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Analysis of \(Trypanosoma\) \(brucei\) motility and the infection process in the tsetse fly vector
(2021)
African trypanosomes are protist pathogens that are infective for a wide spectrum of mammalian hosts. Motility has been shown to be essential for their survival and represents an important virulence factor. Trypanosoma brucei is transmitted by the bite of the bloodsucking tsetse fly, the only vector for these parasites. The voyage through the fly is complex and requires several migration, proliferation and differentiation steps, which take place in a defined order and in specific fly tissues.
The first part of this doctoral thesis deals with the establishment of the trypanosome tsetse system as a new model for microswimmer analysis. There is an increasing interdisciplinary interest in microbial motility, but a lack of accessible model systems. Therefore, this work introduces the first enclosed in vivo host parasite system that is suitable for analysis of diverse microswimmer types in specific microenvironments. Several methods were used and adapted to gain unprecedented insights into trypanosome motion, the fly´s interior architecture and the physical interaction between host and parasite. This work provides a detailed overview on trypanosome motile behavior as a function of development in diverse host surroundings. In additional, the potential use of artificial environments is shown. This can be used to partly abstract the complex fly architecture and analyze trypanosome motion in defined nature inspired geometries.
In the second part of the thesis, the infection of the tsetse fly is under investigation. Two different trypanosome forms exist in the blood: proliferative slender cells and cell cycle arrested stumpy cells. Previous literature states that stumpy cells are pre adapted to survive inside the fly, whereas slender cells die shortly after ingestion. However, infection experiments in our laboratory showed that slender cells were also potentially infective. During this work, infections were set up so as to minimize the possibility of stumpy cells being ingested, corroborating the observation that slender cells are able to infect flies. Using live cell microscopy and fluorescent reporter cell lines, a comparative analysis of the early development following infection with either slender or stumpy cells was performed. The experiments showed, for the first time, the survival of slender trypanosomes and their direct differentiation to the procyclic midgut stage, contradicting the current view in the field of research. Therefore, we can shift perspectives in trypanosome biology by proposing a revised life cycle model of T. brucei, where both bloodstream stages are infective for the vector.
Insight into molecular mechanisms of folding and self-association of spider silk protein domains
(2021)
Spider silk is a biomaterial of extraordinary toughness paired with elasticity. The assembly of silk proteins, so-called spidroins (from “spider” and “fibroin”), generates the silk threads we typically see in our garden or the corners of our houses. Although spider webs from different species vary considerably in geometry and size, many sections of spidroin sequences are conserved. Highly conserved regions, found in all spidroins, relate to the terminal domains of the protein, i.e., the N-terminal (NTD) and C-terminal domains (CTD). Both have an essential function in the silk fibre association and polymerisation.
The NTD is a 14 kDa five-helix bundle, which self-associates via a pH-driven mechanism. This process is critical for starting the polymerisation of the fibre. However, detailed insights into how conserved this mechanism is in different species and the quantitative thermodynamic comparison between homologous NTDs was missing. For this reason, four homologous NTDs of the major ampullate gland (MaSp) from spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus were investigated. I analysed and quantified equilibrium thermodynamics, kinetics of folding, and self-association. Methods involved dynamic light scattering (MALS), stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. The results showed conserved, cooperative two-state folding on a sub-millisecond time scale. All homologous NTDs showed a similarly fast association in the order of 10^9 M^−1 s^−1, while the resulting equilibrium dissociation constants were in the low nanomolar range. Electrostatic forces were found to be of great importance for protein association. Monomeric protein stability increased with salt concentration while enhancing its folding speed. However, due to Debye-Hückel effects, we found intermolecular electrostatics to be shielded, which reduced the NTDs association capacity significantly at high ionic strength. Altogether, the energetics and kinetics of the NTD dimerisation was conserved for all analysed homologs.
Comparable to the NTD, the spider silks CTD is also a α-helix bundle, which covalently links two spidroins. The orientation of the domains predetermines the future fibre geometry. Here again, the detailed quantitative characterisation of the folding and dimerisation was missing. Therefore, the CTD from the E. australis was analysed in-depth. The protein folded via a three-state mechanism and was placed in the family of knotted proteins.
