function of RA during the SD period in embryos and in reproductively active gonads of adults. I generated a transgenic medaka line that reports responsiveness to RA in vivo. With this tool, I compared RA responsiveness with the expression of the main gene involved in the synthesis of RA. My results show that there is a de-correlation between the action of RA with its source. In adults, expression of the RA metabolizing enzymes show sexually dimorphic RA levels, with aldh1a2 levels being higher in testis, and cyp26a1 stronger in female gonad. In ovary, the responsiveness is restricted to the early meiotic oocytes. In testis, RA is acting directly in the pre-meiotic cells, but also in Sertoli and Leydig cells. Treatment experiments on testis organ culture showed that RA pathway activation leads to a decrease in meiosis markers expression levels. During the development, RA responsiveness in the germ cells was observed in both sexes much earlier than the first female meiosis entry. Treatments with RA-synthesis inhibitor show a decrease in meiosis markers expression levels only after the sex differentiation period in female. Expression analyzes of embryos treated with exogenous RA showed induction of dmrt1a at the gonad levels and an increase of amh levels. Both genes are not only involved in male formation, but also in the regulation of germ cell proliferation and differentiation. RA is important in meiosis induction and gametogenesis in adult medaka. However, there is no evidence for a similar role of RA in initiating the first meiosis in female germ cells at the SD stage. Moreover, contrary to common expectation, RA seems to induce sex related genes that are involved indirectly in meiosis inhibition. In this thesis, I showed for the first time that RA can be involved in both induction and inhibition of meiosis entry, depending on the sex and the developmental stage in a stra8-independent model organism.
Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.
Circadian clocks prepare the organism to cyclic environmental changes in light, temperature, or food availability. Here, we characterized the master clock in the brain of a strongly photoperiodic insect, the aphid Acyrthosiphon pisum, immunohistochemically with antibodies against A. pisum Period (PER), Drosophila melanogaster Cryptochrome (CRY1), and crab Pigment-Dispersing Hormone (PDH). The latter antibody detects all so far known PDHs and PDFs (Pigment-Dispersing Factors), which play a dominant role in the circadian system of many arthropods. We found that, under long days, PER and CRY are expressed in a rhythmic manner in three regions of the brain: the dorsal and lateral protocerebrum and the lamina. No staining was detected with anti-PDH, suggesting that aphids lack PDF. All the CRY1-positive cells co-expressed PER and showed daily PER/CRY1 oscillations of high amplitude, while the PER oscillations of the CRY1-negative PER neurons were of considerable lower amplitude. The CRY1 oscillations were highly synchronous in all neurons, suggesting that aphid CRY1, similarly to Drosophila CRY1, is light sensitive and its oscillations are synchronized by light-dark cycles. Nevertheless, in contrast to Drosophila CRY1, aphid CRY1 was not degraded by light, but steadily increased during the day and decreased during the night. PER was always located in the nuclei of the clock neurons, while CRY was predominantly cytoplasmic and revealed the projections of the PER/CRY1-positive neurons. We traced the PER/CRY1-positive neurons through the aphid protocerebrum discovering striking similarities with the circadian clock of D. melanogaster: The CRY1 fibers innervate the dorsal and lateral protocerebrum and putatively connect the different PER-positive neurons with each other. They also run toward the pars intercerebralis, which controls hormone release via the neurohemal organ, the corpora cardiaca. In contrast to Drosophila, the CRY1-positive fibers additionally travel directly toward the corpora cardiaca and the close-by endocrine gland, corpora allata. This suggests a direct link between the circadian clock and the photoperiodic control of hormone release that can be studied in the future.
Background
Gut microbes influence their hosts in many ways, in particular by modulating the impact of diet. These effects have been studied most extensively in humans and mice. In this work, we used whole genome metagenomics to investigate the relationship between the gut metagenomes of dogs, humans, mice, and pigs.
Results
We present a dog gut microbiome gene catalog containing 1,247,405 genes (based on 129 metagenomes and a total of 1.9 terabasepairs of sequencing data). Based on this catalog and taxonomic abundance profiling, we show that the dog microbiome is closer to the human microbiome than the microbiome of either pigs or mice. To investigate this similarity in terms of response to dietary changes, we report on a randomized intervention with two diets (high-protein/low-carbohydrate vs. lower protein/higher carbohydrate). We show that diet has a large and reproducible effect on the dog microbiome, independent of breed or sex. Moreover, the responses were in agreement with those observed in previous human studies.
Conclusions
We conclude that findings in dogs may be predictive of human microbiome results. In particular, a novel finding is that overweight or obese dogs experience larger compositional shifts than lean dogs in response to a high-protein diet.
Background
Shotgun metagenomes contain a sample of all the genomic material in an environment, allowing for the characterization of a microbial community. In order to understand these communities, bioinformatics methods are crucial. A common first step in processing metagenomes is to compute abundance estimates of different taxonomic or functional groups from the raw sequencing data.
Given the breadth of the field, computational solutions need to be flexible and extensible, enabling the combination of different tools into a larger pipeline.
Results
We present NGLess and NG-meta-profiler. NGLess is a domain specific language for describing next-generation sequence processing pipelines. It was developed with the goal of enabling user-friendly computational reproducibility. It provides built-in support for many common operations on sequencing data and is extensible with external tools with configuration files.
Using this framework, we developed NG-meta-profiler, a fast profiler for metagenomes which performs sequence preprocessing, mapping to bundled databases, filtering of the mapping results, and profiling (taxonomic and functional). It is significantly faster than either MOCAT2 or htseq-count and (as it builds on NGLess) its results are perfectly reproducible.
Conclusions
NG-meta-profiler is a high-performance solution for metagenomics processing built on NGLess. It can be used as-is to execute standard analyses or serve as the starting point for customization in a perfectly reproducible fashion.
NGLess and NG-meta-profiler are open source software (under the liberal MIT license) and can be downloaded from https://ngless.embl.de or installed through bioconda.
Since more than two centuries naturalists are fascinated by the profound changes in biodiversity observed along climatic gradients. Although the theories explaining changes in the diversity and the shape of organisms along climatic gradients belong to the foundations of modern ecology, our picture on the spatial patterns and drivers of biodiversity is far from being complete. Ambiguities in theory and data are common and past work has been strongly concentrated on plants and vertebrates. In the last two decades, interest in the fundamental processes structuring diversity along climatic gradients gained new impetus as they are expected to improve our understanding about how ecosystems will respond to global environmental changes. Global temperatures are rising faster than ever before; natural habitats are transformed into agricultural land and existing land use systems get more and more intensified to meet the demands of growing human populations. The fundamental shifts in the abiotic and biotic environment are proclaimed to affect ecosystems all over the world; however, precise predictions about how ecosystems respond to global changes are still lacking. We investigated diversity, traits and ecosystem services of wild bees along climate and land use gradients on Mount Kilimanjaro (Tanzania, East Africa). Wild bees play a major role in ecosystems, as they contribute to the reproduction and performance of wild and crop plants. Their responsiveness to environmental changes is therefore of high ecological and economic importance.
Temperature and energy resources have often been suggested to be the main determinants of global and local species richness, but the mechanisms behind remain poorly understood. In the study described in chapter II we analyzed species richness patterns of wild bees along climate and land use gradients on Mount Kilimanjaro and disentangled the factors explaining most of the changes in bee richness. We found that floral resources had a weak but significant effect on pollinator abundance, which in turn was positively related to species richness. However, temperature was the strongest predictor of species richness, affecting species richness both directly and indirectly by positively influencing bee abundances. We observed higher levels of bee-flower-interactions at higher temperatures, independently of flower and bee abundances. This suggests that temperature restricts species richness by constraining the exploitation of resources by ectotherms. Current land use did not negatively affect species richness. We conclude that the richness of bees is explained by both temperature and resource availability, whereas temperature plays the dominant role as it limits the access of ectotherms to floral resources and may accelerate ecological and evolutionary processes that drive the maintenance and origination of diversity.
Not only species numbers, but also morphological traits like body size are expected to be shaped by both physiological and energetic constraints along elevational gradients. Paradoxically, Bergmann´s rule predicts increases of body sizes in cooler climates resulting from physiological constraints, while species-energy theory suggests declines in the mean body size of species caused by increased extinction probabilities for large-bodied species in low-energy habitats. In chapter III we confronted this ambiguity with field data by studying community-wide body size variation of wild bees on Mt. Kilimanjaro. We found that along a 3680 m elevational gradient bee individuals became on average larger within species, while large species were increasingly absent from high-elevational communities. This demonstrates, on the one hand, how well-established, but apparently contrasting ecological theories can be merged through the parallel consideration of different levels of biological organization. On the other hand it signals that the extinction risk in the course of environmental change is not equally distributed among species within a community.
Land use intensification is known to threaten biodiversity, but the consequences for ecosystem services are still a matter of debate. In chapter IV, we experimentally tested the single and combined contributions of pest predators and pollinators to coffee production along a land use intensification gradient on Mount Kilimanjaro. We found that pest predation increased fruit set by on average 9%, while pollination increased fruit weight of coffee by on average 7.4%. Land use had no significant effect on both ecosystem services. However, we found that in coffee plantations with most intensified land use, pollination services were virtually exclusively provided by the honey bee (Apis mellifera). The reliance on a single pollinator species is risky, as possible declines of that species may directly lower pollination services, resulting in yield losses. In contrast, pollination services in structurally complex homegardens were found to be provided by a diverse pollinator community, increasing the stability of pollination services in a long term.
