Refine
Has Fulltext
- yes (1032)
Is part of the Bibliography
- yes (1032) (remove)
Year of publication
- 2022 (1032) (remove)
Document Type
- Journal article (808)
- Doctoral Thesis (199)
- Working Paper (7)
- Preprint (4)
- Report (3)
- Book (2)
- Book article / Book chapter (2)
- Master Thesis (2)
- Review (2)
- Bachelor Thesis (1)
Language
- English (1032) (remove)
Keywords
- COVID-19 (16)
- SARS-CoV-2 (11)
- machine learning (11)
- virtual reality (11)
- heart failure (10)
- prostate cancer (10)
- inflammation (9)
- remote sensing (9)
- 3D printing (8)
- Germany (8)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (130)
- Graduate School of Life Sciences (79)
- Institut für Informatik (56)
- Medizinische Klinik und Poliklinik I (56)
- Neurologische Klinik und Poliklinik (54)
- Medizinische Klinik und Poliklinik II (50)
- Institut für Geographie und Geologie (48)
- Klinik und Poliklinik für Anästhesiologie (ab 2004) (44)
- Institut für Organische Chemie (41)
- Institut für Psychologie (39)
Schriftenreihe
Sonstige beteiligte Institutionen
- IZKF Nachwuchsgruppe Geweberegeneration für muskuloskelettale Erkrankungen (5)
- Bernhard-Heine-Centrum für Bewegungsforschung (4)
- Zentraleinheit Klinische Massenspektrometrie (3)
- Fraunhofer-Institut für Silicatforschung ISC (2)
- Helmholtz Institute for RNA-based Infection Research (HIRI) (2)
- Krankenhaushygiene und Antimicrobial Stewardship (Universitätsklinikum) (2)
- Wilhelm-Conrad-Röntgen-Forschungszentrum für komplexe Materialsysteme (2)
- ALPARC - The Alpine Network of Protected Areas (1)
- Albert-Ludwigs-Universität Freiburg (1)
- Arizona State University, Tempe, Arizona, USA (1)
The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research.
To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake– feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time.
In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.
In today's world, circumstances, processes, and requirements for systems in general-in this thesis a special focus is given to the context of Cyber-Physical Systems (CPS)-are becoming increasingly complex and dynamic.
In order to operate properly in such dynamic environments, systems must adapt to dynamic changes, which has led to the research area of Self-Adaptive Systems (SAS).
These systems can deal with changes in their environment and the system itself.
In our daily lives, we come into contact with many different self-adaptive systems that are designed to support and improve our way of life.
In this work we focus on the two domains Intelligent Transportation Systems (ITS) and logistics as both domains provide complex and adaptable use cases to prototypical apply the contributions of this thesis.
However, the contributions are not limited to these areas and can be generalized also to other domains such as the general area of CPS and Internet of Things including smart grids or even intelligent computer networks.
In ITS, real-time traffic control is an example adaptive system that monitors the environment, analyzes observations, and plans and executes adaptation actions.
Another example is platooning, which is the ability of vehicles to drive with close inter-vehicle distances.
This technology enables an increase in road throughput and safety, which directly addresses the increased infrastructure needs due to increased traffic on the roads.
In logistics, the Vehicle Routing Problem (VRP) deals with the planning of road freight transport tours.
To cope with the ever-increasing transport volume due to the rise of just-in-time production and online shopping, efficient and correct route planning for transports is important.
Further, warehouses play a central role in any company's supply chain and contribute to the logistical success.
The processes of storage assignment and order picking are the two main tasks in mezzanine warehouses highly affected by a dynamic environment.
Usually, optimization algorithms are applied to find solutions in reasonable computation time.
SASes can help address these dynamics by allowing systems to deal with changing demands and constraints.
For the application of SASes in the two areas ITS and logistics, the definition of adaptation planning strategies is the key success factor.
A wide range of adaptation planning strategies for different domains can be found in the literature, and the operator must select the most promising strategy for the problem at hand.
However, the No-Free-Lunch theorem states that the performance of one strategy is not necessarily transferable to other problems.
Accordingly, the algorithm selection problem, first defined in 1976, aims to find the best performing algorithm for the current problem.
Since then, this problem has been explored more and more, and the machine learning community, for example, considers it a learning problem.
The ideas surrounding the algorithm selection problem have been applied in various use cases, but little research has been done to generalize the approaches.
Moreover, especially in the field of SASes, the selection of the most appropriate strategy depends on the current situation of the system.
Techniques for identifying the situation of a system can be found in the literature, such as the use of rules or clustering techniques.
This knowledge can then be used to improve the algorithm selection, or in the scope of this thesis, to improve the selection of adaptation planning strategies.
In addition, knowledge about the current situation and the performance of strategies in similar previously observed situations provides another opportunity for improvements.
This ongoing learning and reasoning about the system and its environment is found in the research area Self-Aware Computing (SeAC).
In this thesis, we explore common characteristics of adaptation planning strategies in the domain of ITS and logistics presenting a self-aware optimization framework for adaptation planning strategies.
We consider platooning coordination strategies from ITS and optimization techniques from logistics as adaptation planning strategies that can be exchanged during operation to better reflect the current situation.
Further, we propose to integrate fairness and uncertainty handling mechanisms directly into the adaptation planning strategies.
We then examine the complex structure of the logistics use cases VRP and mezzanine warehouses and identify their systems-of-systems structure.
We propose a two-stage approach for vertical or nested systems and propose to consider the impact of intertwining horizontal or coexisting systems.
More specifically, we summarize the six main contributions of this thesis as follows:
First, we analyze specific characteristics of adaptation planning strategies with a particular focus on ITS and logistics.
We use platooning and route planning in highly dynamic environments as representatives of ITS and we use the rich Vehicle Routing Problem (rVRP) and mezzanine warehouses as representatives of the logistics domain.
Using these case studies, we derive the need for situation-aware optimization of adaptation planning strategies and argue that fairness is an important consideration when applying these strategies in ITS.
In logistics, we discuss that these complex systems can be considered as systems-of-systems and this structure affects each subsystem.
Hence, we argue that the consideration of these characteristics is a crucial factor for the success of the system.
Second, we design a self-aware optimization framework for adaptation planning strategies.
The optimization framework is abstracted into a third layer above the application and its adaptation planning system, which allows the concept to be applied to a diverse set of use cases.
Further, the Domain Data Model (DDM) used to configure the framework enables the operator to easily apply it by defining the available adaptation planning strategies, parameters to be optimized, and performance measures.
The framework consists of four components: (i) Coordination, (ii) Situation Detection, (iii) Strategy Selection, and (iv) Parameter Optimization.
While the coordination component receives observations and triggers the other components, the situation detection applies rules or clustering techniques to identify the current situation.
The strategy selection uses this knowledge to select the most promising strategy for the current situation, and the parameter optimization applies optimization algorithms to tune the parameters of the strategy.
Moreover, we apply the concepts of the SeAC domain and integrate learning and reasoning processes to enable ongoing advancement of the framework.
We evaluate our framework using the platooning use case and consider platooning coordination strategies as the adaptation planning strategies to be selected and optimized.
Our evaluation shows that the framework is able to select the most appropriate adaptation strategy and learn the situational behavior of the system.
Third, we argue that fairness aspects, previously identified as an important characteristic of adaptation planning strategies, are best addressed directly as part of the strategies.
Hence, focusing on platooning as an example use case, we propose a set of fairness mechanisms to balance positive and negative effects of platooning among all participants in a platoon.
We design six vehicle sequence rotation mechanisms that continuously change the leader position among all participants, as this is the position with the least positive effects.
We analyze these strategies on roads of different sizes and with different traffic volumes, and show that these mechanisms should also be chosen wisely.
Fourth, we address the uncertainty characteristic of adaptation planning strategies and propose a methodology to account for uncertainty and also address it directly as part of the adaptation planning strategies.
We address the use case of fueling planning along a route associated with highly dynamic fuel prices and develop six utility functions that account for different aspects of route planning.
Further, we incorporate uncertainty measures for dynamic fuel prices by adding penalties for longer travel times or greater distance to the next gas station.
Through this approach, we are able to reduce the uncertainty at planning time and obtain a more robust route planning.
Fifth, we analyze optimization of nested systems-of-systems for the use case rVRP.
Before proposing an approach to deal with the complex structure of the problem, we analyze important constraints and objectives that need to be considered when formulating a real-world rVRP.