By analysing the amino acid composition of the NTD of the MaSp1 of the Euprosthenops australis, we found an unusually high content of methionine residues (Met). To elucidate why this protein exhibits so many Met residues, I mutated all core Mets simultaneously to leucine (Leu). Results revealed a dramatically stabilised NTD, which now folded 50 times faster. After solving the tertiary structure of the mutant by NMR (nuclear magnetic resonance) spectroscopy, the structure of the monomeric mutant was found to be identical with the wild-type protein. However, when probing the dimerisation of the NTD, I could show that the association capacity was substantially impaired for the mutant. Our findings lead to the conclusion that Met provides the NTD with enhanced conformational dynamics and thus mobilises the protein, which results in tightly associated dimers. In additional experiments, I first re-introduced new Met residues into the Met-depleted protein at sequence positions containing native Leu. Hence, the mutated NTD protein was provided with the same number of Leu, which were previously removed by mutation. However, the protein did not regain wild-type characteristics. The functionality was not restored, but its stability was decreased as expected. To probe our hypothesis gained from the MaSp NTD, I transferred the experiment to another protein, namely the Hsp90 chaperone. Therefore, I incorporated methionine residues in the protein, which resulted in a slight improvement of its function.
Finally, trial experiments were performed aiming at the synthesis of shortened spidroin constructs containing less repetitive middle-segments than the wild-type protein. The objective was to study the findings of the terminal domains in the context of an intact spidroin. The synthesis of these engineered spidroins was challenging. Nevertheless, preliminary results encourage the assumption that the characteristics observed in the isolated domains hold true in the context of a full-length spidroin.
Because of its complexity and intricacy, studying the nervous system is often challenging. Fortunately, the small nematode roundworm Caenorhabditis elegans is well established as a model system for basic neurobiological research. The C. elegans model is also the only organism with a supposedly complete connectome, an organism-wide map of synaptic connectivity resolved by electron microscopy, which provides some understanding of how the nervous system works as a whole. However, the number of available data-sets is small and the connectome contains errors and gaps. One example of this concerns electrical synapses. Electrical synapses are formed by gap junctions and difficult to map due to their often ambiguous morphology in electron micrographs, leading to misclassification or omission. On the other hand, chemical synapses are more easily mapped, but many aspects of their mode of operation remain elusive and their role in the C. elegans connectome is oversimplified. A comprehensive understanding of signal transduction of neurons between each other and other cells will be indispensable for a comprehensive understanding of the nervous system. In this thesis, I approach these challenges with a combination of advanced light and electron microscopy techniques.
First, this thesis describes a strategy to increase synaptic specificity in connectomics. Specifically, I classify gap junctions with a high degree of confidence. To achieve this, I utilized array tomography (AT). In this thesis, AT is adapted for high-pressure freezing to optimize for structure preservation and for super-resolution light microscopy; in this manner, I aim to bridge the gap between light and electron microscopy resolutions. I call this adaptation super-resolution array tomography (srAT). The srAT approach made it possible to clearly identify and map gap junctions with high precision and accuracy. The results from this study showcased the feasibility of incorporating electrical synapses into connectomes in a systematic manner, and subsequent studies have used srAT for other models and questions.
As mentioned above, the C. elegans connectomic model suffers from a shortage of datasets. For most larval stages, including the special dauer larval stage, connectome data is completely missing up to now. To obtain the first partial connectome data-set of the C. elegans dauer larva, we used focused ion-beam scanning electron microscopy (FIB-SEM). This technique offers an excellent axial resolution and is useful for acquiring large volumes for connectomics. Together with our collaborators, I acquired several data-sets which enable the analysis of dauer stage-specific “re-wiring” of the nervous system and thus offer valuable insights into connectome plasticity/variability.
While chemical synapses are easy to map relative to electrical synapses, signal transduction via chemical transmitters requires a large number of different proteins and molecular processes acting in conjunction in a highly constricted space. Because of the small spatial scale of the synapse, investigating protein function requires very high resolution, which electron tomography provides. I analyzed electron tomograms of a worm-line with a mutant synaptic protein, the serine/threonine kinase SAD-1, and found remarkable alterations in several architectural features. My results confirm and re-contextualize previous findings and provide new insight into the functions of this protein at the chemical synapse.
Finally, I investigated the effectiveness of our methods on “malfunctioning,” synapses, using an amyotrophic lateral sclerosis (ALS) model. In the putative synaptopathy ALS, the mechanisms of motor neuron death are mostly unknown. However, mutations in the gene FUS (Fused in Sarcoma) are one known cause of the disease. The expression of the mutated human FUS in C. elegans was recently shown to produce an ALS-like phenotype in the worms, rendering C. elegans an attractive disease model for ALS. Together with our collaboration partners, I applied both srAT and electron tomography methods to “ALS worms” and found effects on vesicle docking. These findings help to explain electrophysiological recordings that revealed a decrease in frequency of mini excitatory synaptic currents, but not amplitudes, in ALS worms compared to controls. In addition, synaptic endosomes appeared larger and contained electron-dense filaments in our tomograms. These results substantiate the idea that mutated FUS impairs vesicle docking and also offer new insights into further molecular mechanisms of disease development in FUS-dependent ALS. Furthermore, we demonstrated the broader applicability of our methods by successfully using them on cultured mouse motor neurons.