We showed that on Mount Kilimanjaro pollinator communities changed along elevational gradients in terms of species richness (chapter II) and trait composition (chapter III). Temperature and the temperature-mediated accessibility of resources were identified as important predictors of these patterns, which contributes to our fundamental understanding about the factors that shape ectothermic insect communities along climatic gradients. The strong temperature-dependence of pollinators suggests that temperature shifts in the course of global change are likely to affect pollinator communities. Pollinators might either profit from rising temperatures, or shift to higher elevations, which could result in related biotic attrition in the lowland with consequences for the provision of ecosystem services in cropping systems. Up to now, land use intensification had no significant impact on the diversity of pollinator communities and their ecosystem services. Pollinators might profit from the strong landscape heterogeneity in the region and from the amount of flower resources in the understory of cropping systems. However,progressing homogenization of the landscape and the pronounced application of pesticides could result in reduced diversity and dominance of single species, as we already found in sun coffee plantations. Such shifts in community compositions could threaten the stability of ecosystem services within cropping and natural systems in a long term.
ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach.
In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops.
Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90.
Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy.
Monoclonal antlbodies (MAbs) directed against Xiphophorus melanoma cells were deve(oped and tested by lndirect immunofluorescence and Immunoperoxidase staining for reactivity with a panel of I 5 allogeneic tissues and 12 allogeneic cell llnes. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. ln addition, all embryonie cells of all histiotypes from developmental stages later than mld·organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. ln contrast, normal tissues and organs from adult fish dlsplayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens.
Specialization of plant-pollinator interactions increases with temperature at Mt. Kilimanjaro
(2020)
Aim: Species differ in their degree of specialization when interacting with other species, with significant consequences for the function and robustness of ecosystems. In order to better estimate such consequences, we need to improve our understanding of the spatial patterns and drivers of specialization in interaction networks.
Methods: Here, we used the extensive environmental gradient of Mt. Kilimanjaro (Tanzania, East Africa) to study patterns and drivers of specialization, and robustness of plant–pollinator interactions against simulated species extinction with standardized sampling methods. We studied specialization, network robustness and other network indices of 67 quantitative plant–pollinator networks consisting of 268 observational hours and 4,380 plant–pollinator interactions along a 3.4 km elevational gradient. Using path analysis, we tested whether resource availability, pollinator richness, visitation rates, temperature, and/or area explain average specialization in pollinator communities. We further linked pollinator specialization to different pollinator taxa, and species traits, that is, proboscis length, body size, and species elevational ranges.
Results: We found that specialization decreased with increasing elevation at different levels of biological organization. Among all variables, mean annual temperature was the best predictor of average specialization in pollinator communities. Specialization differed between pollinator taxa, but was not related to pollinator traits. Network robustness against simulated species extinctions of both plants and pollinators was lowest in the most specialized interaction networks, that is, in the lowlands.
Conclusions: Our study uncovers patterns in plant–pollinator specialization along elevational gradients. Mean annual temperature was closely linked to pollinator specialization. Energetic constraints, caused by short activity timeframes in cold highlands, may force ectothermic species to broaden their dietary spectrum. Alternatively or in addition, accelerated evolutionary rates might facilitate the establishment of specialization under warm climates. Despite the mechanisms behind the patterns have yet to be fully resolved, our data suggest that temperature shifts in the course of climate change may destabilize pollination networks by affecting network architecture.
Characterization of a novel putative factor involved in host adaptation in Trypanosoma brucei
(2016)
Trypanosomes are masters of adaptation to different host environments
during their complex life cycle. Large-scale proteomic approaches provide information on changes at
the cellular level in a systematic way. However, a detailed work on single components is necessary
to understand the adaptation mechanisms on a molecular level. Here we have performed a detailed
characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation
factor (Tb927.11.2400) identified previously in a SILAC-based comparative proteome study.
Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form
(PCF) stage specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin like
(TbFlabarinL) and demonstrate that it is a result of a gene duplication event, which occurred in
African trypanosomes. TbFlabarinL is not essential for growth of the parasites under cell culture
conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We
generated a TbFlabarinL-specific antibody and showed that it localizes in the flagellum. The
co-immunoprecipitation experiment together with a biochemical cell fractionation indicated a dual
association of TbFlabarinL with the flagellar
membrane and the components of the paraflagellar rod.
Healthy functioning of the female reproductive tract (FRT) depends on balanced and dynamic regulation by hormones during the menstrual cycle, pregnancy and childbirth. The mucosal epithelial lining of different regions of the FRT—ovaries, fallopian tubes, uterus, cervix and vagina—facilitates the selective transport of gametes and successful transfer of the zygote to the uterus where it implants and pregnancy takes place. It also prevents pathogen entry. Recent developments in three-dimensional (3D) organoid systems from the FRT now provide crucial experimental models that recapitulate the cellular heterogeneity and physiological, anatomical and functional properties of the organ in vitro. In this review, we summarise the state of the art on organoids generated from different regions of the FRT. We discuss the potential applications of these powerful in vitro models to study normal physiology, fertility, infections, diseases, drug discovery and personalised medicine.
In social groups, infections have the potential to spread rapidly and cause disease outbreaks. Here, we show that in a social insect, the ant Lasius neglectus, the negative consequences of fungal infections (Metarhizium brunneum) can be mitigated by employing an efficient multicomponent behaviour, termed destructive disinfection, which prevents further spread of the disease through the colony. Ants specifically target infected pupae during the pathogens non-contagious incubation period, utilising chemical 'sickness cues' emitted by pupae. They then remove the pupal cocoon, perforate its cuticle and administer antimicrobial poison, which enters the body and prevents pathogen replication from the inside out. Like the immune system of a metazoan body that specifically targets and eliminates infected cells, ants destroy infected brood to stop the pathogen completing its lifecycle, thus protecting the rest of the colony. Hence, in an analogous fashion, the same principles of disease defence apply at different levels of biological organisation.
The first goal of this study was to develop cell lines with a stable expression of bio-fluorescent topo II and topo I. This was successfully achieved using a bicistronic vector system. Control experiments showed that proteins of expected size were expressed, and that GFP-tagged topos I, IIa, and IIb were active in the cells and fully integrated in the endogenous pools of the enzymes. These cell-lines provided a novel tool for investigating the cell biology of human DNA topoisomerases. Our most important finding was, that both types of mammalian topoisomerases are entirely mobile proteins that are in continuous and rapid flux between all compartments of the nucleus and between the cytososl and the chromosomes of mitotic cells. This was particularly surprising with regard to topo II, which is considered to be a structural component of the nuclear matrix and the chromosome scaffold. We must conclude that if this was the case, then these architectural structures appear to be much more dynamic than believed until now. In this context it should also be mentioned, that the alignment of topo II with the central axes of the chromosome arms, which has until now been considered a hall-mark of the enzyme’s association with the chromosomal scaffold, is not seen in vivo and can be demonstrated to be to some extent an artefact of immunohistochemistry. Furthermore, we show that the two isoforms of topo II (a and b) have a different localisation during mitotic cell division, supporting the general concept that topo II functions at mitosis are exclusively assigned to the a-form, whereas at interphase the two isoenzymes work in concert. Despite unrestricted mobility within the entire nuclear space, topoisomerases I and II impose as mostly nucleolar proteins. We show that this is due to the fact that in the nucleoli they are moving slower than in the nucleoplasm. The decreased nucleolar mobility cannot be due to DNA-interactions, because compounds that fix topoisomerases to the DNA deplete them from the nucleoli. Interestingly, the subnucleolar distribution of topoisomerases I and II was complementary. The type II enzyme filled the entire nucleolar space, but excluded the fibrial centers, whereas topo I accumulated at the fibrial centers, an allocation directed by the enzyme’s N-terminus. During mitosis, it also mediates association with the nucleolar organising regions of the acrocentric chromosomes. Thus, topo I stays associated with the rDNA during the entire cell-cycle and consistently colocalizes there with RNA-polymerase I. Finally, we show that certain cancer drugs believed to act by stabilising covalent catalytic DNA-intermediates of topoisomerases, do indeed immobilize the enzymes in living cells. Interestingly, these drugs do not target topoisomerases in the nucleoli but only in the nucleoplasm.
Background: Patterns that arise from an ecological process can be driven as much from the landscape over which the process is run as it is by some intrinsic properties of the process itself. The disentanglement of these effects is aided if it possible to run models of the process over artificial landscapes with controllable spatial properties. A number of different methods for the generation of so-called ‘neutral landscapes’ have been developed to provide just such a tool. Of these methods, a particular class that simulate fractional Brownian motion have shown particular promise. The existing methods of simulating fractional Brownian motion suffer from a number of problems however: they are often not easily generalisable to an arbitrary number of dimensions and produce outputs that can exhibit some undesirable artefacts. Methodology: We describe here an updated algorithm for the generation of neutral landscapes by fractional Brownian motion that do not display such undesirable properties. Using Monte Carlo simulation we assess the anisotropic properties of landscapes generated using the new algorithm described in this paper and compare it against a popular benchmark algorithm. Conclusion/Significance: The results show that the existing algorithm creates landscapes with values strongly correlated in the diagonal direction and that the new algorithm presented here corrects this artefact. A number of extensions of the algorithm described here are also highlighted: we describe how the algorithm can be employed to generate landscapes that display different properties in different dimensions and how they can be combined with an environmental gradient to produce landscapes that combine environmental variation at the local and macro scales.