Then, we propose a two-stage workflow to optimize both systems individually, flexibly, and interchangeably.
We apply Genetic Algorithms and Ant Colony Optimization (ACO) to both nested systems and compare the performance of our workflow with state-of-the-art optimization algorithms for this use case.
In our evaluation, we show that the proposed two-stage workflow is able to handle the complex structure of the problem and consider all real-world constraints and objectives.
Finally, we study coexisting systems-of-systems by optimizing typical processes in mezzanine warehouses.
We first define which ergonomic and economic constraints and objectives must be considered when addressing a real-world problem.
Then, we analyze the interrelatedness of the storage assignment and order picking problems; we identify opportunities to design optimization approaches that optimize all objectives and aim for a good overall system performance, taking into account the interdependence of both systems.
We use the NSGA-II for storage assignment and Ant Colony Optimization (ACO) for order picking and adapt them to the specific requirements of horizontal systems-of-systems.
In our evaluation, we compare our approaches to state-of-the-art approaches in mezzanine warehouses and show that our proposed approaches increase the system performance.
Our proposed approaches provide important contributions to both academic research and practical applications.
To the best of our knowledge, we are the first to design a self-aware optimization framework for adaptation planning strategies that integrates situation-awareness, algorithm selection, parameter tuning, as well as learning and reasoning.
Our evaluation of platooning coordination shows promising results for the application of the framework.
Moreover, our proposed strategies to compensate for negative effects of platooning represent an important milestone, which could lead to higher acceptance of this technology in society and support its future adoption in the real world.
The proposed methodology and utility functions that address uncertainty are an important step to improving the capabilities of SAS in an increasingly turbulent environment.
Similarly, our contributions to systems-of-systems optimization are major contributions to the state of logistics and systems-of-systems research.
Finally, we select real-world use cases for the application of our approaches and cooperate with industrial partners, which highlights the practical relevance of our contributions.
The reduction of manual effort and required expert knowledge in our self-aware optimization framework is a milestone in bridging the gap between academia and practice.
One of our partners integrated the two-stage approach to tackling the rVRP into its software system, improving both time to solution and solution quality.
In conclusion, the contributions of this thesis have spawned several research projects such as a long-term industrial project on optimizing tours and routes in parcel delivery funded by Bayerisches Verbundforschungsprogramm (BayVFP) – Digitalisierung and further collaborations, opening up many promising avenues for future research.
Microbial rhodopsins are abundant membrane proteins often capable of ion transport and are found in all three domains of life. Thus, many fungi, especially phyto-associated or phyto-pathogenic ones, contain these green-light-sensing photoreceptors. Proteins that perceive other wavelengths are often well characterized in terms of their impact on fungal biology whereas little is known about the function of fungal rhodopsins. In this work, five fungal rhodopsins, UmOps1 and UmOps2 from the corn smut Ustilago maydis as well as ApOps1, ApOps2 and ApOps3 from the black yeast Aureobasidium pullulans, were characterized electrophysiologically using mammalian expression systems and the patch-clamp technique to explore their ion transport properties. The latter three were modified using a membrane trafficking cassette, termed “2.0” that consists of the lucy rho motif, two Kir2.1 Golgi apparatus trafficking signals and a Kir2.1 endoplasmic reticulum export signal, what resulted in better plasma membrane localization. Rhodopsin mutants were created to identify amino acid residues that are key players in the ion transport process. Current enhancement in the presence of weak organic acids, that was already described before for the fungal rhodopsin CarO from Fusarium fujikuroi (García-Martínez et al., 2015; Adam et al., 2018), was investigated for the U. maydis rhodopsins as well as for ApOps2 by supplementing acetate in the patch-clamp electrolyte solutions. All five rhodopsins were found to be proton pumps unidirectionally transporting protons out of the cytosol upon green-light exposure with every rhodopsin exhibiting special features or unique characteristics in terms of the photocurrents. To name just a few, UmOps1, for example, showed a striking pH-dependency with massive enhancement of pump currents in the presence of extracellular acidic pH. Moreover, especially ApOps2 and ApOps3 showed very high current densities, however, the ones of ApOps3 were impaired when exchanging intracellular sodium to cesium. Concerning the mutations, it was found, that the electron releasing group in UmOps1 seems to be involved in the striking pH effect and that the mutation of the proton donor site resulted in almost unfunctional proteins. Moreover, a conserved arginine inside ApOps2 was mutated to turn the proton pump into a channel. Regarding the effect of weak organic acids, acetate was able to induce enhanced pump currents in UmOps1 and ApOps2, but not in UmOps2. Due to the capability of current production upon light illumination, microbial rhodopsins are used in the research field of optogenetics that aims to control neuronal activity by light. ApOps2 was used to test its functionality in differentiated NG108-15 cells addressing the question whether it is a promising candidate that can be used as an optogenetic tool. Indeed, this rhodopsin could be functionally expressed in this experimental system. Furthermore, microscopic studies were done to elucidate the localization of selected rhodopsins in fungal cells. Therefore, conventional (confocal laser scanning or structured illumination microscopy) as well as novel super-resolution techniques (expansion or correlated light and electron microscopy) were used. This was done on U. maydis sporidia, the yeast-like form of this fungus, via eGFP-tagged UmOps1 or UmOps2 expressing strains. Moreover, CarO-eYFP expressing F. fujikuroi was imaged microscopically to confirm the plasma membrane and tonoplast localization (García-Martínez et al., 2015) with the help of counterstaining experiments. UmOps1 was found to reside in the plasma membrane, UmOps2 localized to the tonoplast and CarO was indeed found in both of these localizations. This work gains further insight into rhodopsin functions and paves the way for further research in terms of the biological role of rhodopsins in fungal life cycles.
In this work, the influence of aromatic structures on drug encapsulation, self-assembly and hydrogel formation was investigated. The physically crosslinked gelling systems were characterized and optimized for the use in biofabrication and applied in initial (bio)printing experiments.
Chapter I: The cytocompatible (first in vitro and in vivo studies) amphiphile PMeOx-b-PBzOx-b- PMeOx (A-PBzOx-A) was used for the solubilization of PTX, schizandrin A (SchA), curcumin (CUR), niraparib and HS-173.
Chapter II: Compared to the polymers A-PPheOx-A, A-PBzOx-A and A-PBzOzi-A, only the polymer A-PPheOzi-A showed a reversible temperature- and concentration-dependent inverse thermogelation, which is accompanied by a morphology change from long wormlike micelles in the gel to small spherical micelles in solution. The worm formation results from novel interactions between the hydrophilic and hydrophobic aromatic polymer blocks. Changes in the hydrophilic block significantly alter the gel system. Rheological properties can be optimized by concentration and temperature, which is why the hydrogel was used to significantly improve the printability and stability of Alg in a blend system.
Chapter III: By extending the side chain of the aromatic hydrophobic block, the inverse thermogelling polymer A-poly(2-phenethyl-2-oxazoline)-A (A-PPhenEtOx-A) is obtained. Rapid gelation upon cooling is achieved by inter-correlating spherical micelles. Based on ideal rheological properties, first cytocompatible bioprinting experiments were performed in combination with Alg. The polymers A- poly(2-benzhydryl-2-oxazoline)-A (A-PBhOx-A) and A-poly(2-benzhydryl-2-oxazine) (A-PBhOzi-A) are characterized by two aromatic benzyl units per hydrophobic repeating unit. Only the polymer A- PBhOzi-A exhibited inverse thermogelling behavior. Merging micelles could be observed by electron microscopy. The system was rheologically characterized and discussed with respect to an application in 3D printing.
Chapter IV: The thermogelling POx/POzi system, in particular the block copolymer PMeOx-b- PnPrOzi, was used in different applications in the field of biofabrication. The introduction of maleimide and furan units along the hydrophilic polymer part ensured additional stabilization by Diels-Alder crosslinking after the printing process.
∆Np63 is a master regulator of squamous cell identity and regulates several signaling pathways that crucially
contribute to the development of squamous cell carcinoma (SCC) tumors. Its contribution to coordinating the
expression of genes involved in oncogenesis, epithelial identity, DNA repair, and genome stability has been
extensively studied and characterized. For SCC, the expression of ∆Np63 is an essential requirement to
maintain the malignant phenotype. Additionally, ∆Np63 functionally contributes to the development of cancer
resistance toward therapies inducing DNA damage.