Overall, using the C. elegans model and a combination of light and electron microscopy methods, this thesis helps to elucidate the structure and function of neuronal synapses, towards the aim of obtaining a comprehensive model of the nervous system.
The human pathogen Chlamydia trachomatis is the main cause of sexually transmitted infections worldwide. The obligate intracellular bacteria are the causative agent of several diseases that reach from conjunctivitis causing trachoma and blindness as well as salpingitis and urethritis which can lead to infertility if left untreated.
In order to gain genetically engineered Chlamydia that inducible knock down specific gene expression, the CRISPRi system was established in C. trachomatis. In a proof of principle experiment it was shown that C. trachomatis pCRISPRi:gCdu1III target ChlaDUB1 expression and reduce the protein amount up to 50 %. Knock-down of the DUB did not influence protein levels of anti-apoptotic Mcl-1 and did not make cells susceptible for apoptosis. However, reduced dCas9 protein size, bacterial growth impairment and off target effects interfering with the GFP signal, form obstacles in CRISPRi system in Chlamydia. For routinely use of the CRISPRi method in C. trachomatis further investigation is needed.
Since the bacterial life cycle includes two morphological and functional distinct forms, it is essential for chlamydial spread to complete the development cycle and form infectious progeny. Therefore, Chlamydia has evolved strategies to evade the host immune system in order to stay undetected throughout the developmental cycle. The bacteria prevent host cell apoptosis via stabilization of anti-apoptotic proteins like Mcl-1, Survivin and HIF-1α and activate pro-survival pathways, inhibiting invasion of immune cells to the site of infection. The host cell itself can destroy intruders via cell specific defense systems that involve autophagy and recruitment of professional immune cells. In this thesis the role of the chlamydial deubiuqitinase ChlaDUB1 upon immune evasion was elucidated. With the mutant strain Ctr Tn-cdu1 that encodes for a truncated DUB due to transposon insertion, it was possible to identify ChlaDUB1 as a potent opponent of the autophagic system. Mutant inclusions were targeted by K48 and K63 chain ubiquitination. Subsequently the inclusion was recognized by autophagic receptors like p62, NBR1 and NDP52 that was reversed again by complementation with the active DUB. Xenophagy was promoted so far as LC3 positive phagosomes formed around the inclusion of Ctr Tn-cdu1, which did not fuse with the lysosome. The detected growth defect in human primary cells of Chlamydia missing the active DUB was not traced back to autophagy, but was due to impaired development and replication. It was possible to identify Ankib1, the E3 ligase, that ubiquitinates the chlamydial inclusion in a siRNA based screen. The activating enzyme Ube1 and the conjugating enzyme Ube2L3 are also essential in this process. Chlamydia have a reduced genome and depend on lipids and nutrients that are translocated from the host cell to the inclusion to proliferate. Recruitment of fragmented Golgi stacks to the inclusion surface was prevented when ChlaDUB1 was inactive, probably causing diminished bacterial growth. Additionally, the modification of the inclusion by Ankib1 and subsequent decoration by autophagic markers was not only present in human but also murine cells. Comparison of other Chlamydia strains and species revealed Ankib1 to be located at the proximity of the inclusion in C. trachomatis strains only but not in C. muridarum or C. pneumoniae, indicating that Ankib1 is specifically the E3 ligase of C. trachomatis. Moreover, the role of ChlaDUB1 in infected tissue was of interest, since ChlaDUB1 protein was also found in early EB stage and so might get in contact with invading immune cells after cell lysis. While bacteria spread and infect new host cells, Chlamydia can also infect immune cells. Infection of human neutrophils with Ctr Tn-cdu1 shows less bacterial survival and affirms the importance of the DUB for bacterial fitness in these cells.
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.
Neuroblastoma is the most abundant, solid, extracranial tumor in early childhood and the leading cause of cancer-related childhood deaths worldwide. Patients with high-risk neuroblastoma often show MYCN-amplification and elevated levels of Aurora-A. They have a low overall survival and despite multimodal therapy options a poor therapeutic prognosis. MYCN-amplified neuroblastoma cells depend on Aurora-A functionality. Aurora-A stabilizes MYCN and prevents it from proteasomal degradation by competing with the E3 ligase SCFFBXW7. Interaction between Aurora-A and MYCN can be observed only in S phase of the cell cycle and activation of Aurora-A can be induced by MYCN in vitro. These findings suggest the existence of a profound interconnection between Aurora-A and MYCN in S phase. Nevertheless, the details remain elusive and were investigated in this study.