A fundamental problem in deciding between mutually exclusive options is that the decision needs to be categorical although the properties of the options often differ but in grade. We developed an experimental handle to study this aspect of behavior organization. Larval Drosophila were trained such that in one set of animals odor A was rewarded, but odor B was not (A+/B), whereas a second set of animals was trained reciprocally (A/B+). We then measured the preference of the larvae either for A, or for B, or for “morphed” mixtures of A and B, that is for mixtures differing in the ratio of the two components. As expected, the larvae showed higher preference when only the previously rewarded odor was presented than when only the previously unrewarded odor was presented. For mixtures of A and B that differed in the ratio of the two components, the major component dominated preference behavior—but it dominated less than expected from a linear relationship between mixture ratio and preference behavior. This suggests that a minor component can have an enhanced impact in a mixture, relative to such a linear expectation. The current paradigm may prove useful in understanding how nervous systems generate discrete outputs in the face of inputs that differ only gradually.
Similarity between odours is notoriously difficult to measure. Widely used behavioural approaches in insect olfaction research are cross-adaptation, masking, as well as associative tasks based on olfactory learning and the subsequent testing for how specific the established memory is. A concern with such memory-based approaches is that the learning process required to establish an odour memory may alter the way the odour is processed, such that measures of perception taken at the test are distorted. The present study was therefore designed to see whether behavioural judgements of perceptual distance are different for two different memory-based tasks, namely generalization and discrimination. We used odour-reward learning in larval Drosophila as a study case. In order to challenge the larvae's olfactory system, we chose to work with binary mixtures and their elements (1-octanol, n-amyl acetate, 3-octanol, benzaldehyde and hexyl acetate). We determined the perceptual distance between each mixture and its elements, first in a generalization task, and then in a discrimination task. It turns out that scores of perceptual distance are correlated between both tasks. A re-analysis of published studies looking at element-to-element perceptual distances in larval reward learning and in adult punishment learning confirms this result. We therefore suggest that across a given set of olfactory stimuli, associative training does not grossly alter the pattern of perceptual distances.
Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order “cortical” brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.
OGEE is an Online GEne Essentiality database. To enhance our understanding of the essentiality of genes, in OGEE we collected experimentally tested essential and non-essential genes, as well as associated gene properties known to contribute to gene essentiality. We focus on large-scale experiments, and complement our data with text-mining results. We organized tested genes into data sets according to their sources, and tagged those with variable essentiality statuses across data sets as conditionally essential genes, intending to highlight the complex interplay between gene functions and environments/experimental perturbations. Developments since the last public release include increased number of species and gene essentiality data sets, inclusion of non-coding essential sequences and genes with intermediate essentiality statuses. In addition, we included 16 essentiality data sets from cancer cell lines, corresponding to 9 human cancers; with OGEE, users can easily explore the shared and differentially essential genes within and between cancer types. These genes, especially those derived from cell lines that are similar to tumor samples, could reveal the oncogenic drivers, paralogous gene expression pattern and chromosomal structure of the corresponding cancer types, and can be further screened to identify targets for cancer therapy and/or new drug development. OGEE is freely available at http://ogee.medgenius.info.
Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.
Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par
In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par
Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par
The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par
In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{WÜ} cells by using newly generated \textit{Mip\textsuperscript{WÜ}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{WÜ} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par
My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}$>$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par
In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par
After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par
The hematopoietic-specific Rho-family GTP exchange factor (GEF) Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, I show that Vav-1 is also expressed in murine NK cells. Vav-1-/- mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as cells from wild type mice. Unexpectedly, IL-2-activated Vav-1-/- NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1-/- NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, these results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK cell natural cytotoxicity. Vav-2, a protein homologous to Vav-1 has also been implicated in NK cell functions. However, NK cells from Vav-2-/- mice have normal cytotoxic activities and NK cells that lack both Vav-1 and Vav-2 exhibit similar defect as Vav-1-/- cells. Thus Vav-2 has no apparent function in the development and the activation of NK cells. Although NK cell development is normal in Vav-1-/- mice, their numbers of NKT cells were dramatically diminished. Furthermore, NKT cells from Vav-1 mutant mice failed to produce IL-4 and IFNg following in vivo CD3 stimulation. A similar loss of NKT cells was observed in Vav-1-/-Vav-2-/- mice, but not in Vav-2-/- mice, suggesting that only Vav-1, and not Vav-2, is an essential regulator of NKT cell development and NK cell cytotoxicity. Similar to Vav-1, Lsc is a Rho GEF that is expressed specifically in the hematopoietic system. It contains a regulator of G-protein signaling (RGS) domain which negatively regulates the Ga12 and Ga13 subunits of G-protein coupled receptors (GPCRs). This study shows that NK and NKT cell development are normal in Lsc-/- mice. However, NK cells from mutant mice display enhanced cytotoxic responses towards a panel of tumor cells. These data implicate for the first time a RGS-containing Rho GEF in cytotoxic responses and suggest that Lsc down-modulate NK cell activation.
The idea that populations are spatially structured has become a very powerful concept in ecology, raising interest in many research areas. However, despite dispersal being a core component of the concept, it typically does not consider the movement behavior underlying any dispersal. Using individual‐based simulations in continuous space, we explored the emergence of a spatially structured population in landscapes with spatially heterogeneous resource distribution and with organisms following simple area‐concentrated search (ACS); individuals do not, however, perceive or respond to any habitat attributes per se but only to their foraging success. We investigated the effects of different resource clustering pattern in landscapes (single large cluster vs. many small clusters) and different resource density on the spatial structure of populations and movement between resource clusters of individuals. As results, we found that foraging success increased with increasing resource density and decreasing number of resource clusters. In a wide parameter space, the system exhibited attributes of a spatially structured populations with individuals concentrated in areas of high resource density, searching within areas of resources, and “dispersing” in straight line between resource patches. “Emigration” was more likely from patches that were small or of low quality (low resource density), but we observed an interaction effect between these two parameters. With the ACS implemented, individuals tended to move deeper into a resource cluster in scenarios with moderate resource density than in scenarios with high resource density. “Looping” from patches was more likely if patches were large and of high quality. Our simulations demonstrate that spatial structure in populations may emerge if critical resources are heterogeneously distributed and if individuals follow simple movement rules (such as ACS). Neither the perception of habitat nor an explicit decision to emigrate from a patch on the side of acting individuals is necessary for the emergence of such spatial structure.
A metacommunity approach will be a useful framework to assess and predict changes in biodiversity in spatially structured landscapes and changing environments. However, the relationship between two core elements of metacommunity dynamics, dispersal and species interaction are not well understood. Most theoretical studies on dispersal evolution assume that target species are in isolation and do not interact with other species although the species interactions and community structure should have strong interdependence with dispersal. On the one hand, a species interaction can change the cost and benefit structure of dispersing in relation to non-dispersing individuals. On the other hand, with dispersal, an individual can follow respectively avoid species partners. Moreover, it is also important to explore the interdependence between dispersal and species interaction with spatial and temporal heterogeneity of environment because it would allow us to gain more understanding about responses of community to disturbances such as habitat destruction or global climate change, and this aspect is up to now not well-studied. In this thesis, I focus on the interactive and evolutionary feedback effects between dispersal and various types of interspecific interactions in different environmental settings. More specifically, I contrast dispersal evolution in scenarios with different types of interactions (chapter 2), explore the concurrent evolution of dispersal and habitat niche width (specialization) in spatial heterogeneous landscape (chapter 3) and consider (potential) multidimensional evolutionary responses under climate change (chapter 4). Moreover, I investigate consequences of different dispersal probability and group tolerance on group formation respectively group composition and the coexistence of ‘marker types’ (chapter 5). For all studies, I utilize individual-based models of single or multiple species within spatially explicit (grid-based) landscapes. In chapter 5, I also use an analytical model in addition to an individual-based model to predict phenomenon in group recognition and group formation. ...
Purpose
Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series.
Materials and Methods
WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest.
Results
One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P<.001; hazard ratio, 2.33) and OS (P=.01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss.
Conclusion
Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage.
Here we describe a novel conditional mouse lung tumor model for investigation of the pathogenesis of human lung cancer. On the basis of the frequent involvement of the Ras-RAF-MEK-ERK signaling pathway in human non-small cell lung carcinoma (NSCLC), we have explored the target cell availability, reversibility, and cell type specificity of transformation by oncogenic C-RAF. Targeting expression to alveolar type II cells or to Clara cells, the two likely precursors of human NSCLC, revealed differential tumorigenicity between these cells. Whereas expression of oncogenic C-RAF in alveolar type II cells readily induced multifocal macroscopic lung tumors independent of the developmental state, few tumors with type II pneumocytes features and incomplete penetrance were found when targeted to Clara cells. Induced tumors did not progress and were strictly dependent on the initiating oncogene. Deinduction of mice resulted in tumor regression due to autophagy rather than apoptosis. Induction of autophagic cell death in regressing lung tumors suggests the use of autophagy enhancers as a treatment choice for patients with NSCLC.
Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color (n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients.