SCC patients are currently treated with the same conventional Cisplatin therapy as they would have been
treated 30 years ago. In contrast to patients with other tumor entities, the survival of SCC patients is limited,
and the efficacy of the current therapies is rather low. Considering the rising incidences of these tumor entities,
the development of novel SCC therapies is urgently required. Targeting ∆Np63, the transcription factor, is a
potential alternative to improve the therapeutic response and clinical outcomes of SCC patients.
However, ∆Np63 is considered “undruggable.” As is commonly observed in transcription factors, ∆Np63 does
not provide any suitable domains for the binding of small molecule inhibitors. ∆Np63 regulates a plethora of
different pathways and cellular processes, making it difficult to counteract its function by targeting
downstream effectors. As ∆Np63 is strongly regulated by the ubiquitin–proteasome system (UPS), the
development of deubiquitinating enzyme inhibitors has emerged as a promising therapeutic strategy to target
∆Np63 in SCC treatment.
This work involved identifying the first deubiquitinating enzyme that regulates ∆Np63 protein stability. Stateof-the-art SCC models were used to prove that USP28 deubiquitinates ∆Np63, regulates its protein stability,
and affects squamous transcriptional profiles in vivo and ex vivo. Accordingly, SCC depends on USP28 to
maintain essential levels of ∆Np63 protein abundance in tumor formation and maintenance. For the first time,
∆Np63, the transcription factor, was targeted in vivo using a small molecule inhibitor targeting the activity of
USP28. The pharmacological inhibition of USP28 was sufficient to hinder the growth of SCC tumors in
preclinical mouse models.
Finally, this work demonstrated that the combination of Cisplatin with USP28 inhibitors as a novel therapeutic
alternative could expand the limited available portfolio of SCC therapeutics. Collectively, the data presented
within this dissertation demonstrates that the inhibition of USP28 in SCC decreases ∆Np63 protein abundance,
thus downregulating the Fanconi anemia (FA) pathway and recombinational DNA repair. Accordingly, USP28
inhibition reduces the DNA damage response, thereby sensitizing SCC tumors to DNA damage therapies, such
as Cisplatin.
Our current data demonstrate that besides the known risk factors, including apical aneurysm, reduced left ventricular longitudinal systolic function (MAPSE) and advanced diastolic dysfunction, Right ventricular dysfunction as determined by reduced tricuspid annular plane systolic excursion (TAPSE) or right ventricular fractional area change (RV_FAC) is independently associated with left ventricular thrombus formation in acute anterior myocardial infarction patients, especially in the setting of anterior myocardial infarction without the formation of an apical aneurysm. This study suggests that besides left ventricular abnormalities, right ventricular dysfunction likewise contributes LVT formation in patients with acute anterior myocardial infarction.
Cancer is one of the major causes of mortality in developed countries. In 2020, there were more than 19.3 million new cases of tumor malignancies worldwide, with more than 10 million deaths. The high rates of cancer cases and mortality necessitate extensive research and the development of novel cancer treatments and antitumor agents. In most cases, conventional treatment strategies for tumor therapy are based on chemotherapeutic treatment, which is supplemented with radiotherapy and/or surgical resection of solid tumors [1]. The use of chemotherapy for the treatment of cancer has significant side effects, the most dangerous of which is toxicity [2] [3].
Modern methods of treating tumors focus on specific drug delivery to the tumor site, actively targeting the tumor cells, as well as the reduction of side effects. One of the most promising current approaches is based on oncolytic viruses. Antitumor properties of viruses were documented at the beginning of the 20th century when some cancer patients recovered after acute viral infections, particularly influenza [4]. Vaccinia virus (VACV) is a member of the Poxviridae family, has natural antitumor properties, and provides a good basis for generating efficient recombinant oncolytic strains. Furthermore, VACV has never been shown to integrate into the host genome [5]. VACV is likely one of the safest and well-studied viruses due to extensive research being done in molecular biology and pathophysiology to investigate its potential as a vaccine for smallpox eradication programs. It has been administered to over 200 million people worldwide. VACV antitumor therapeutic effectiveness has been established in xenograft models with a variety of tumor types for human and canine cancers. Furthermore, recombinant oncolytic VACVs expressing genes encoding light-emitting proteins are a big improvement in a treatment strategy that combines tumor-specific therapies and diagnostics.
Oncolytic virus treatments are effective in xenograft cancer models in mice, however, the significant improvements found in mice do not always translate to human cancer patients. These therapies should be tested in dogs with spontaneous cancer not only to offer well translatable information regarding the possible efficiency of viral therapy for human cancers but also to improve the health of our household pets as well. Spontaneous canine tumors are starting to be regarded as an essential model of human cancers that can reproduce the tumor microenvironment and immune response of cancer patients [6]. Just as data obtained in dog experiments can improve cancer therapy for human patients, these findings can also be used to improve treatment protocols in canine patients.
Hundreds of studies and dozens of reviews have been published regarding the antitumor effects of various recombinants of VACV, but information on the anticancer features of initial, genetically-unmodified “naïve” VACV is still limited. In the first studies, we compared different wild-type, non-modified strains of VACV and tested their oncolytic properties on a panel of various cancer cells derived from different organs. In addition, we also tested a protection system based on the “Trojan horse” concept - using a combination of human Adipose tissue-derived Stem Cells (hADSC) and three different wild-type single plaque purified Vaccinia virus strains: W1, L1, and T1. We showed that all tested human cell lines (FaDu, MDA MB 231, HNT-13, HNT-35, and PC-3) are permissive to L0, W0, T0, L1, W1, and L1 infection. Furthermore, we tested the cytotoxicity of VACV in different cancer cell lines (A549, PC-3, MDA-MB 231, FaDu, HNT-13, HNT-25, and HNT-35). All strains lysed the cells, which was most visible at 96 hpi. We also showed that all tested strains could efficiently infect and multiply in hADSC at a high level. In our in vivo study, we tested the therapeutic efficacy of the wild-type Vaccinia viruses L1, W1, and T1 alone or in combination with hADSC. Wild-type VACV strains were tested for their oncolytic efficiency in human lung adenocarcinoma (A549) in a xenograft model. Treatment of A549 tumors with different doses of L1 and W1 as well as with a L1/ADSC or W1/ADSC combination led to significant tumor regression compared to the PBS control. Additionally, the treatment with L1 and W1 and the combination of L1/ADSC and W1/ADSC was well tolerated by the animals. In the case of the wild-type Tian Tan strain, results were not obtained due to the high cytotoxicity of this strain. Therefore, it should be attenuated for further studies.
In the second part of the current study, we investigated the oncolytic effect of C1-opt1, W1 opt1, and L3-opt1 strains based on the wild-type Copenhagen, Wyeth, and Lister vaccines with additional expression of turboFP635. Replication and cytotoxicity assays demonstrated that all 3 viruses were able to infect, replicate in and kill canine tumor cell lines STSA-1 and CT1258 in a virus dose- and time- dependent fashion. Cytotoxicity and replication assays were also performed on cultured canine Adipose-derived Mesenchymal Stem Cells (cAdMSC). The results showed that the cells were lysed much slower than the tumor cells. It suggests that these cells can harbour the virus for a long-term period, allowing the virus to spread into the body and there is enough time to reach the primary tumor or metastases before the cell carrier is destroyed. The viral replication in cAdMSC in our study was lower than in canine cancer cells (STSA-1 and CT1258) at the same MOI. After being studied in cell culture, C1 opt1 and their combination with cAdMSC (C1-opt1/cAdMSC) were used in canine STSA 1 tumor bearing nude mice. We tested the oncolytic effect of the C1-opt1 virus alone and in combination with cAdMSC in the canine STSA-1 xenograft mouse model. Altogether, our findings have shown that both C1-opt1 and cAdMSC/C1-opt1 significantly reduced tumor size or eliminated the tumor. There was no significant difference between C1-opt1 alone and cAdMSC/C1-opt1. The virus particles were mostly found within the tumor after 24 dpi, some amount of virus particles were found in the lungs of mice injected with a combination of cAdMSC/C1-opt1 but not in the group injected with virus alone (cAdMSC might get stuck in the lungs and cause virus propagation there).
Taken together, this study provided a proof-of-concept that hADSC/cAdMSC can be used as a carrier system for the “Trojan horse” concept. However, it should be confirmed in another experimental model system, such as canine patients. Moreover, these findings suggest that wild-type, non-modified strains of Vaccinia virus isolates can be considered promising candidates for oncolytic virotherapy, especially in combination with mesenchymal stem cells.