Fractionation experiments show that Aurora-A is recruited to chromatin in S phase in a MYCN-dependent manner. Albeit being unphosphorylated on the activating T288 residue, Aurora-A kinase activity was still present in S phase and several putative, novel targets were identified by phosphoproteomic analysis. Particularly, eight phosphosites dependent on MYCN-activated Aurora-A were identified. Additionally, phosphorylation of serine 10 on histone 3 was verified as a target of this complex in S phase. ChIP-sequencing experiments reveal that Aurora-A regulates transcription elongation as well as histone H3.3 variant incorporation in S phase. 4sU-sequencing as well as immunoblotting demonstrated that Aurora-A activity impacts splicing. PLA measurements between the transcription and replication machinery revealed that Aurora-A prevents the formation of transcription-replication conflicts, which activate of kinase ATR.
Aurora-A inhibitors are already used to treat neuroblastoma but display dose-limiting toxicity. To further improve Aurora-A based therapies, we investigated whether low doses of Aurora-A inhibitor combined with ATR inhibitor could increase the efficacy of the treatment albeit reducing toxicity. The study shows that the combination of both drugs leads to a reduction in cell growth as well as an increase in apoptosis in MYCN-amplified neuroblastoma cells, which is not observable in MYCN non-amplified neuroblastoma cells. This new approach was also tested by a collaboration partner in vivo resulting in a decrease in tumor burden, an increase in overall survival and a cure of 25% of TH-MYCN mice. These findings indicate indeed a therapeutic window for targeting MYCN-amplified neuroblastoma.
Die Regulation der Genexpression steht am Anfang vieler zellbiologischer Prozesse wie beispielsweise dem Zellwachstum oder der Differenzierung. Gene werden an Promotoren transkribiert, wobei ein Promotor selbst aus vielen logischen Einheiten aufgebaut ist, den Transkriptionsfaktorbindestellen (TFBSs). Diese können sehr nah beieinander liegen, aber auch weit entfernt voneinander sein. Sie werden spezifisch von Transkriptionsfaktoren (TFs) gebunden, die die Transkritptionsrate z.B. verstärken (Enhancer) oder schwächen (Silencer) können. Zwei oder mehr dieser TFBSs mit bestimmtem Abstand werden als "Module" zusammengefasst, die über Spezies hinweg konserviert sein können. Typischerweise findet man Module in Zellen mit einem Zellkern. Spezies mit gemeinsamen Modulen können ein Hinweis auf die gemeinsame phylogenetische Abstammung darstellen, aber auch gemeinsame Funktionsmechanismen von TFs über Gene hinweg aufdecken. Heutzutage sind verschiedene Anwendungen verfügbar, mit denen nach TFBSs in DNA gesucht werden kann. Zum Zeitpunkt des Verfassens dieser Arbeit sind aber nur zwei kommerzielle Produkte bekannt, die nicht nur TFBSs, sondern auch Module erkennen. Deshalb stellen wir hier die freie und quelloffene Lösung "AIModules" vor, die diese Lücke füllt und einen Webservice zur Verfügung stellt, der es erlaubt nach TFBSs sowie nach Modulen auf DNA- und auf RNA-Abschnitten zu suchen. Für die Motivesuche werden entweder Matrizen aus der Jaspar Datenbank oder Matrizen vom Anwender verwendet. Darüberhinaus zeigen wir, dass unser Tool für die TF Suche nur Sekunden benötigt, wohingegen conTraV3 mindestens eine Stunde für dieselbe Analyse braucht. Zusätzlich kann der Anwender bei unserem Tool den Grad der Konserviertheit für TFs mit angeben und wir zeigen, dass wir mit unserer Lösung, die die Jaspar Datenbank heranzieht, mehr Module finden, als ein kommerziell verfügbares Produkt. Weiterhin kann mit unserer Lösung auch auf RNA-Sequenzen nach regulatorischen Motiven gesucht werden, wenn der Anwender die dafür nötigen Matrizen liefert. Wir zeigen dies am Beispiel von Polyadenylierungsstellen. Zusammenfassend stellen wir ein Werkzeug vor, das erstens frei und quelloffen ist und zweitens entweder auf Servern veröffentlicht werden kann oder On-Site auf einem Notebook läuft. Unser Tool erlaubt es Promotoren zu analysieren und nach konservierten Modulen sowie TFBSs in Genfamilien sowie nach regulatorischen Elementen in mRNA wie z.B. Polyadenylierungsstellen oder andere regulatorische Elemente wie beispielsweise Enhancern oder Silencern in genomischer DNA zu suchen.