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
When bovine or human growth hormones (GH) were injected into 6 months old (about 10 g) gilthead seabream, the growth rate of the fish, as measured by changes in their weight, increased by only about 15% compared with the saline-injected control. No effect or even slight inhibition of the growth rate was obtained when chicken or porcine GHs were injected. In a preliminary experiment, it was found that injection ofthe native GH increased the growth rate ofthe fish by about 20% after treatment for only 2 weeks. An expression vector, using the pRE1 plasmid and transformation into MZl cells, produced the gilthead seabream GH, providing a supply for further experiments on the effect of the homologaus GH on growth. Two reporter genes, ß-galactosidase (lacZ) and melanoma oncogene of Xiphophorus (mrk YY), were microinjected into fertilized eggs of S. aurata. Expression of these two genes could be demonstrated in 2-day-old embryos, the lacZ gene by staining of its enzymatic product, and the mrk YY gene by its phenotypic expression.
Traps baited with attractive lures are increasingly used at entry-points and surrounding natural areas to intercept exotic wood-boring beetles accidentally introduced via international trade. Several trapping variables can affect the efficacy of this activity, including trap color. In this study, we tested whether species richness and abundance of jewel beetles (Buprestidae), bark and ambrosia beetles (Scolytinae), and their common predators (i.e., checkered beetles, Cleridae) can be modified using trap colors different to those currently used for surveillance of jewel beetles and bark and ambrosia beetles (i.e., green or black). We show that green and black traps are generally efficient, but also that many flower-visiting or dark-metallic colored jewel beetles and certain bark beetles are more attracted by other colors. In addition, we show that checkered beetles have color preferences similar to those of their Scolytinae preys, which limits using trap color to minimize their inadvertent removal. Overall, this study confirmed that understanding the color perception mechanisms in wood-boring beetles can lead to important improvements in trapping techniques and thereby increase the efficacy of surveillance programs.
Background
Brood parasites can exert strong selection pressure on their hosts. Many brood parasites escape their detection by mimicking sensory cues of their hosts. However, there is little evidence whether or not the hosts are able to escape the parasites’ mimicry by changing these cues. We addressed this question by analyzing cuticular hydrocarbon (CHC) profiles of Cerceris and Philanthus wasps and their brood parasites, cuckoo wasps mimicking the CHC profiles of their hosts. Some of these hosts use hydrocarbons to preserve their prey against fungal infestation and thus, they cannot significantly change their CHC composition in response to chemical mimicry by Hedychrum brood parasites.
Results
We found that the CHC overlap between brood parasites and their hosts was lower in case of host wasps not preserving their prey than in case of prey-preserving host wasps, whose CHC evolution is constrained. Furthermore, the CHC profiles in non-preserving host wasps is more strongly diversified in females than in males, thus in the sex that is chemically mimicked by brood parasites.
Conclusion
Our results provide evidence for a chemical arms race between those hosts that are liberated from stabilizing selection on their chemical template and their parasites.
Cuticular hydrocarbons (CHC) abound on the surface of arthropods. In spite of their simple structure (molecules of carbon and hydrogen atoms), they provide pivotal functions in insects: their hydrophobic properties confer the insects a means to regulate water balance and avoid desiccation, whereas their diversity has enhanced their use as signals and cues in a wide range of communication and recognition processes. Although the study of CHC in insects over the past two decades has provided great insight into the wide range of functions they play, there is still a gap in understanding how they diversify and evolve. In this thesis, I have used members of the family Chrysididae to explore patterns of diversification of CHC. Most of the species of cuckoo wasps in this study are specialized parasitoids or kleptoparasites of mainly solitary hymenopteran hosts. Other hosts of the family include butterflies or stick insects. Cuckoo wasps are a particular interesting model to study the evolution of cuticular hydrocarbons because of their chemical adaptations that allow them to remain unrecognized by their hosts. Chemical insignificance (the reduction of the total amount of CHC on the cuticle) and chemical mimicry (the de novo production of CHC profiles resembling those of their female host) have been described in some representatives of the family and unpublished evidence suggests chemical deception is widespread in Chrysididae (Chapter 2). Nonetheless, to trace the evolution of any trait of interest, a reliable phylogenetic reconstruction of the family is required. Therefore, the first study of this thesis constitutes the largest and to-date most reliable phylogenetic reconstruction of the family Chrysididae, which includes representatives of 186 species of cuckoo wasps. While the results of this phylogenetic reconstruction are consistent with previous ideas on the relationships of subfamilies and tribes, it shows the existence of several non-monophyletic genera (Chapter 3). CHC are involved in intraspecific recognition, often acting as contact sex pheromones. Nevertheless, it is not yet understood to what extent CHC profiles differ between the two sexes and whether some compound classes are more prevalent in one or the other sex. So far, no comparison of CHC profiles of males and females has been done for more than a dozen of related species. In Chapter 4, I describe and compare CHC profiles of females and males of 58 species of cuckoo wasps in order to evaluate whether and to what extent CHC profiles of these species differ between the sexes. I demonstrated that CHC profiles of cuckoo wasps are frequently (more than 90% of the species analyzed) and strongly dimorphic (both sexes of a given species tend to produce very different CHC compounds). Methyl-branched compounds tend to be more prevalent in males (especially dimethyl-branched compounds) and unsaturated compounds prevail in females. Moreover, a sex-specific pattern in the distribution of the double bond position of alkenes was evident: internal double bond positions (> 11) occur predominantly in males, whereas alkenes with the doublé bond at position 9 were more abundant and frequent in females (Chapter4). In Chapter5, I investigated how CHC profiles of cuckoo wasps differ across species. Are CHC profiles of cuckoo wasps species-specific, enabling their use as cues for species recognition? How do CHC profiles resemble phylogenetic relatedness? In Chapter 5, I try to answer these questions by comparing CHC profiles of 59 species of cuckoo wasps. CHC profiles of cuckoo wasps are shown to be species (and sex-) specific. I show that CHC profiles are useful as a complementary tool to help delimiting taxonomically difficult sibling species. Moreover, the evaluation of CHC profiles of five commonly occurring species within a genus, showed little or no geographical variation. However, CHC profiles of closely related species may differ strongly among each other, not being useful to track the evolutionary history of species (Chapter 5). Sexual selection is generally credited for generating striking sexual dimorphism by causing changes in male traits. Most often, sexual selection has a stronger effect on males, who compete for access to and may be selected by females, thus male traits may rapidly evolve. Nevertheless, in cuckoo wasps, it appears that it is the female sex the one evolving faster changes, with females of very closely related species showing extremely divergent profiles. One plausible reason for this disparity is that natural selection acting on female’s CHC profiles may be stronger than sexual selection on males (Chapter 6). Since females of cuckoo wasps are most probably engaged in an evolutionary arms race with their female hosts, CHC profiles of female cuckoo wasps are likely rapidly evolving, thus explaining part of the strong observed sexual dimorphism of CHC (Chapter 6). In fact, Chapter 7 shows evidence of a possible ongoing evolutionary arms race between five cuckoo wasps of the genus Hedychrum and their hosts. Hedychrum species parasitize either Coleoptera-hunting or Hymenoptera-hunting digger wasps. Since the coleopteran prey of the former digger wasps is naturally better protected against fungus infestation, these wasps do not embalm their prey with alkene-enriched secretions as do the Hymenoptera-hunting digger wasps. Thus, Coleoptera-hunting digger wasps can apparently diversify their profiles to escape chemical mimicry. Interestingly, only female cuckoo wasps of these hosts have started producing the same compound classes and even the same CHC compounds as those of their hosts. Male cuckoo wasps, however retain an alkene-enriched CHC profile that reflects the molecular phylogeny of the genus (Chapter 7). Whereas, a larger number of parasite-host comparisons may be needed to further conclude that an arms race between cuckoo wasps and their hosts is capable of generating sexual dimorphism of cuckoo wasps, this thesis constitutes the first effort towards this, providing a starting point for further studies. Finally, I provide some methodological tools that may help in speeding up the sometimes cumbersome process of analyzing and identifying CHC profiles. One of the most time-demanding steps in the processing of CHC data is the alignment of CHC chromatograms. This process is often done manually, because alignment programs are mostly designed for metabolomics or are just recently being developed. I analyzed CHC profiles using a combined approach with two freely available programs. I used AMDIS (Automated Mass Spectral Deconvolution and Identification System, http://chemdata.nist.gov/mass-spc/amdis/) to deconvolute and automatically identify all CHC of interest present in a chromatogram. I then developed a series of R scripts to correct for potential, unavoidable errors while processing CHC chromatograms with AMDIS. Chapter 8 explains this procedure. In the next chapter, I developed a program that helps in the identification of one commonly occurring class of hydrocarbons. The limited number of linear alkanes (only one per carbon atom) and their characteristic diagnostic ion allows a rapid and unambigous identification of these substances. In opposition, unsaturated and methyl-branched compounds are more difficult to identify, as a result of the much larger diversity of existing compounds. To identify unsaturated compounds a derivatization is necessary to determine the position of the double bond. Methyl-branched alkanes, however can be identified from the original chromatogram if their diagnostic ions are known. Nonetheless, polymethyl-branched alkanes (e.g., compounds with two or more methyl groups along the chain) are often difficult to identify, because they may appear in mixes (e.g., 3,7 diMeC27 and 3,9 diMeC27), and tables containing the diagnostic ions are not easily available. Therefore, I developed a program that creates a table with all possiblemethyl-branched compounds containing up to 4 methyl groups, and that provides their diagnostic ions and a calculated retention index. This may allow a much faster identification of the methyl-branched compound a researcher is dealing with, without having to lose time in the tedious calculations by hand. The program is able to correctly identify, or at least, greatly reduce the number of possible options for the identification of an unknown methyl-branched compound. Thus, using this tool, most methyl-branched compounds can be readily identified (Chapter 9). This thesis ends with a general discussion (Chapter 10). Overall, this work provides a comprehensive overview of the diversity of cuticular hydrocarbons of cuckoo wasps. The analyses presented here shed light on the emergence and evolution of interspecific diversity and intraspecific sexual dimorphism of CHC profiles. In addition, two technical methods have been developed that could greatly facilitate the CHC analysis of insects.