For a large fraction of the proteins expressed in the human brain only the primary
structure is known from the genome project. Proteins conserved in evolution can
be studied in genetic models such as Drosophila. In this doctoral thesis monoclonal
antibodies (mAbs) from the Wuerzburg Hybridoma library are produced and
characterized with the aim to identify the target antigen. The mAb ab52 was found
to be an IgM which recognized a cytosolic protein of Mr ~110 kDa on Western
blots. The antigen was resolved by two-dimensional gel electrophoresis (2DE) as a
single distinct spot. Mass spectrometric analysis of this spot revealed EPS-15
(epidermal growth factor receptor pathway substrate clone 15) to be a strong
candidate. Another mAb from the library, aa2, was already found to recognize
EPS-15, and comparison of the signal of both mAbs on Western blots of 1D and
2D electrophoretic separations revealed similar patterns, hence indicating that both
antigens could represent the same protein. Finally absence of the wild-type signal
in homozygous Eps15 mutants in a Western blot with ab52 confirmed the ab52
antigen to be EPS-15. Thus both the mAbs aa2 and ab52 recognize the Drosophila
homologue of EPS-15. The mAb aa2, being an IgG, is more suitable for
applications like immunoprecipitation (IP). It has already been submitted to the
Developmental Studies Hybridoma Bank (DSHB) to be easily available for the
entire research community.
The mAb na21 was also found to be an IgM. It recognizes a membrane associated
antigen of Mr ~10 kDa on Western blots. Due to the membrane associated nature
of the protein, it was not possible to resolve it by 2DE and due to the IgM nature of
the mAb it was not possible to enrich the antigen by IP. Preliminary attempts to
biochemically purify the endogenously expressed protein from the tissue, gave
99
promising results but could not be completed due to lack of time. Thus
biochemical purification of the protein seems possible in order to facilitate its
identification by mass spectrometry. Several other mAbs were studied for their
staining pattern on cryosections and whole mounts of Drosophila brains. However,
many of these mAbs stained very few structures in the brain, which indicated that
only a very limited amount of protein would be available as starting material.
Because these antibodies did not produce signals on Western blots, which made it
impossible to enrich the antigens by electrophoretic methods, we did not attempt
their purification. However, the specific localization of these proteins makes them
highly interesting and calls for their further characterization, as they may play a
highly specialized role in the development and/or function of the neural circuits
they are present in. The purification and identification of such low expression
proteins would need novel methods of enrichment of the stained structures.
During natural behavior, cognitive processes constantly coincide with body movements such as head or eye movements or blinks. However, during experimental investigations of cognitive processes, movements are often highly restricted which is rather unnatural. In order to improve our understanding of natural behavior, this thesis investigates the interaction between cognition and movements by focusing on spontaneous blinks, which naturally interact with other body movements.
Spontaneous blinks are inevitably connected to vision as they shut out incoming visual information. Both sensory-based and cognitive factors, for example, stimulus occurrence and evaluation, were reported to influence blink behavior. Our first study investigated if such influences are comparable for visual and non-visual input. The chosen experimental design allowed dissociating sensory-driven and cognitive influences, which then could be compared between the visual and auditory domain. Our results show that blinks are more strongly modulated during passive observation of visual input compared to auditory input. This modulation is however enhanced for both input modalities by an increased attentional demand. In addition, the cognitively defined meaning of a stimulus changes blink latency independent of the sensory domain. Overall, our findings show that spontaneous blinks and cognitive processes are linked beyond vision. Moreover, the underlying cognitive processes that influence blinks are largely the same across different sensory input indicating that blinks are profoundly integrated into our system.
When investigating natural behavior, it is important to consider that movements rarely occur in isolation, but are executed side by side. As these movements interact and have a link to cognitive processes, the complexity of our system increases. In order to take this complexity into account, the second part of the experimental research focused on movement interactions, more specifically on the interactions between blinks, pupil size and speaking. Our results reveal that speech-related motor activity increases blink rate and pupil size as well as modulates blink timing. This is in line with previous research that described a relation between different body and eye movements. Importantly, each bodily-induced change in eye movements affects visual information intake. Therefore, different movements can be tightly linked to perceptual processes through complex interactions.
Altogether, the work of this thesis provides rich evidence that movements and cognitive processes are deeply intertwined. Therefore, movements should be seen as an integral part of our system. Taking the relevance of movements and their interactions into account during experimental investigations is necessary in order to reveal a more realistic and complete picture of human natural behavior.
Aims
There is an ongoing discussion whether the categorization of patients with heart failure according to left ventricular ejection fraction (LVEF) is scientifically justified and clinically relevant. Major efforts are directed towards the identification of appropriate cut-off values to correctly allocate heart failure-specific pharmacotherapy. Alternatively, an LVEF continuum without definite subgroups is discussed. This study aimed to evaluate the natural distribution of LVEF in patients presenting with acutely decompensated heart failure and to identify potential subgroups of LVEF in male and female patients.
Methods and results
We identified 470 patients (mean age 75 ± 11 years, n = 137 female) hospitalized for acute heart failure in whom LVEF could be quantified by Simpson's method in an in-hospital echocardiogram. Non-parametric modelling revealed a bimodal shape of the LVEF distribution. Parametric modelling identified two clusters suggesting two LVEF peaks with mean (variance) of 61% (9%) and 31% (10%), respectively. Sub-differentiation by sex revealed a sex-specific bimodal clustering of LVEF. The respective threshold differentiating between ‘high’ and ‘low’ LVEF was 45% in men and 52% in women.
Conclusions
In patients presenting with acute heart failure, LVEF clustered in two subgroups and exhibited profound sex-specific distributional differences. These findings might enrich the scientific process to identify distinct subgroups of heart failure patients, which might each benefit from respectively tailored (pharmaco)therapies.
Background
Influenza virus infections in immunologically naïve children (primary infection) may be more severe than in children with re-infections who are already immunologically primed. We compared frequency and severity of influenza virus primary and re-infections in pre-school children requiring outpatient treatment.
Methods
Influenza-unvaccinated children 1–5 years of age presenting at pediatric practices with febrile acute respiratory infection < 48 h after symptom onset were enrolled in a prospective, cross-sectional, multicenter surveillance study (2013–2015). Influenza types/subtypes were PCR-confirmed from oropharyngeal swabs. Influenza type/subtype-specific IgG antibodies serving as surrogate markers for immunological priming were determined using ELISA/hemagglutination inhibition assays. The acute influenza disease was defined as primary infection/re-infection by the absence/presence of influenza type-specific immunoglobulin G (IgG) and, in a second approach, by the absence/presence of subtype-specific IgG. Socio-demographic and clinical data were also recorded.
Results
Of 217 influenza infections, 178 were due to influenza A (87 [49%] primary infections, 91 [51%] re-infections) and 39 were due to influenza B (38 [97%] primary infections, one [3%] re-infection). Children with “influenza A primary infections” showed fever with respiratory symptoms for a shorter period than children with “influenza A re-infections” (median 3 vs. 4 days; age-adjusted p = 0.03); other disease characteristics were similar. If primary infections and re-infections were defined based on influenza A subtypes, 122 (87%) primary infections (78 “A(H3N2) primary infections”, 44 “A(H1N1)pdm09 primary infections”) and 18 (13%) re-infections could be classified (14 “A(H3N2) re-infections” and 4 “A(H1N1)pdm09 re-infections”). Per subtype, primary infections and re-infections were of similar disease severity. Children with re-infections defined on the subtype level usually had non-protective IgG titers against the subtype of their acute infection (16 of 18; 89%). Some patients infected by one of the influenza A subtypes showed protective IgG titers (≥ 1:40) against the other influenza A subtype (32/140; 23%).
Conclusions
Pre-school children with acute influenza A primary infections and re-infections presented with similar frequency in pediatric practices. Contrary to expectation, severity of acute “influenza A primary infections” and “influenza A re-infections” were similar. Most “influenza A re-infections” defined on the type level turned out to be primary infections when defined based on the subtype. On the subtype level, re-infections were rare and of similar disease severity as primary infections of the same subtype. Subtype level re-infections were usually associated with low IgG levels for the specific subtype of the acute infection, suggesting only short-time humoral immunity induced by previous infection by this subtype. Overall, the results indicated recurring influenza virus infections in this age group and no or only limited heterosubtypic antibody-mediated cross-protection.