Im Zuge der Bemühungen um neue, tumorspezifische Therapieansätze für die Myelomerkrankung hat sich der C-X-C-Chemokinrezeptor 4 (CXCR4) aufgrund seiner zentralen Rolle in der Tumorgenese als vielversprechender Angriffspunkt hervorgetan. Im Sinne eines theranostischen Konzepts wird der Rezeptor mithilfe eines radioaktiv markierten Liganden quantifiziert und anschließend von rezeptorspezifischen Radiotherapeutika als Zielstruktur genutzt. Die CXCR4-Expression ist allerdings ein höchst dynamischer Prozess mit großer inter- und intraindividueller Heterogenität, der u.a. durch eine begleitende Chemotherapie beeinflusst werden kann. Ob sich therapieinduzierte Veränderungen der Rezeptorexpression gezielt nutzen lassen, um die CXCR4-Expression zu optimieren und so die Effektivität der CXCR4-gerichteten Strategien zu steigern, wurde bislang nicht untersucht.
Vor diesem Hintergrund wurden in der vorliegenden Arbeit verschiedene, in der Myelomtherapie etablierte Substanzen sowohl einzeln als auch in Kombination hinsichtlich ihres Einflusses auf die CXCR4-Expression von MM-Zelllinien und primären MM-Zellen unter in vitro Bedingungen analysiert.
In den durchgeführten Experimenten zeigte sich eine hohe Variabilität der CXCR4-Expression der MM-Zellen nach Therapieinduktion, die sich als substanz-, dosis- und zeitabhängig herausstellte. Die Ergebnisse bestätigten das große Potenzial der therapieinduzierten Modulation der CXCR4-Expression. Im weiteren Verlauf sind translationale Forschungsansätze gerechtfertigt, die die Übertragbarkeit der in vitro gewonnenen Ergebnisse auf die komplexen Vorgänge im lebenden Organismus überprüfen. Langfristiges Ziel ist der Entwurf eines patientenzentrierten, multimodalen Therapiekonzepts, welches das CXCR4-gerichtete theranostische Konzept mit einer individuell angepassten, medikamentösen MM-Therapie kombiniert.
Comparing the appetitive learning performance of six European honeybee subspecies in a common apiary
(2021)
The Western honeybee (Apis mellifera L.) is one of the most widespread insects with numerous subspecies in its native range. How far adaptation to local habitats has affected the cognitive skills of the different subspecies is an intriguing question that we investigate in this study. Naturally mated queens of the following five subspecies from different parts of Europe were transferred to Southern Germany: A. m. iberiensis from Portugal, A. m. mellifera from Belgium, A. m. macedonica from Greece, A. m. ligustica from Italy, and A. m. ruttneri from Malta. We also included the local subspecies A. m. carnica in our study. New colonies were built up in a common apiary where the respective queens were introduced. Worker offspring from the different subspecies were compared in classical olfactory learning performance using the proboscis extension response. Prior to conditioning, we measured individual sucrose responsiveness to investigate whether possible differences in learning performances were due to differential responsiveness to the sugar water reward. Most subspecies did not differ in their appetitive learning performance. However, foragers of the Iberian honeybee, A. m. iberiensis, performed significantly more poorly, despite having a similar sucrose responsiveness. We discuss possible causes for the poor performance of the Iberian honeybees, which may have been shaped by adaptation to the local habitat.
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.
The nuclear envelope serves as important mRNA surveillance system. In yeast and humans, several control mechanisms act in parallel to prevent nuclear export of unprocessed mRNAs. However, trypanosomes lack homologues to most of the proteins involved. In addition, gene expression in trypanosomes relies almost completely on post-transcriptional regulation as they transcribe mRNAs as long polycistrons, which are subsequently processed into individual mRNA molecules by trans-splicing. As trans-splicing is not error-free, unspliced mRNAs may be recognized and prevented from reaching the cytoplasm by a yet unknown mechanism.