Background: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and are present in eukaryotes of all eukaryotic super-groups; still, only two proteins have been functionally characterised. One is ALPH1 from the Kinetoplastid Trypanosoma brucei that we recently found to be the mRNA decapping enzyme of the parasite. mRNA decapping by ALPHs is unprecedented in eukaryotes, which usually use nudix hydrolases, but the bacterial ancestor protein ApaH was recently found to decap non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or more widespread among eukaryotes.
Results: We screened 824 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to refine phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of ALPHs. We found that most eukaryotes have either no ALPH (500/824) or very short ALPHs, consisting almost exclusively of the catalytic domain. These ALPHs had mostly predicted non-cytoplasmic localisations, often supported by the presence of transmembrane helices and signal peptides and in two cases (one in this study) by experimental data. The only exceptions were ALPH1 homologues from Kinetoplastida, that all have unique C-terminal and mostly unique N-terminal extension, and at least the T. brucei enzyme localises to the cytoplasm. Surprisingly, despite of these non-cytoplasmic localisations, ALPHs from all eukaryotic super-groups had in vitro mRNA decapping activity.
Conclusions: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme since, or use it exclusively outside the cytoplasm in organelles in a version consisting of the catalytic domain only. While our data provide no evidence for the presence of further mRNA decapping enzymes among eukaryotic ALPHs, the broad substrate range of ALPHs that includes mRNA caps provides an explanation for the selection against the presence of a cytoplasmic ALPH protein as a mean to protect mRNAs from unregulated degradation. Kinetoplastida succeeded to exploit ALPH as their mRNA decapping enzyme, likely using the Kinetoplastida-unique N- and C-terminal extensions for regulation.
N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated.
To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif.
Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.
While our knowledge about the roles of microbes and viruses in the ocean has increased tremendously due to recent advances in genomics and metagenomics, research on marine microbial eukaryotes and zooplankton has benefited much less from these new technologies because of their larger genomes, their enormous diversity, and largely unexplored physiologies. Here, we use a metatranscriptomics approach to capture expressed genes in open ocean Tara Oceans stations across four organismal size fractions. The individual sequence reads cluster into 116 million unigenes representing the largest reference collection of eukaryotic transcripts from any single biome. The catalog is used to unveil functions expressed by eukaryotic marine plankton, and to assess their functional biogeography. Almost half of the sequences have no similarity with known proteins, and a great number belong to new gene families with a restricted distribution in the ocean. Overall, the resource provides the foundations for exploring the roles of marine eukaryotes in ocean ecology and biogeochemistry.
Non–Small-Cell Lung Cancer (NSCLC) is the most frequent human lung cancer and a major cause of death due to its high rate of metastasis1. These facts emphasize the urgent need for the investigation of new targets for anti-metastatic therapy. Up to now a number of genes and gene products have been identified that positively or negatively affect the probability of established human tumor cell lines to metastasize2. Previously, together with the group of Professor Ulf Rapp, we have described the first conditional mouse model for metastasis of NSCLC and identified a gene, c-MYC, that is able to orchestrate all steps of this process. We could identify potential markers for detection of metastasis and highlighted GATA4, which is exclusively expressed during lung development, as a target for future therapeutic intervention2. However, the mechanism underlying this metastatic conversion remained to be identified, and was therefore the focus of the present work. Here, GATA4 is identified as a MYC target in the development of metastasis and epigenetic alterations at the GATA4 promoter level are shown after MYC expression in NSCLC in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. This work identifies a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in NSCLC, suggesting novel potential targets for the development of anti-metastatic therapy.
In the fast-evolving landscape of biomedical research, the emergence of big data has presented researchers with extraordinary opportunities to explore biological complexities. In biomedical research, big data imply also a big responsibility. This is not only due to genomics data being sensitive information but also due to genomics data being shared and re-analysed among the scientific community. This saves valuable resources and can even help to find new insights in silico. To fully use these opportunities, detailed and correct metadata are imperative. This includes not only the availability of metadata but also their correctness. Metadata integrity serves as a fundamental determinant of research credibility, supporting the reliability and reproducibility of data-driven findings. Ensuring metadata availability, curation, and accuracy are therefore essential for bioinformatic research. Not only must metadata be readily available, but they must also be meticulously curated and ideally error-free. Motivated by an accidental discovery of a critical metadata error in patient data published in two high-impact journals, we aim to raise awareness for the need of correct, complete, and curated metadata. We describe how the metadata error was found, addressed, and present examples for metadata-related challenges in omics research, along with supporting measures, including tools for checking metadata and software to facilitate various steps from data analysis to published research.
Highlights
• Data awareness and data integrity underpins the trustworthiness of results and subsequent further analysis.
• Big data and bioinformatics enable efficient resource use by repurposing publicly available RNA-Sequencing data.
• Manual checks of data quality and integrity are insufficient due to the overwhelming volume and rapidly growing data.
• Automation and artificial intelligence provide cost-effective and efficient solutions for data integrity and quality checks.
• FAIR data management, various software solutions and analysis tools assist metadata maintenance.
Since ancient times aging has also been regarded as a disease, and humankind has always strived to extend the natural lifespan. Analyzing the genes involved in aging and disease allows for finding important indicators and biological markers for pathologies and possible therapeutic targets. An example of the use of omics technologies is the research regarding aging and the rare and fatal premature aging syndrome progeria (Hutchinson-Gilford progeria syndrome, HGPS). In our study, we focused on the in silico analysis of differentially expressed genes (DEGs) in progeria and aging, using a publicly available RNA-Seq dataset (GEO dataset GSE113957) and a variety of bioinformatics tools. Despite the GSE113957 RNA-Seq dataset being well-known and frequently analyzed, the RNA-Seq data shared by Fleischer et al. is far from exhausted and reusing and repurposing the data still reveals new insights. By analyzing the literature citing the use of the dataset and subsequently conducting a comparative analysis comparing the RNA-Seq data analyses of different subsets of the dataset (healthy children, nonagenarians and progeria patients), we identified several genes involved in both natural aging and progeria (KRT8, KRT18, ACKR4, CCL2, UCP2, ADAMTS15, ACTN4P1, WNT16, IGFBP2). Further analyzing these genes and the pathways involved indicated their possible roles in aging, suggesting the need for further in vitro and in vivo research. In this paper, we (1) compare “normal aging” (nonagenarians vs. healthy children) and progeria (HGPS patients vs. healthy children), (2) enlist genes possibly involved in both the natural aging process and progeria, including the first mention of IGFBP2 in progeria, (3) predict miRNAs and interactomes for WNT16 (hsa-mir-181a-5p), UCP2 (hsa-mir-26a-5p and hsa-mir-124-3p), and IGFBP2 (hsa-mir-124-3p, hsa-mir-126-3p, and hsa-mir-27b-3p), (4) demonstrate the compatibility of well-established R packages for RNA-Seq analysis for researchers interested but not yet familiar with this kind of analysis, and (5) present comparative proteomics analyses to show an association between our RNA-Seq data analyses and corresponding changes in protein expression.
Machine learning techniques are excellent to analyze expression data from single cells. These techniques impact all fields ranging from cell annotation and clustering to signature identification. The presented framework evaluates gene selection sets how far they optimally separate defined phenotypes or cell groups. This innovation overcomes the present limitation to objectively and correctly identify a small gene set of high information content regarding separating phenotypes for which corresponding code scripts are provided. The small but meaningful subset of the original genes (or feature space) facilitates human interpretability of the differences of the phenotypes including those found by machine learning results and may even turn correlations between genes and phenotypes into a causal explanation. For the feature selection task, the principal feature analysis is utilized which reduces redundant information while selecting genes that carry the information for separating the phenotypes. In this context, the presented framework shows explainability of unsupervised learning as it reveals cell-type specific signatures. Apart from a Seurat preprocessing tool and the PFA script, the pipeline uses mutual information to balance accuracy and size of the gene set if desired. A validation part to evaluate the gene selection for their information content regarding the separation of the phenotypes is provided as well, binary and multiclass classification of 3 or 4 groups are studied. Results from different single-cell data are presented. In each, only about ten out of more than 30000 genes are identified as carrying the relevant information. The code is provided in a GitHub repository at https://github.com/AC-PHD/Seurat_PFA_pipeline.
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.
By promoting ceramide release at the cytosolic membrane leaflet, the neutral sphingomyelinase 2 (NSM) is capable of organizing receptor and signalosome segregation. Its role in T cell receptor (TCR) signaling remained so far unknown. We now show that TCR-driven NSM activation is dispensable for TCR clustering and initial phosphorylation, but of crucial importance for further signal amplification. In particular, at low doses of TCR stimulatory antibodies, NSM is required for Ca\(^{2+}\) mobilization and T cell proliferation. NSM-deficient T cells lack sustained CD3ζ and ZAP-70 phosphorylation and are unable to polarize and stabilize their microtubular system. We identified PKCζ as the key NSM downstream effector in this second wave of TCR signaling supporting dynamics of microtubule-organizing center (MTOC). Ceramide supplementation rescued PKCζ membrane recruitment and MTOC translocation in NSM-deficient cells. These findings identify the NSM as essential in TCR signaling when dynamic cytoskeletal reorganization promotes continued lateral and vertical supply of TCR signaling components: CD3ζ, Zap70, and PKCζ, and functional immune synapses are organized and stabilized via MTOC polarization.