Motives motivate human behavior. Most behaviors are driven by more than one motive, yet it is unclear how different motives interact and how such motive combinations affect the neural computation of the behaviors they drive. To answer this question, we induced two prosocial motives simultaneously (multi-motive condition) and separately (single motive conditions). After the different motive inductions, participants performed the same choice task in which they allocated points in favor of the other person (prosocial choice) or in favor of themselves (egoistic choice). We used fMRI to assess prosocial choice-related brain responses and drift diffusion modeling to specify how motive combinations affect individual components of the choice process. Our results showed that the combination of the two motives in the multi-motive condition increased participants' choice biases prior to the behavior itself. On the neural level, these changes in initial prosocial bias were associated with neural responses in the bilateral dorsal striatum. In contrast, the efficiency of the prosocial decision process was comparable between the multi-motive and the single-motive conditions. These findings provide insights into the computation of prosocial choices in complex motivational states, the motivational setting that drives most human behaviors .
Chorioamnionitis is associated with an increased risk of preterm birth and aggravates adverse outcomes such as BPD. Development of BPD is associated with chronic inflammatory reactions and oxidative stress in the airways which may be antenatally initiated by chorioamnionitis. A20 is an immunomodulatory protein involved in the negative feedback regulation of inflammatory reactions and is a possible regulator protein in oxidative stress reactions. The influence of chorioamnionitis on A20 gene regulation in the fetal lung is unknown. We characterized the influence of LPS and proinflammatory cytokines on A20 expression in human lung endothelial (HPMEC-ST1.6R) and epithelial (A549) cells in vitro by real-time PCR and/or western blotting and used a sheep model of LPS-induced chorioamnionitis for in vivo studies. To study the functional role of A20, endogenous A20 was overexpressed in HPMEC-ST1.6R and A549 cells. LPS induced proinflammatory cytokines in HPMEC-ST1.6R and A549 cells. Both LPS and/or proinflammatory cytokines elevated A20 at transcriptional and translational levels. Intra-amniotic LPS transiently increased IL-1β, IL-6, IL-8, and TNF-α mRNA levels in fetal lamb lungs, associated with an increase in A20 mRNA and protein levels. Overexpression of A20 reduced proinflammatory cytokines in vitro. Repeated LPS exposure induced LPS tolerance for proinflammatory cytokines and A20 in vitro and in vivo. Antenatal inflammation induced a transient increase in proinflammatory cytokines in the preterm fetal lung. The expression of proinflammatory cytokines increased expression of A20. Elevated A20 may have a protective role by downregulating chorioamnionitis-triggered fetal lung inflammation. A20 may be a novel target for pharmacological interventions to prevent chorioamnionitis-induced airway inflammation and lung damage, which can result in BPD later in life.
Background
Non-suicidal self-injury (NSSI) has become a substantial public health problem. NSSI is a high-risk marker for the development and persistence of mental health problems, shows high rates of morbidity and mortality, and causes substantial health care costs. Thus, there is an urgent need for action to develop universal prevention programs for NSSI before adolescents begin to show this dangerous behavior. Currently, however, universal prevention programs are lacking.
Methods
The main objective of the present study is to evaluate a newly developed universal prevention program (“DUDE – Du und deine Emotionen / You and your emotions”), based on a skills-based approach in schools, in 3200 young adolescents (age 11–14 years). The effectiveness of DUDE will be investigated in a cluster-randomized controlled trial (RCT) in schools (N = 16). All groups will receive a minimal intervention called “Stress-free through the school day” as a mental health literacy program to prevent burnout in school. The treatment group (N = 1600; 8 schools) will additionally undergo the universal prevention program DUDE and will be divided into treatment group 1 (DUDE conducted by trained clinical psychologists; N = 800; 4 schools) and treatment group 2 (DUDE conducted by trained teachers; N = 800; 4 schools). The active control group (N = 1600; 8 schools) will only receive the mental health literacy prevention. Besides baseline assessment (T0), measurements will occur at the end of the treatment (T1) and at 6- (T2) and 12-month (T3) follow-up evaluations. The main outcome is the occurrence of NSSI within the last 6 months assessed by a short version of the Deliberate Self-Harm Inventory (DSHI-9) at the 1-year follow-up (primary endpoint; T3). Secondary outcomes are emotion regulation, suicidality, health-related quality of life, self-esteem, and comorbid psychopathology and willingness to change.
Discussion
DUDE is tailored to diminish the incidence of NSSI and to prevent its possible long-term consequences (e.g., suicidality) in adolescents. It is easy to access in the school environment. Furthermore, DUDE is a comprehensive approach to improve mental health via improved emotion regulation.
In ischemic stroke (IS) impairment of the blood-brain barrier (BBB) has an important role in the secondary deterioration of neurological function. BBB disruption is associated with ischemia-induced inflammation, brain edema formation, and hemorrhagic infarct transformation, but the underlying mechanisms are incompletely understood. Dysfunction of endothelial cells (EC) may play a central role in this process. Although neuronal NLR-family pyrin domain-containing protein 3 (NLRP3) inflammasome upregulation is an established trigger of inflammation in IS, the contribution of its expression in EC is unclear. We here used brain EC, exposed them to oxygen and glucose deprivation (OGD) in vitro, and analyzed their survival depending on inflammasome inhibition with the NLRP3-specific drug MCC950. During OGD, EC death could significantly be reduced when targeting NLRP3, concomitant with diminished endothelial NLRP3 expression. Furthermore, MCC950 led to reduced levels of Caspase 1 (p20) and activated Gasdermin D as markers for pyroptosis. Moreover, inflammasome inhibition reduced the secretion of pro-inflammatory chemokines, cytokines, and matrix metalloproteinase-9 (MMP9) in EC. In a translational approach, IS was induced in C57Bl/6 mice by 60 mins transient middle cerebral artery occlusion and 23 hours of reperfusion. Stroke volume, functional outcome, the BBB integrity, and-in good agreement with the in vitro results-MMP9 secretion as well as EC survival improved significantly in MCC950-treated mice. In conclusion, our results establish the NLRP3 inflammasome as a critical pathogenic effector of stroke-induced BBB disruption by activating inflammatory signaling cascades and pyroptosis in brain EC.
Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues.
In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration.
The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.
To date, no consensus exists regarding the best surgical management of isolated, micro-traumatic long thoracic nerve (LTN) paresis. Our hypothesis was that a combined decompression of the LTN at two potential locations for entrapment would be effective in the management of dynamic LTN paresis. We report on twelve patients with isolated LTN parersis, with tenderness at two entrapment sites, who underwent bifocal LTN decompression after undergoing unsuccessful conservative treatment for at least 6 months; all patients had preoperative electrodiagnostic studies that confirmed the paresis and ruled out peripheral neuritis. Clinical and electrical improvements were observed in eight patients (67%) regarding shoulder flexion, shoulder abduction, and Quick-DASH scores. Four patients (33%) did not improve after surgery. The results corroborate our hypothesis that a bifocal LTN decompression can be an effective and reliable therapeutic option in more than half of a very selective patient population suffering from serratus anterior muscle deficiency.
Introduction
Detailed and up-to-date data on the epidemiology and healthcare costs of Influenza are fundamental for public health decision-making. We analyzed inpatient data on Influenza-associated hospitalizations (IAH), selected complications and risk factors, and their related direct costs for Germany during ten consecutive years.
Methods
We conducted a retrospective cost-of-illness study on patients with laboratory-confirmed IAH (ICD-10-GM code J09/J10 as primary diagnosis) by ICD-10-GM-based remote data query using the Hospital Statistics database of the German Federal Statistical Office. Clinical data and associated direct costs of hospital treatment are presented stratified by demographic and clinical variables.