When trans-splicing is inhibited in trypanosomes, the formation of a novel RNA granule type at the cytoplasmic periphery of the nucleus, so called nuclear periphery granules (NPGs) was previously observed. To identify potential regulators of nuclear export control, changes in protein localization which occur when trans-splicing is inhibited, were globally analyzed during this work. For this, trypanosome nuclei were purified under conditions maintaining NPG attachment to the nucleus, in the absence and presence of trans-splicing. Mass spectrometry analyses identified 128 proteins which are specifically enriched in nuclear preparations of cells inhibited for trans-splicing. Amongst them are proteins, which change their localization to the nucleus or to the nuclear pores as well as many proteins that move into NPGs. Some of these proteins are promising candidates for nuclear export control proteins, as the changes in localization (to the nucleus or nuclear pores) were specific to the accumulation of unspliced mRNAs. The NPG proteome almost exclusively contains proteins involved in mRNA metabolism, mostly unique to trypanosomes, notably major translation initiation factors were absent. These data indicate that NPGs are RNP complexes which have started or completed nuclear export, but not yet entered translation. As a byproduct of these proteomic studies, a high-quality dataset of the yet unknown T. brucei nuclear proteome is provided, closing an important gap in knowledge to study trypanosome biology, in particular nuclear related processes.
NPGs were characterized in more detail by microscopy. The granules are cytoplasmic and present in at least two different trypanosome life cycle stages. There are at least two distinct granule subsets, with differences in protein composition. A closer analysis of NPGs by electron microscopy revealed that the granules are electron dense structures, which are connected to nuclear pores by string-like structures.
In order to approach the function of NPGs, on the one hand, the hypothesis that NPGs might be related to perinuclear germ granules of adult gonads of C. elegans was tested: we found no relation between the two granule types. On the other hand, initial single molecule mRNA FISH experiments performed in trypanosomes showed no accumulation of unspliced transcripts in NPGs, arguing against an involvement of the granules in mRNA quality control.
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.
Olfactory circuits change structurally and physiologically during development and adult life. This allows insects to respond to olfactory cues in an appropriate and adaptive way according to their physiological and behavioral state, and to adapt to their specific abiotic and biotic natural environment. We highlight here findings on olfactory plasticity and modulation in various model and non-model insects with an emphasis on moths and social Hymenoptera. Different categories of plasticity occur in the olfactory systems of insects. One type relates to the reproductive or feeding state, as well as to adult age. Another type of plasticity is context-dependent and includes influences of the immediate sensory and abiotic environment, but also environmental conditions during postembryonic development, periods of adult behavioral maturation, and short- and long-term sensory experience. Finally, plasticity in olfactory circuits is linked to associative learning and memory formation. The vast majority of the available literature summarized here deals with plasticity in primary and secondary olfactory brain centers, but also peripheral modulation is treated. The described molecular, physiological, and structural neuronal changes occur under the influence of neuromodulators such as biogenic amines, neuropeptides, and hormones, but the mechanisms through which they act are only beginning to be analyzed.
The transcription factor NRF2 is considered as the master regulator of cytoprotective and ROS-detoxifying gene expression. Due to their vulnerability to accumulating reactive oxygen species, melanomas are dependent on an efficient oxidative stress response, but to what extent melanomas rely on NRF2 is only scarcely investigated so far. In tumor entities harboring activating mutations of NRF2, such as lung adenocarcinoma, NRF2 activation is closely connected to therapy resistance. In melanoma, activating mutations are rare and triggers and effectors of NRF2 are less well characterized.
This work revealed that NRF2 is activated by oncogenic signaling, cytokines and pro-oxidant triggers, released cell-autonomously or by the tumor microenvironment. Moreover, silencing of NRF2 significantly reduced melanoma cell proliferation and repressed well-known NRF2 target genes, indicating basal transcriptional activity of NRF2 in melanoma. Transcriptomic analysis showed a large set of deregulated gene sets, besides the well-known antioxidant effectors. NRF2 suppressed the activity of MITF, a marker for the melanocyte lineage, and induced expression of epidermal growth factor receptor (EGFR), thereby stabilizing the dedifferentiated melanoma phenotype and limiting pigmentation markers and melanoma-associated antigens. In general, the dedifferentiated melanoma phenotype is associated with a reduced tumor immunogenicity. Furthermore, stress-inducible cyclooxygenase 2 (COX2) expression, a crucial immune-modulating gene, was regulated by NRF2 in an ATF4-dependent manner. Only in presence of both transcription factors was COX2 robustly induced by H2O2 or TNFα. COX2 catalyzes the first step of the prostaglandin E2 (PGE2) synthesis, which was described to be associated with tumor immune evasion and reduction of the innate immune response.