Chlamydia are Gram-negative obligate intracellular bacteria responsible for a wide spectrum of relevant diseases. Due to their biphasic developmental cycle Chlamydia depend on an intact host cell for replication and establishment of an acute infection. Chlamydia have therefore evolved sophisticated strategies to inhibit programmed cell death (PCD) induced by a variety of stimuli and to subvert the host immune system. This work aimed at elucidating whether an infection with C. trachomatis can influence the cellular response to double-stranded RNA (dsRNA). The synthesis of dsRNA is a prominent feature of viral replication inside infected cells that can induce both PCD and the activation of a cellular innate immune response. In order to mimic chlamydial and viral co-infections, Chlamydia-infected cells were transfected with polyinosinic:polycytidylic acid (polyI:C), a synthetic dsRNA. In the first part of this work it was investigated whether C. trachomatis-infected host cells could resist apoptosis induced by polyI:C. A significant reduction in apoptosis, determined by PARP cleavage and DNA fragmentation, could be observed in infected cells. It could be shown that processing of the initiator caspase-8 was inhibited in infected host cells. This process was dependent on early bacterial protein synthesis and was specific for dsRNA because apoptosis induced by TNFalpha was not blocked at the level of caspase-8. Interestingly, the activation of cellular factors involved in apoptosis induction by dsRNA, most importantly PKR and RNase L, was not abrogated in infected cells. Instead, RNA interference experiments revealed the crucial role of cFlip, a cellular caspase-8 inhibitor, for chlamydial inhibition of dsRNA-induced apoptosis. First data acquired by co-immunoprecipitation experiments pointed to an infection-induced concentration of cFlip in the dsRNA-induced death complex of caspase-8 and FADD. In the second part of this work, the chlamydial influence on the first line of defense against viral infections, involving expression of interferons and interleukins, was examined. Activation of the interferon regulatory factor 3 (IRF-3) and the NF-kappaB transcription factor family member p65, both central regulators of the innate immune response to dsRNA, was altered in Chlamydia-infected epithelial cells. polyI:C-induced degradation of IkappaB-alpha, the inhibitor of NF-kappaB, was accelerated in infected cells which was accompanied by a change in nuclear translocation of the transcription factor. Translocation of IRF-3, in contrast, was significantly blocked upon infection. Together the data presented here demonstrate that infection with C. trachomatis can drastically alter the cellular response to dsRNA and imply an impact of chlamydial infections on the outcome of viral super-infections.
Although macroecology is a well‐established field, much remains to be learned about the large‐scale variation of fungal traits. We conducted a global analysis of mean fruit body size of 59 geographical regions worldwide, comprising 5340 fungal species exploring the response of fruit body size to latitude, resource availability and temperature. The results showed a hump‐shaped relationship between mean fruit body size and distance to the equator. Areas with large fruit bodies were characterised by a high seasonality and an intermediate mean temperature. The responses of mutualistic species and saprotrophs were similar. These findings support the resource availability hypothesis, predicting large fruit bodies due to a seasonal resource surplus, and the thermoregulation hypothesis, according to which small fruit bodies offer a strategy to avoid heat and cold stress and therefore occur at temperature extremes. Fruit body size may thus be an adaptive trait driving the large‐scale distribution of fungal species.
DNA microarrays have become a standard technique to assess the mRNA levels for complete genomes. To identify significantly regulated genes from these large amounts of data a wealth of methods has been developed. Despite this, the functional interpretation (i.e. deducing biological hypothesis from the data) still remains a major bottleneck in microarray data analysis. Most available methods display the set of significant genes in long lists, from which common functional properties have to be extracted. This is not only a tedious and time-consuming task, which becomes less and less feasible with increasing numbers of experimental conditions, but is also prone to errors, since it is commonly done by eye. In the course of this work methods have been developed and tested, that allow for a computerbased analysis of functional properties being relevant in the given experimental setting. To this end the Gene Ontology was chosen as an appropriate source of annotation data, because it combines human-readability with computer-accessibility of the annotations term and thus allows for a statistical analysis of functional properties. Here the gene-annotations are integrated in a Correspondence Analysis which allows to visualize genes, hybridizations and functional categories in a single plot. Due to the increasing amounts of available annotations and the fact that in most settings only few functional processes are differentially regulated, several filter criteria have been developed to reduce the number of displayed annotations to a set being relevant in the given experimental setting. The applicability of the presented visualization and filtering have both been validated on datasets of varying complexity. Starting from the well studied glucose-pathway in S. cerevisiae up to the comparison of different tumor types in human. In both settings the method generated well interpretable plots, which allowed for an immediate identification of the major functional differences between the experimental conditions [90]. While the integration of annotation data like GO facilitates functional interpretation, it lacks the capability to identify key regulatory elements. To facilitate such an analysis, the occurrence of transcription factor binding sites in upstream regions of genes has been integrated to the analysis as well. Again this methodology was biologically validated on S. cerevisiae as well human cancer data sets. In both settings TFs known to exhibit central roles for the observed transcriptional changes were plotted in marked positions and thus could be immediately identified [206]. In essence, integration of supplementary information in Correspondence Analysis visualizes genes, hybridizations and annotation data in a single, well interpretable plot. This allows for an intuitive identification of relevant annotations even in complex experimental settings. The presented approach is not limited to the shown types of data, but is generalizable to account for the majority of the available annotation data.
Peritonitis is a common disease in man, frequently caused by fungi, such as Candida albicans; however, in seldom cases opportunistic infections with Saccharomyces cerevisiae are described. Resident peritoneal macrophages (prMΦ) are the major group of phagocytic cells in the peritoneum. They express a broad range of surface pattern recognition receptors (PRR) to recognize invaders. Yeast infections are primarily detected by the Dectin-1 receptor, which triggers activation of NFAT and NF-κB pathways.
The transcription of the Nfatc1 gene is directed by the two alternative promoters, inducible P1 and relatively constitutive P2 promoter. While the role of P1-directed NFATc1α-isoforms to promote survival and proliferation of activated lymphocytes is well-established, the relevance of constitutively generated NFATc1β-isoforms, mainly expressed in resting lymphocytes, myeloid and non-lymphoid cells, remains unclear. Moreover, former work at our department indicated different roles for NFATc1α- and NFATc1β-proteins in lymphocytes.
Our data revealed the functional role of NFATc1 in peritoneal resident macrophages. We demonstrated that the expression of NFATc1β is required for a proper immune response of prMΦ during fungal infection-induced acute peritonitis. We identified Ccl2, a major chemokine produced in response to fungal infections by prMΦ, as a novel NFATc1 target gene which is cooperatively regulated through the NFAT- and canonical NF-κB pathways. Consequently, we showed that NFATc1β deficiency in prMΦ results in a decreased infiltration of inflammatory monocytes, leading to a delayed clearance of peritoneal fungal infection.
We could further show that the expression of NFATc1β-isoforms is irrelevant for homeostasis of myeloid and adaptive immune system cells and that NFATc1α- (but not β-) isoforms are required for a normal development of peritoneal B1a cells. In contrast to the situation in myeloid cells, NFATc1β deficiency is compensated by increased expression of NFATc1α-isoforms in lymphoid cells. As a consequence, NFATc1ß is dispensable for activation of the adaptive immune system.
Taken together our results illustrate the redundancy and indispensability of NFATc1-isoforms in the adaptive and innate immune system, indicating a complex regulatory system for Nfatc1 gene expression in different compartments of the immune system and likely beyond that.
Background: Because most human stroke victims are elderly, studies of experimental stroke in the aged rather than the young rat model may be optimal for identifying clinically relevant cellular responses, as well for pinpointing beneficial interventions.
Methodology/Principal Findings: We employed the Affymetrix platform to analyze the whole-gene transcriptome following temporary ligation of the middle cerebral artery in aged and young rats. The correspondence, heat map, and dendrogram analyses independently suggest a differential, age-group-specific behaviour of major gene clusters after stroke. Overall, the pattern of gene expression strongly suggests that the response of the aged rat brain is qualitatively rather than quantitatively different from the young, i.e. the total number of regulated genes is comparable in the two age groups, but the aged rats had great difficulty in mounting a timely response to stroke. Our study indicates that four genes related to neuropathic syndrome, stress, anxiety disorders and depression (Acvr1c, Cort, Htr2b and Pnoc) may have impaired response to stroke in aged rats. New therapeutic options in aged rats may also include Calcrl, Cyp11b1, Prcp, Cebpa, Cfd, Gpnmb, Fcgr2b, Fcgr3a, Tnfrsf26, Adam 17 and Mmp14. An unexpected target is the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 in aged rats, a key enzyme in the cholesterol synthesis pathway. Post-stroke axonal growth was compromised in both age groups.
Conclusion/Significance: We suggest that a multi-stage, multimodal treatment in aged animals may be more likely to produce positive results. Such a therapeutic approach should be focused on tissue restoration but should also address other aspects of patient post-stroke therapy such as neuropathic syndrome, stress, anxiety disorders, depression, neurotransmission and blood pressure.
Background
Cuticular hydrocarbons (CHC) have been documented to play crucial roles as species- and sex-specific cues in the chemical communication systems of a wide variety of insects. However, whether they are sufficient by themselves as the sole cue triggering sexual behavior as well as preference of con- over heterospecific mating partners is rarely assessed. We conducted behavioral assays in three representative species of parasitoid wasps (Hymenoptera: Pteromalidae) to determine their reliance on CHC as species-specific sexual signaling cues.