Results
Between January 2010 to December 2019, 156,097 persons were hospitalized due to laboratory-confirmed Influenza (J09/J10 primary diagnosis). The annual cumulative incidence was low in 2010, 2012 and 2014 (1.3 to 3.1 hospitalizations per 100,000 persons) and high in 2013 and 2015-2019 (12.6 to 60.3). Overall direct per patient hospitalization costs were mean (SD) 3521 EUR (± 8896) and median (IQR) 1805 EUR (1502; 2694), with the highest mean costs in 2010 (mean 8965 EUR ± 26,538) and the lowest costs in 2012 (mean 2588 EUR ± 6153). Mean costs were highest in 60-69 year olds, and in 50-59, 70-79 and 40-49 year olds; they were lowest in 10-19 year olds. Increased costs were associated with conditions such as diabetes (frequency 15.0%; 3.45-fold increase compared to those without diabetes), adiposity (3.3%; 2.09-fold increase) or immune disorders (5.6%; 1.88-fold increase) and with Influenza-associated complications such as Influenza pneumonia (24.3%; 1.95-fold), bacterial pneumonia (6.3%; 3.86-fold), ARDS (1.2%; 10.90-fold increase) or sepsis (2.3%; 8.30-fold). Estimated overall costs reported for the 10-year period were 549.6 Million euros (95% CI 542.7-556.4 million euros).
Conclusion
We found that the economic burden of IAH in Germany is substantial, even when considering solely laboratory-confirmed IAH reported as primary diagnosis. The highest costs were found in the elderly, patients with certain underlying risk factors and patients who required advanced life support treatment, and median and mean costs showed considerable variations between single years. Furthermore, there was a relevant burden of disease in middle-aged adults, who are not covered by the current vaccination recommendations in Germany.
Protein kinase D2 drives chylomicron-mediate lipid transport in the intestine and promotes obesity
(2022)
Obesity and associated metabolic syndrome are growing concerns in modern society due to the negative consequences for human health and well-being. Cardiovascular diseases and type 2 diabetes are only some of the pathologies associated to overweight. Among the main causes are decreased physical activity and food availability and composition. Diets with high content of fat are energy-dense and their overconsumption leads to an energy imbalance, which ultimately promotes energy storage as fat and obesity. Aberrant activation of signalling cascades and hormonal imbalances are characteristic of this disease and members of the Protein Kinase D (PKD) family have been found to be involved in several mechanisms mediating metabolic homeostasis. Therefore, we aimed to investigate the role of Protein Kinase D2 (PKD2) in the regulation of metabolism. Our investigation initiated with a mice model for global PKD2 inactivation, which allowed us to prove a direct involvement of this kinase in lipids homeostasis and obesity. Inactivation of PKD2 protected the mice from high-fat diet-induced obesity and improved their response to glucose, insulin and lipids. Furthermore, the results indicated that, even though there were no changes in energy intake or expenditure, inactivation of PKD2 limited the absorption of fat from the intestine and promoted energy excretion in feces. These results were verified in a mice model for specific deletion of intestinal PKD2. These mice not only displayed an improved metabolic fitness but also a healthier gut microbiome profile. In addition, we made use of a small-molecule inhibitor of PKD in order to prove that local inhibition of PKD2 in the intestine was sufficient to inhibit lipid absorption. The usage of the inhibitor not only protected the mice from obesity but also was efficient in avoiding additional body-weight gain after obesity was pre-established in mice. Mechanistically, we determined that PKD2 regulates lipids uptake in enterocytes by phosphorylation of Apolipoprotein A4 (APOA4) and regulation of chylomicron-mediated triglyceride absorption. PKD2 deletion or inactivation increased abundance of APOA4 and decreased the size of chylomicrons and therefore lipids absorption from the diet. Moreover, intestinal activation of PKD2 in human obese patients correlated with higher levels of triglycerides in circulation and a detrimental blood profile. In conclusion, we demonstrated that PKD2 is a key regulator of dietary fat absorption in murine and human context, and its inhibition might contribute to the treatment of obesity.
RNA represents one of the most abundant macromolecules in both eukaryotic and prokaryotic cells. Since the discovery that RNA could play important gene regulatory functions in the physiology of a cell, small regulatory RNAs (sRNAs) have been at the center of molecular biology studies. Functional sRNAs can be independently transcribed or derived from processing of mRNAs and other non-coding regions and they often associate with RNA-binding proteins (RBPs). Ever since the two major bacterial RBPs, Hfq and ProQ, were identified, the way we approach the identification and characterization of sRNAs has drastically changed. Initially, a single sRNA was annotated and its function studied with the use of low-throughput biochemical techniques. However, the development of RNA-seq techniques over the last decades allowed for a broader identification of sRNAs and their functions. The process of studying a sRNA mainly focuses on the characterization of its interacting RNA partner(s) and the consequences of this binding. By using RNA interaction by ligation and sequencing (RIL-seq), the present thesis aimed at a high-throughput mapping of the Hfq-mediated RNA-RNA network in the major human pathogen Salmonella enterica.
RIL-seq was at first performed in early stationary phase growing bacteria, which enabled the identification of ~1,800 unique interactions. In- depth analysis of such complex network was performed with the aid of a newly implemented RIL-seq browser. The interactome revealed known and new interactions involving sRNAs and genes part of the envelope regulon. A deeper investigation led to the identification of a new RNA sponge of the MicF sRNA, namely OppX, involved in establishing a cross-talk between the permeability at the outer membrane and the transport capacity at the periplasm and the inner membrane. Additionally, RIL-seq was applied to Salmonella enterica grown in SPI-2 medium, a condition that mimicks the intracellular lifestyle of this pathogen, and finally extended to in vivo conditions during macrophage infection. Collectively, the results obtained in the present thesis helped unveiling the complexity of such RNA networks.
This work set the basis for the discovery of new mechanisms of RNA-based regulation, for the identification of a new physiological role of RNA sponges and finally provided the first resource of RNA interactions during infection conditions in a major human pathogen.
The propounded thesis investigated fear learning including fear conditioning, its generalization as well as its extinction in 133 healthy children and adolescents aged 8 to 17 years. The main goal was to analyze these processes also in the course of childhood and adolescence due to far less research in this age span compared to adults. Of note, childhood is the typical period for the onset of anxiety disorders. To achieve this, an aversive discriminative fear conditioning, generalization and extinction paradigm, which based on the “screaming lady paradigm” from Lau et al. (2008) and was adapted by Schiele & Reinhard et al. (2016), was applied. All probands traversed the pre-acquisition (4 x CS-, 4 x CS+, no US), the acquisition (12 x CS-, 12 x CS+, reinforcement rate: 83%), the generalization (12 x CS-, 12 x GS4, 12 x GS3, 12 x GS2, 12 x GS1, 12 x CS+, reinforcement rate: 50%) and the extinction (18 x CS-, 18 x CS+, no US). The generalization stimuli, i.e. GS1-GS4, were built out of CS- and CS+ in different mixtures on a percentage basis in steps of 20% from CS- to CS+. Pictures of faces of two actresses with a neutral expression were used for the discriminative conditioning, whereby the CS+ was paired with a 95-dB loud female scream at the same time together with a fearful facial expression (US). CS- and GS1-GS4 were never followed by the US. Subjective ratings (arousal, valence and US expectancy) were collected and further the psychophysiological measure of the skin conductance response (SCR). The hypotheses were 1) that underage probands show a negative correlation between age and overgeneralization and 2) that anxiety is positively correlated with overgeneralization in the same sample. ANOVAs with repeated measures were conducted for all four dependent variables with phase (pre-acquisition phase, 1. + 2. acquisition phase, 1. + 2. generalization phase, 1. - 3. extinction phase) and stimulus type
(CS-, CS+, GS1-GS4) as within-subject factors. For the analyses of the modulatory effects of age and anxiety in additional separate ANCOVAs were conducted including a) age, b) the STAIC score for trait anxiety and c) the CASI score for anxiety sensitivity as covariates. Sex was always included as covariate of no interest. On the one hand, findings indicated that the general extent of the reactions (arousal, valence and US expectancy ratings and the SCR) decreased with growing age, i.e. the older the probands the lower their reactions towards the stimuli regardless of the type of dependent variable. On the other hand, ratings of US expectancy, i.e. the likelihood that a stimulus is followed by a US (here: female scream coupled with a fearful facial expression), showed better discrimination skills the older the probands were, resulting in a smaller overgeneralization within older probands. It must be emphasized very clearly that no causality can be derived. Thus, it was only an association revealed between
15
age and generalization of conditioned fear, which is negative. Furthermore, no obvious impact of trait anxiety could be detected on the different processes of fear learning. Especially, no overgeneralization was expressed by the probands linked to higher trait anxiety. In contrast to trait anxiety, for anxiety sensitivity there was an association between its extent and the level of fear reactions. This could be described best with a kind of parallel shifts: the higher the anxiety sensitivity, the stronger the fear reactions. Likewise, for anxiety sensitivity no overgeneralization due to a stronger extent of anxiety sensitivity could be observed.