In accordance with these potentially immune-suppressive features, immunocompetent mice injected with NRF2 knockout melanoma cells had a strikingly longer tumor-free survival compared to NRF2-proficient cells. In line with the in vitro data, NRF2-deficient tumors showed suppression of COX2 and induction of MITF. Furthermore, transcriptomic analyses of available tumors revealed a strong induction of genes belonging to the innate immune response, such as RSAD2 and IFIH1. The expression of these genes strongly correlated with immune evasion parameters in human melanoma datasets and NRF2 activation or PGE2 supplementation limited the innate immune response in vitro.
In summary, the stress dependent NRF2 activation stabilizes the dedifferentiated melanoma phenotype and facilitates the synthesis of PGE2. As a result, NRF2 reduces gene expression of the innate immune response and promotes the generation of an immune-cold tumor microenvironment. Therefore, NRF2 not only elevated the ROS resilience, but also strongly contributed to tumor growth, maintenance, and immune control in cutaneous melanoma.
Background: The chemokine receptor CCR7 is crucial for an intact immune function, but its expression is also associated with clinical outcome in several malignancies. No data exist on the expression of CCR7 in adrenocortical tumors. Methods: CCR7 expression was investigated by qRT-PCR and immunohistochemistry in 4 normal adrenal glands, 59 adrenocortical adenomas, and 181 adrenocortical carcinoma (ACC) samples. Results: CCR7 is highly expressed in the outer adrenocortical zones and medulla. Aldosterone-producing adenomas showed lower CCR7 protein levels (H-score 1.3 ± 1.0) compared to non-functioning (2.4 ± 0.5) and cortisol-producing adenomas (2.3 ± 0.6), whereas protein expression was variable in ACC (1.8 ± 0.8). In ACC, CCR7 protein expression was significantly higher in lymph node metastases (2.5 ± 0.5) compared to primary tumors (1.8±0.8) or distant metastases (2.0 ± 0.4; p < 0.01). mRNA levels of CCR7 were not significantly different between ACCs, normal adrenals, and adrenocortical adenomas. In contrast to other tumor entities, neither CCR7 protein nor mRNA expression significantly impacted patients' survival. Conclusion: We show that CCR7 is expressed on mRNA and protein level across normal adrenals, benign adrenocortical tumors, as well as ACCs. Given that CCR7 did not influence survival in ACC, it is probably not involved in tumor progression, but it could play a role in adrenocortical homeostasis.
The unusual occurrence and developmental diversity of asexual eukaryotes remain a puzzle. De novo formation of a functioning asexual genome requires a unique assembly of sets of genes or gene states to disrupt cellular mechanisms of meiosis and gametogenesis, and to affect discrete components of sexuality and produce clonal or hemiclonal offspring. We highlight two usually overlooked but essential conditions to understand the molecular nature of clonal organisms, that is, a nonrecombinant genomic assemblage retaining modifiers of the sexual program, and a complementation between altered reproductive components. These subtle conditions are the basis for physiologically viable and genetically balanced transitions between generations. Genomic and developmental evidence from asexual animals and plants indicates the lack of complementation of molecular changes in the sexual reproductive program is likely the main cause of asexuals' rarity, and can provide an explanatory frame for the developmental diversity and lability of developmental patterns in some asexuals as well as for the discordant time to extinction estimations.
How diversity of life is generated, maintained, and distributed across space and time is the central question of community ecology. Communities are shaped by three assembly processes: (I) dispersal, (II) environ-mental, and (III) interaction filtering. Heterogeneity in environmental conditions can alter these filtering processes, as it increases the available niche space, spatially partitions the resources, but also reduces the effective area available for individual species. Ultimately, heterogeneity thus shapes diversity. However, it is still unclear under which conditions heterogeneity has positive effects on diversity and under which condi-tions it has negative or no effects at all. In my thesis, I investigate how environmental heterogeneity affects the assembly and diversity of diverse species groups and whether these effects are mediated by species traits.
In Chapter II, I first examine how much functional traits might inform about environmental filtering pro-cesses. Specifically, I examine to which extent body size and colour lightness, both of which are thought to reflect the species thermal preference, shape the distribution and abundance of two moth families along elevation. The results show, that assemblages of noctuid moths are more strongly driven by abiotic filters (elevation) and thus form distinct patterns in colour lightness and body size, while geometrid moths are driven by biotic filters (habitat availability), and show no decline in body size nor colour lightness along elevation. Thus, one and the same functional trait can have quite different effects on community assembly even between closely related taxonomic groups.