Results
We found a surprising degree of either unspecific or insufficient sexual signaling when CHC are singled out as recognition cues. Most strikingly, the cosmopolitan species Nasonia vitripennis, expected to experience enhanced selection pressure to discriminate against other co-occurring parasitoids, did not discriminate against CHC of a partially sympatric species from another genus, Trichomalopsis sarcophagae. Focusing on the latter species, in turn, it became apparent that CHC are even insufficient as the sole cue triggering conspecific sexual behavior, hinting at the requirement of additional, synergistic sexual cues particularly important in this species. Finally, in the phylogenetically and chemically most divergent species Muscidifurax uniraptor, we intriguingly found both CHC-based sexual signaling as well as species discrimination behavior intact although this species is naturally parthenogenetic with sexual reproduction only occurring under laboratory conditions.
Conclusions
Our findings implicate a discrepancy in the reliance on and specificity of CHC as sexual cues in our tested parasitioid wasps. CHC profiles were not sufficient for unambiguous discrimination and preference behavior, as demonstrated by clear cross-attraction between some of our tested wasp genera. Moreover, we could show that only in T. sarcophagae, additional behavioral cues need to be present for triggering natural mating behavior, hinting at an interesting shift in signaling hierarchy in this particular species. This demonstrates the importance of integrating multiple, potentially complementary signaling modalities in future studies for a better understanding of their individual contributions to natural sexual communication behavior.
1. Protection against desiccation and chemical communication are two fundamental functions of cuticular hydrocarbons (CHCs) in insects. In the parasitoid jewel wasp Nasonia vitripennis (Walker), characterised by a cosmopolitan distribution through largely different environments, CHCs function as universally recognised female sex pheromones. However, CHC uniformity as basis for sexual recognition may conflict with the desiccation protection function, expected to display considerable flexibility through adaptation to different environmental conditions.
2. We compared male and female CHC profiles of N. vitripennis across a wide latitudinal gradient in Europe and correlated their CHC variation with climatic factors associated with desiccation. Additionally, we tested male mate discrimination behaviour between populations to detect potential variations in female sexual attractiveness.
3. Results did not conform to the general expectation that longer, straight‐chain CHCs occur in higher proportions in warmer and drier climates. Instead, unexpected environmental correlations of intermediate chain‐length CHCs (C31) were found exclusively in females, potentially reflecting the different life histories of the sexes in N. vitripennis.
4. Furthermore, we found no indication of population‐specific male mate preference, confirming the stability of female sexual attractiveness, likely conveyed through their CHC profiles. C31 mono‐ and C33 di‐methyl‐branched alkanes were consistently and most strongly associated with sexual dimorphism, suggesting their potential role in encoding the female‐specific sexual signalling function.
5. Our study sheds light on how both adaptive flexibility and conserved sexual attractiveness can potentially be integrated and encoded in CHC profiles of N. vitripennis females across a wide distribution range in Europe.
Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells.
Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.
Background:
Chloroplast-encoded genes (matK and rbcL) have been formally proposed for use in DNA barcoding efforts targeting embryophytes. Extending such a protocol to chlorophytan green algae, though, is fraught with problems including non homology (matK) and heterogeneity that prevents the creation of a universal PCR toolkit (rbcL). Some have advocated the use of the nuclear-encoded, internal transcribed spacer two (ITS2) as an alternative to the traditional chloroplast markers. However, the ITS2 is broadly perceived to be insufficiently conserved or to be confounded by introgression or biparental inheritance patterns, precluding its broad use in phylogenetic reconstruction or as a DNA barcode. A growing body of evidence has shown that simultaneous analysis of nucleotide data with secondary structure information can overcome at least some of the limitations of ITS2. The goal of this investigation was to assess the feasibility of an automated, sequence-structure approach for analysis of IT2 data from a large sampling of phylum Chlorophyta.
Methodology/Principal Findings:
Sequences and secondary structures from 591 chlorophycean, 741 trebouxiophycean and 938 ulvophycean algae, all obtained from the ITS2 Database, were aligned using a sequence structure-specific scoring matrix. Phylogenetic relationships were reconstructed by Profile Neighbor-Joining coupled with a sequence structure-specific, general time reversible substitution model. Results from analyses of the ITS2 data were robust at multiple nodes and showed considerable congruence with results from published phylogenetic analyses.
Conclusions/Significance:
Our observations on the power of automated, sequence-structure analyses of ITS2 to reconstruct phylum-level phylogenies of the green algae validate this approach to assessing diversity for large sets of chlorophytan taxa. Moreover, our results indicate that objections to the use of ITS2 for DNA barcoding should be weighed against the utility of an automated, data analysis approach with demonstrated power to reconstruct evolutionary patterns for highly divergent lineages.
Chronic Obstructive Pulmonary Disease (COPD) exacerbations are a considerable reason for increased morbidity and mortality in patients. Infections with influenza virus (H1N1), respiratory syncytial virus (RSV) or nontypeable Haemophilus influenzae (NTHi) are important triggers of exacerbations. To date, no treatments are available which can stop the progression of COPD. Novel approaches are urgently needed. Pre-clinical models of the disease are crucial for the development of novel therapeutic options.
In order to establish pre-clinical models which mimic aspects of human COPD exacerbations, mice were exposed to cigarette smoke (CS) and additionally infected with H1N1, RSV and/or NTHi. Clinically relevant treatments such as the corticosteroids Fluticasone propionate and Dexamethasone, the phosphodiesterase-4 (PDE-4) inhibitor Roflumilast and the long-acting muscarinic receptor antagonist Tiotropium were tested in the established models. Furthermore, a novel treatment approach using antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1 was examined in the established CS/H1N1 model. Levels of IFN-γ, IL-1β, IL-2, IL-6, KC, TNF-α, RANTES, IL-17, MCP-1, MIP 1α and MIP-1β were measured in lung homogenate. Numbers of total cells, neutrophils and macrophages were assessed in bronchoalveolar lavage (BAL) fluid. Hematoxylin- and eosin- (H&E-) stained lung slices were analyzed to detect pathological changes. Quantitative polymerase-chain-reaction (qPCR) was used to investigate gene expression of ICAM-1 and MUC5 A/C. The viral/bacterial load was investigated in lung homogenate or BAL fluid. In addition to the in vivo studies, the effects of the above mentioned treatments were investigated in vitro in H1N1, RSV or NTHi-infected (primary) human bronchial epithelial cells using submerged or air-liquid-interface (ALI) cell culture systems.
Four pre-clinical models (CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi) were established depicting clinically relevant aspects of COPD exacerbations such as increased inflammatory cells and cytokines in the airways and impaired lung function.
In the CS/H1N1 model, Tiotropium improved lung function and was superior in reducing inflammation in comparison to Fluticasone or Roflumilast. Moreover, Fluticasone increased the loss of body-weight, levels of IL-6, KC and TNF-α and worsened lung function. In CS/RSV-exposed mice Tiotropium but not Fluticasone or Roflumilast treatment reduced neutrophil numbers and IL-6 and TNF α levels in the lung. The viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after Fluticasone and Dexamethasone treatment. The results from these studies demonstrate that Tiotropium has anti-inflammatory effects on CS/virus-induced inflammation and might help to explain the observed reduction of exacerbation rates in Tiotropium-treated COPD patients. Furthermore, the findings from this work indicate that treatment with Fluticasone or Dexamethasone might not be beneficial to reduce inflammation in the airways of COPD patients and supports clinical studies that link treatment with corticosteroids to an increased risk for pneumonia.
Testing of anti-IL-1α, anti-IL-1β or anti-IL-1R1 Abs in the CS/H1N1 model suggests that, in line with clinical data, antagonization of IL-1β is not sufficient to reduce pulmonary inflammation and indicates a predominant role of IL-1α in CS/virus-induced airway inflammation. In line with the in vivo findings, anti-IL-1α but not anti-IL-1β Abs reduced levels of TNF-α and IL-6 in H1N1-infected primary human bronchial epithelial ALI cell culture. Blocking the IL-1R1 provided significant inhibitory effects on inflammatory cells in vivo but was inferior compared to inhibiting both its soluble ligands IL-1α and IL-1β. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 mRNA expression was attenuated. These results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce inflammation and exacerbations in COPD patients. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to inhibition of the IL-1R1.
As in the CS/virus models, corticosteroid treatment failed to reduce inflammatory cells in the CS/NTHi and CS/H1N1/NTHi models, increased the loss of body-weight and the bacterial load. Furthermore, Roflumilast administration had no significant effects on cell counts or cytokines. However, it improved compliance in the CS/NTHi model. Treatment with Azithromycin reduced the bacterial load in the CS/NTHi model and reduced numbers of total cells, neutrophils, macrophages and levels of KC and TNF-α in the CS/H1N1/NTHi model.
In conclusion, the established CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi models depict clinically relevant aspects of human COPD exacerbations in mice and provide the opportunity to investigate underlying disease mechanisms and to test novel therapies.