Longitudinal follow-up examinations and, furthermore, neurobiological investigations are needed for replication purposes and purposes of gaining more supporting or opposing insights, but also for the profound exploration of the impact of hormonal changes during puberty and of the maturation processes of different brain structures. Finally, the question whether enhanced generalization of conditioned fear facilitates the development of anxiety disorders or vice versa remains unsolved yet.
Avocado (Persea americana Mill.) is a major horticultural crop that relies on insect mediated pollination. In avocado production, a knowledge gap exists as to the importance of insect pollination, especially in East African smallholder farms. Although it is evident that pollination improves the yield of avocado fruits, it is still unclear if pollination has benefits on fruit quality and the nutritional profile, particularly oils. Prior studies have shown that honey bees increase avocado’s fruit set and yield. However, an avocado flower is being visited by various insect species. Therefore, determining pollination efficiency will allow a comparison of the relative importance of the different insect species to optimize crop pollination for increased fruit set and crop yield and pollinator conservation. This study was conducted in a leading smallholder avocado production region in Kenya, first I assessed the dependence of avocado fruit set on insect pollination and whether current smallholder production systems suffer from a deficit in pollination services. Furthermore, I assessed if supplementation with colonies of the Western honey bee (Apis mellifera L.) to farms mitigated potential pollination deficits. The results revealed a very high reliance of avocado on insect pollinators, with a significantly lower fruit set observed for self- and wind-pollinated (17.4%) or self-pollinated flowers (6.4%) in comparison with insect-pollinated flowers (89.5%). I found a significant pollination deficit across farms, with hand-pollinated flowers on average producing 20.7% more fruits than non-treated open flowers prior to fruit abortion. This pollination deficit could be compensated by the supplementation of farms with A. mellifera colonies. These findings suggest that pollination is limiting fruit set in avocado and that A. mellifera supplementation on farms is a potential option to increase fruit yield. Secondly, I investigated the contribution of insect pollination to fruit and seed weight, oil, protein, carbohydrate, and phytochemicals contents (flavonoids and phenolics), and whether supplementation with pollinators (honey bee) could improve these fruit parameters was assessed. This was through pollinator-manipulative pollination treatments: hand, open, pollinator exclusion experiments. The results showed that avocado fruit weight was significantly higher in open and hand-pollinated than pollinator exclusion treatments, indicating that flower visitors/pollinators contribute to avocado yields and enhance marketability. Furthermore, insect pollination resulted in heavier seeds and higher oil contents, indicating that insect pollination is beneficial for the fruit’s high seed yield and quantity of oil. Honey bee supplementation also enhanced the avocado fruit weight by 18% more than in control farms and slightly increased the avocado oil content (3.6%). Contrarily, insect pollination did not influence other assayed fruit quality parameters (protein, carbohydrates, and phytochemicals). These results indicate that insect pollinators are essential for optimizing avocado yields, nutritional quality (oils), and thus marketability, underscoring the value of beehive supplementation to achieve high-quality avocado fruits and improved food security. Thirdly, pollinator efficiency based on pollen deposition after single visits by different pollinator species in avocado flowers was tested, and their frequency was recorded. The estimated pollination efficiency was highest in honey bees (Apis mellifera), followed by the hoverfly species (Phytomia incisa). These two species had the highest pollen deposition and more pollen grains on their bodies. In addition, honey bees were the most frequent avocado flower visitors, followed by flies. The findings from this study highlight the higher pollination efficiency of honey bees and Phytomia incisa. Hence, management practices supporting these species will promote increased avocado fruit yield. Additionally, these results imply that managed honey bees can be maintained to improve avocado pollination, particularly in areas lacking sufficient wild pollinators.
Mesenchymal stem/stromal cells (MSCs) are a rare subpopulation of cells first identified in bone marrow with the potential to proliferate in plastic-adherent colonies and to generate de novo bone marrow stroma and its environment upon serial transplantation to heterotopic anatomical sites. Given their multipotency and self renewal competence, MSCs are prime prospective candidates for most modern musculoskeletal-tissue engineering and regenerative medicine approaches. Still, their envisioned therapeutic use is being questioned with concerns regarding their definition, characterization and integrative functions in vivo. It is well established that microenvironmental cues such as the extracellular matrix (ECM)-chemistry, the mechanical environment and local cellular and/or paracrine interactions critically control MSCs behavior. Yet, most of the scientific knowledge regarding the biology and therapeutic effect of MSCs originates from mechanistic in vitro studies where microenvironmental cues are hardly addressed. Therefore, manifestable changes in cell proliferation behavior and multilineage differentiation potential might be triggered that eventually compromise the translation of results to clinics. This thesis aims to address the complexity of MSCs interactions within the skeletal niche microenvironment in order to provide alternative methods to bypass the current MSCs in vitro culture limitations. Firstly, the influence of ECM-chemistry on MSCs behavior in vitro was explored by means of decellularized human bone models here established. Basal or osteogenic tailored cell-derived decellularized 2D matrices (dECM), proved to be suitable culture substrates for MSCs expansion by providing close-to-native cell-ECM interactions. Moreover, quantified morphological shape changes suggested a material osteo supportive potential, further functionally validated by observable spontaneous mineralization of MSCs. Aiming to identify novel intrinsic ECM regulatory features specific to the skeletal niche, 3D decellularized human trabecular bone scaffolds (dBone) were additionally developed and comprehensively characterized. Remarkably, the MSCs cultured on dBone scaffolds exhibit upregulation of genes associated with stemness as well as niche-related protein expression advocating for the conservation of the naïve MSCs phenotype. vi On the other hand, the effect of biomimetic mineralization on MSCs osteogenic lineage differentiation potential was further addressed by hydroxyapatite functionalization of type-I collagen in presence of magnesium. Mineralized scaffolds exhibited higher cell viability and a clear trend of osteogenic genes upregulation comparing with non-mineralized scaffolds. Lastly, in order to mimic the complexity of the native MSCs environment, a dynamic culture system was applied to the 3D decellularized bone constructs, previously studied in single static conditions. Mechanical stimuli generated by (1) continuous perfusion of cell culture medium at 1.7 mL/min and (2) compressive stress from 10% uniaxial load at 1 Hz, resulted in an improved cell repopulation within the scaffold and boosting of de novo ECM production. The stress-induced gene expression pattern suggested early MSCs commitment towards the osteogenic lineage mediated by integrin matrix adhesion, therefore further corroborating the recapitulation of a reliable in vitro bone niche model in dBone scaffolds. To conclude, the here developed in vitro models provide a progressive increased biomimicking complexity through which significant insights regarding MSC interactions with microenvironmental features in the skeletal niche can be obtained, thus surely paving the way for a better understanding of the role of MSCs in bone homeostasis and regeneration.
RNA sequencing (RNA-seq) has become a transformative method to profile genome-wide gene expression and whole transcriptome analysis over the last decade. In recent years, with the development of new technologies, it has become possible to study gene expression at single-cell level. This new advances in single-cell RNA-sequencing has revolutionized the way scientists study biological processes. Single-cell RNA-sequencing has been used in different areas to better understand the underlying mechanisms of biological processes.
In particular, single-RNA-sequencing is a suitable method to study infectious diseases. Infection is composed of heterogeneous mechanisms on either the host or pathogen side and the best way to understand the heterogeneity of these mechanisms and how they interact with each other is to study infectious diseases at the single-cell level. Studying infection processes at the single-cell level can reveal not only the heterogeneity but also the dynamics of infection and the interplay between the host and pathogen at the molecular level.
In this thesis, we implemented and applied different single-cell RNA-seq technologies to better understand infectious diseases. In the present work, we conducted four independent but related research works to shed light on different aspects of infection biology:
● We took advantage of this novel technology to study the consequences of RSV infection on primary human epithelial cells. The primary human epithelial cells were collected from six donors and cultured in air liquid interface (ALI) cell culture inoculated with respiratory syncytial virus (RSV). In this project, we discovered ciliated cells as the susceptible cell types in RSV infection. We applied viral load as an indicator of infection progression and used it to reconstruct the dynamics of host response to RSV infection. Reconstruction of the dynamics of infection revealed many host genes and pathways that were suppressed or induced as a result of RSV infection. Pathways related to innate immune response and interferon response were suppressed during the progression of infection and on the other hand pathways like protein targeting to endoplasmic reticulum and apoptosis were induced.