In Chapter III, I elucidate how traits shift the relative importance of dispersal and environmental filtering in determining beta diversity between forests. Environmental filtering via forest heterogeneity had on aver-age higher independent effects than dispersal filtering within and among regions, suggesting that forest heterogeneity determines species turnover even at country-wide extents. However, the relative importance of dispersal filtering increased with decreasing dispersal ability of the species group. From the aspects of forest heterogeneity covered, variations in herb or tree species composition had overall stronger influence on the turnover of species than forest physiognomy. Again, this ratio was influenced by species traits, namely trophic position, and body size, which highlights the importance of ecological properties of a taxo-nomic group in community assembly.
In Chapter IV, I assess whether such ecological properties ultimately determine the level of heterogeneity which maximizes species richness. Here, I considered several facets of heterogeneity in forests. Though the single facets of heterogeneity affected diverse species groups both in positive and negative ways, we could not identify any generalizable mechanism based on dispersal nor the trophic position of the species group which would dissolve these complex relationships.
In Chapter V, I examine the effect of environmental heterogeneity of the diversity of traits itself to evalu-ate, whether the effects of environmental heterogeneity on species richness are truly based on increases in the number of niches. The results revealed that positive effects of heterogeneity on species richness are not necessarily based on an increased number of niches alone, but proposedly also on a spatially partition of resources or sheltering effects. While ecological diversity increased overall, there were also negative trends which indicate filtering effects via heterogeneity.
In Chapter VI, I present novel methods in measuring plot-wise heterogeneity of forests across continental scales via Satellites. The study compares the performance of Sentinel-1 and LiDar-derived measurements in depicting forest structures and heterogeneity and to their predictive power in modelling diversity. Senti-nel-1 could match the performance of Lidar and shows high potential to assess free yet detailed infor-mation about forest structures in temporal resolutions for modelling the diversity of species.
Overall, my thesis supports the notion that heterogeneity in environmental conditions is an important driv-er of beta-diversity, species richness, and ecological diversity. However, I could not identify any general-izable mechanism which direction and form this effect will have.
Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads.
We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function.
Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations.
Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.
Breed predispositions to canine digital neoplasms are well known. However, there is currently no statistical analysis identifying the least affected breeds. To this end, 2912 canine amputated digits submitted from 2014–2019 to the Laboklin GmbH & Co. KG for routine diagnostics were statistically analyzed. The study population consisted of 155 different breeds (most common: 634 Mongrels, 411 Schnauzers, 197 Labrador Retrievers, 93 Golden Retrievers). Non-neoplastic processes were present in 1246 (43%), tumor-like lesions in 138 (5%), and neoplasms in 1528 cases (52%). Benign tumors (n = 335) were characterized by 217 subungual keratoacanthomas, 36 histiocytomas, 35 plasmacytomas, 16 papillomas, 12 melanocytomas, 9 sebaceous gland tumors, 6 lipomas, and 4 bone tumors. Malignant neoplasms (n = 1193) included 758 squamous cell carcinomas (SCC), 196 malignant melanomas (MM), 76 soft tissue sarcomas, 52 mast cell tumors, 37 non-specified sarcomas, 29 anaplastic neoplasms, 24 carcinomas, 20 bone tumors, and 1 histiocytic sarcoma. Predisposed breeds for SCC included the Schnauzer (log OR = 2.61), Briard (log OR = 1.78), Rottweiler (log OR = 1.54), Poodle (log OR = 1.40), and Dachshund (log OR = 1.30). Jack Russell Terriers (log OR = −2.95) were significantly less affected by SCC than Mongrels. Acral MM were significantly more frequent in Rottweilers (log OR = 1.88) and Labrador Retrievers (log OR = 1.09). In contrast, Dachshunds (log OR = −2.17), Jack Russell Terriers (log OR = −1.88), and Rhodesian Ridgebacks (log OR = −1.88) were rarely affected. This contrasted with the well-known predisposition of Dachshunds and Rhodesian Ridgebacks to oral and cutaneous melanocytic neoplasms. Further studies are needed to explain the underlying reasons for breed predisposition or “resistance” to the development of specific acral tumors and/or other sites.
A viral infection involves entry and replication of viral nucleic acid in a host organism, subsequently leading to biochemical and structural alterations in the host cell. In the case of SARS-CoV-2 viral infection, over-activation of the host immune system may lead to lung damage. Albeit the regeneration and fibrotic repair processes being the two protective host responses, prolonged injury may lead to excessive fibrosis, a pathological state that can result in lung collapse. In this review, we discuss regeneration and fibrosis processes in response to SARS-CoV-2 and provide our viewpoint on the triggering of alveolar regeneration in coronavirus disease 2019 (COVID-19) patients.