In this thesis, synaptic transmission was studied electrophysiologically at an invertebrate model synapse, the neuromuscular junction of the Drosophila 3rd instar wandering larvae. In the first part, synaptic function is characterized at the neuromuscular junction in fly lines which are null mutants for the synaptic proteins “the synapse associated protein of 47 kDa” (Sap-47156), Synapsin (Syn97), the corresponding double mutant (Sap-47156, Syn97), a null mutant for an as yet uncharacterized Drosophila SR protein kinase, the Serine-Arginine protein kinase 3 (SRPK3), and the Löchrig (Loe) mutant which shows a strong neurodegenerative phenotype. Intracellular voltage recordings from larval body wall muscles 6 and 7 were performed to measure amplitude and frequency of spontaneous single vesicle fusion events (miniature excitatory junction potentials or mEJPs). Evoked excitatory junction potentials (eEJPs) at different frequencies and calcium concentrations were also measured to see if synaptic transmission was altered in mutants which lacked these synaptic proteins. In addition, structure and morphology of presynaptic boutons at the larval neuromuscular junction were examined immunohistochemically using monoclonal antibodies against different synaptic vesicle proteins (SAP-47, CSP, and Synapsin) as well as the active zone protein Bruchpilot. Synaptic physiology and morphology was found to be similar in all null mutant lines. However, Löchrig mutants displayed an elongated bouton morphology, a significant shift towards larger events in mEJP amplitude frequency histograms, and increased synaptic facilitation during a 10 Hz tetanus. These deficits suggest that Loe mutants may have a defect in some aspect of synaptic vesicle recycling. The second part of this thesis involved the electrophysiological characterization of heterologously expressed light activated proteins at the Drosophila neuromuscular junction. Channelrhodopsin-2 (ChR2), a light gated ion channel, and a photoactivated adenylate cyclase (PAC) were expressed in larval motor neurons using the UAS-Gal4 system. Single EJPs could be recorded from muscles 15, 16, and 17 when larva expressing ChR2 were illuminated with short (100 ms) light pulses, whereas long light pulses (10 seconds) resulted in trains of EJPs with a frequency of around 25 Hz. Larva expressing PAC in preparations where motor neurons were cut from the ventral ganglion displayed a significant increase in mEJP frequency after a 1 minute exposure to blue light. Evoked responses in low (.2 mM) calcium were also significantly increased when PAC was stimulated with blue light. When motor nerves were left intact, PAC stimulation resulted in light evoked EJPs in muscles 6 and 7 in a manner consistent with RP3 motor neuron activity. ChR2 and PAC are therefore useful and reliable tools for manipulating neuronal activity in vivo.
Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression.
At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor.
Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.
Large parts of the tropical lowland rain forests of Sabah (Malaysia) were transformed into secondary forests due to heavy logging. Additionally the remaining forest remnants are isolated from each other by large scale oil palm plantations. Biodiversity patterns and responses of the community of leaf litter ants were studied in anthropogenically disturbed habitats and primary forests of different size. In logged over forests, only 70 per cent of the species of a primary forest were present even 25 years after timber extraction. The ant communities were thinned and could be described by a lower species density producing lower species numbers and a different community composition. The similarity in species number and community composition between logged over forests of different degrees of disturbance was explained by source-sink dynamics within a heterogeneous forest matrix. Rain forest fragments displayed even higher reductions in species density, numbers and diversity due to a more pronounced thinning effect. Even forest isolates exceeding 4 000 ha in size did not support more than 50 per cent of the species of the leaf litter ant community of a contiguous primary rain forest. Additionally, an increase in tramp species was recorded with decreasing size of the forest fragments, leading to a very different community composition. Regarding the leaf litter ant community, the remaining rain forest fragments of Sabah are effectively isolated by a barrier of oil palm plantation, now stretching all over the lowlands of the east coast. Only 13 species, which belonged to the forest ant community in highly disturbed areas were collected in these plantations. Some of the 10 other species of the highly reduced ground-dwelling ant community in the plantations are known as invasive tramp species, forming large exclusive territories. Correlative evidence and a field experiment implied, that leaf litter humidity, volume and temperature affect the distribution and community composition of forest leaf litter ant species. The smaller primary forests and the most disturbed logged over forests in this study revealed higher temperatures and lower humidity levels and a reduction in leaf litter volume compared to a large primary forest or forests affected by a lower impact of timber harvesting. If the pattern for leaf litter ants is confirmed for other taxa, the implications for any efficient management design aiming to preserve the majority of the biodiversity of the country are tremendous and current concepts need rethinking.
Abstract
Background
HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases.
Results
As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde.
Conclusion
The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.
HyphaTracker: An ImageJ toolbox for time-resolved analysis of spore germination in filamentous fungi
(2018)
The dynamics of early fungal development and its interference with physiological signals and environmental factors is yet poorly understood. Especially computational analysis tools for the evaluation of the process of early spore germination and germ tube formation are still lacking. For the time-resolved analysis of conidia germination of the filamentous ascomycete Fusarium fujikuroi we developed a straightforward toolbox implemented in ImageJ. It allows for processing of microscopic acquisitions (movies) of conidial germination starting with drift correction and data reduction prior to germling analysis. From the image time series germling related region of interests (ROIs) are extracted, which are analysed for their area, circularity, and timing. ROIs originating from germlings crossing other hyphae or the image boundaries are omitted during analysis. Each conidium/hypha is identified and related to its origin, thus allowing subsequent categorization. The efficiency of HyphaTracker was proofed and the accuracy was tested on simulated germlings at different signal-to-noise ratios. Bright-field microscopic images of conidial germination of rhodopsin-deficient F. fujikuroi mutants and their respective control strains were analysed with HyphaTracker. Consistent with our observation in earlier studies the CarO deficient mutant germinated earlier and grew faster than other, CarO expressing strains.
Whole Genome Duplications Shaped the Receptor Tyrosine Kinase Repertoire of Jawed Vertebrates
(2016)
The receptor tyrosine kinase (RTK) gene family, involved primarily in cell growth and differentiation, comprises proteins with a common enzymatic tyrosine kinase intracellular domain adjacent to a transmembrane region. The amino-terminal portion of RTKs is extracellular and made of different domains, the combination of which characterizes each of the 20 RTK subfamilies among mammals. We analyzed a total of 7,376 RTK sequences among 143 vertebrate species to provide here the first comprehensive census of the jawed vertebrate repertoire. We ascertained the 58 genes previously described in the human and mouse genomes and established their phylogenetic relationships. We also identified five additional RTKs amounting to a total of 63 genes in jawed vertebrates. We found that the vertebrate RTK gene family has been shaped by the two successive rounds of whole genome duplications (WGD) called 1R and 2R (1R/2R) that occurred at the base of the vertebrates. In addition, the Vegfr and Ephrin receptor subfamilies were expanded by single gene duplications. In teleost fish, 23 additional RTK genes have been retained after another expansion through the fish-specific third round (3R) of WGD. Several lineage-specific gene losses were observed. For instance, birds have lost three RTKs, and different genes are missing in several fish sublineages. The RTK gene family presents an unusual high gene retention rate from the vertebrate WGDs (58.75% after 1R/2R, 64.4% after 3R), resulting in an expansion that might be correlated with the evolution of complexity of vertebrate cellular communication and intracellular signaling.
Background: In most trypanosomes, endo and exocytosis only occur at a unique organelle called the flagellar pocket (FP) and the flagellum exits the cell via the FP. Investigations of essential cytoskeleton-associated structures located at this site have revealed a number of essential proteins. The protein TbBILBO1 is located at the neck of the FP in a structure called the flagellar pocket collar (FPC) and is essential for biogenesis of the FPC and parasite survival. TbMORN1 is a protein that is present on a closely linked structure called the hook complex (HC) and is located anterior to and overlapping the collar. TbMORN1 is essential in the bloodstream form of T. brucei. We now describe the location and function of BHALIN, an essential, new FPC-HC protein. Methodology/Principal Findings: Here, we show that a newly characterised protein, BHALIN (BILBO1 Hook Associated LINker protein), is localised to both the FPC and HC and has a TbBILBO1 binding domain, which was confirmed in vitro. Knockdown of BHALIN by RNAi in the bloodstream form parasites led to cell death, indicating an essential role in cell viability. Conclusions/Significance: Our results demonstrate the essential role of a newly characterised hook complex protein, BHALIN, that influences flagellar pocket organisation and function in bloodstream form T. brucei parasites.
(1) Background: The recurrence of glioblastoma multiforme (GBM) is mainly due to invasion of the surrounding brain tissue, where organic solutes, including glucose and inositol, are abundant. Invasive cell migration has been linked to the aberrant expression of transmembrane solute-linked carriers (SLC). Here, we explore the role of glucose (SLC5A1) and inositol transporters (SLC5A3) in GBM cell migration. (2) Methods: Using immunofluorescence microscopy, we visualized the subcellular localization of SLC5A1 and SLC5A3 in two highly motile human GBM cell lines. We also employed wound-healing assays to examine the effect of SLC inhibition on GBM cell migration and examined the chemotactic potential of inositol. (3) Results: While GBM cell migration was significantly increased by extracellular inositol and glucose, it was strongly impaired by SLC transporter inhibition. In the GBM cell monolayers, both SLCs were exclusively detected in the migrating cells at the monolayer edge. In single GBM cells, both transporters were primarily localized at the leading edge of the lamellipodium. Interestingly, in GBM cells migrating via blebbing, SLC5A1 and SLC5A3 were predominantly detected in nascent and mature blebs, respectively. (4) Conclusion: We provide several lines of evidence for the involvement of SLC5A1 and SLC5A3 in GBM cell migration, thereby complementing the migration-associated transportome. Our findings suggest that SLC inhibition is a promising approach to GBM treatment.