● We developed a new method which is capable of sequencing the transcriptome of a bacterium at the single-cell level and potentially can help us to characterize the bacterial heterogeneity during the course of infection. In this research project, bacteria were cultured in three different culture conditions namely Late stationary phase, Anaerobic shock and NaCl shock and we used a poly(A)-independent single-cell RNA-sequencing protocol to sequence bacteria at the single-cell level. In this work, we report the faithful capture of growth-dependent gene expression patterns in individual Salmonella and Pseudomonas bacteria. The results of our analysis showed that not only we could capture transcripts across different RNA classes but also our method is capable of discerning the transcriptome of bacteria across different culture conditions.
● We used single-cell RNA-sequencing technology to characterize the immune cells landscape over the course of atherosclerosis. Atherosclerosis is considered a cardiac disease which is highly related to infections and previous infections with bacteria or viruses is considered as a risk factor for atherosclerosis. We performed single-cell RNA sequencing of aortic CD45+ cells extracted from healthy and atherosclerotic aorta of mice. We managed to find certain cell populations which were specifically present in atherosclerotic mice. One of the atheroschelorotic populations was previously undescribed TREM2high macrophages showing enrichment in Trem2 gene expression. This population of macrophages seemed to be involved in functions like lipid metabolism and catabolism and lesion calcification. This work revealed the phenotypic heterogeneity and immune cells landscape of different immune cell populations at different stages of atherosclerosis. Our work paves the way to better describe the relation between different infectious diseases and cardiovascular diseases.
● We developed a web-based platform called Infection Atlas to browse and visualize single-cell RNA-sequencing data. Infection Atlas platform provides a user-friendly interface to study different aspects of infectious diseases at the single-cell level and can potentially promote targeted approaches to intervene in infectious diseases. This platform which is available at infection-atlas.org in the short term provides a user-friendly interface to browse and visualize different aspects of infectious diseases and in the long-term is expected to be a comprehensive atlas of infection in human and mouse across different tissues and different pathogens.
Overall, in this thesis we provide a framework to study infectious diseases at the single cell level with providing novel data analysis methods and this thesis paves the way for future studies to study host-pathogen encounters at the single-cell level.
The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed.
In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the “gold standard” in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs.
In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes.
Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture.
Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive.
With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies.
Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.
The transcription factor MYC is a onco-protein, found to be deregulated in many human cancers. High MYC levels correlate with an aggressive tumor outcome and poor survival rates. Despite MYC being discovered as an oncogene already in the 1970s, how MYC regulates transcription of its target genes, which are involved in cellular growth and proliferation, is not fully understood yet.
In this study, the question how MYC influences factors interacting with the RNA polymerase II ensuring productive transcription of its target genes was addressed using quantitative mass spectrometry. By comparing the interactome of RNA polymerase II under varying MYC levels, several potential factors involved in transcriptional elongation were identified. Furthermore, the question which of those factors interact with MYC was answered by employing quantitative mass spectrometry of MYC itself. Thereby, the direct interaction of MYC with the transcription elongation factor SPT5, a subunit of the DRB-sensitivity inducing factor, was discovered and analyzed in greater detail. SPT5 was shown to be recruited to chromatin by MYC. In addition, the interaction site of MYC on SPT5 was narrowed down to its evolutionary conserved NGN-domain, which is the known binding site for SPT4, the earlier characterized second subunit of the DRB-sensitivity inducing factor. This finding suggests a model in which MYC and SPT4 compete for binding the NGN-domain of SPT5.
Investigations of the SPT5-interacting region on MYC showed binding of SPT5 to MYC’s N-terminus including MYC-boxes 0, I and II.
In order to analyze proteins interacting specifically with the N-terminal region of MYC, a truncated MYC-mutant was used for quantitative mass spectrometric analysis uncovering reduced binding for several proteins including the well-known interactor TRRAP and TRRAP-associated complexes.
Summarized, ...
Laser spectroscopic gas sensing has been applied for decades for several applications
as atmospheric monitoring, industrial combustion gas analysis or fundamental research.
The availability of new laser sources in the mid-infrared opens the spectral fingerprint
range to the technology where multiple molecules possess their fundamental ro-vibrational
absorption features that allow very sensitive detection and accurate discrimination of
the species. The increasing maturity of quantum cascade lasers that cover this highly
interesting spectral range motivated this research to gain fundamental knowledge about
the spectra of hydrocarbon gases in pure composition and in complex mixtures as they
occur in the petro-chemical industry. The long-term target of developing accurate and fast
hydrocarbon gas analyzers, capable of real-time operation while enabling feedback-loops,
would lead to a paradigm change in this industry.
This thesis aims to contribute to a higher accuracy and more comprehensive understanding
of the sensing of hydrocarbon gas mixtures. This includes the acquisition of yet
unavailable high resolution and high accuracy reference spectra of the respective gases,
the investigation of their spectral behavior in mixtures due to collisional broadening of
their transitions and the verification of the feasibility to quantitatively discriminate the
spectra when several overlapping species are simultaneously measured in gas mixtures.
To achieve this knowledge a new laboratory environment was planned and built up to
allow for the supply of the individual gases and their arbitrary mixing. The main element
was the development of a broadly tunable external-cavity quantum cascade laser based
spectrometer to record the required spectra. This also included the development of a new
measurement method to obtain highly resolved and nearly gap-less spectral coverage as
well as a sophisticated signal post-processing that was crucial to achieve the high accuracy
of the measurements. The spectroscopic setup was used for a thorough investigation of
the spectra of the first seven alkanes as of their mixtures. Measurements were realized
that achieved a spectral resolution of 0.001 cm-1 in the range of 6-11 µm while ensuring an
accuracy of 0.001 cm-1 of the spectra and attaining a transmission sensitivity of 2.5 x 10-4
for long-time averaging of the acquired spectra.
These spectral measurements accomplish a quality that compares to state-of-the art
spectral databases and revealed so far undocumented details of several of the investigated
gases that have not been measured with this high resolution before at the chosen measurement
conditions. The results demonstrate the first laser spectroscopic discrimination of a
seven component gas mixture with absolute accuracies below 0.5 vol.% in the mid-infrared
provided that a sufficiently broad spectral range is covered in the measurements. Remaining
challenges for obtaining improved spectral models of the gases and limitations of the
measurement accuracy and technology are discussed.
The fate and behavior of bone marrow mesenchymal stem/stromal cells (BM-MSC) is bidirectionally influenced by their microenvironment, the stem cell niche, where a magnitude of biochemical and physical cues communicate in an extremely orchestrated way. It is known that simplified 2D in vitro systems for BM-MSC culture do not represent their naïve physiological environment. Here, we developed four different 2D cell-based decellularized matrices (dECM) and a 3D decellularized human trabecular-bone scaffold (dBone) to evaluate BM-MSC behavior. The obtained cell-derived matrices provided a reliable tool for cell shape-based analyses of typical features associated with osteogenic differentiation at high-throughput level. On the other hand, exploratory proteomics analysis identified native bone-specific proteins selectively expressed in dBone but not in dECM models. Together with its architectural complexity, the physico-chemical properties of dBone triggered the upregulation of stemness associated genes and niche-related protein expression, proving in vitro conservation of the naïve features of BM-MSC.
Continued reports over the past decades of unknown aerial phenomena (short UAP) have given high relevance to the investigation and research of these. Especially reports by US Navy pilots and official investigations by the US Office of the director of national intelligence have emphasized the value of such efforts. Due to the inherently limited scope of earth based observations, a satellite based instrument for detection of such phenomena may prove especially useful. This paper as such investigates the possible viability of such an instrument on a nano satellite mission.
Purpose
The goal of our study was to conduct an online survey that highlights patterns of practice during total hip arthroplasty (THA).
Methods
The survey was conducted in June and August 2020. Three hundred thirteen members of the German Society for Endoprosthesis participated in the survey.
Results
The anterolateral approach is by far the most popular approach used for primary total hip arthroplasty, followed by the anterior approach during minimally invasive (55% for the anterolateral and 29% for the anterior) and regular surgery (52% for the anterolateral and 20% for the anterior). Two-thirds of the orthopaedic surgeons do not use drainages during THA. Moreover, 80% of the survey participants routinely apply tranexamic acid during surgery. Surgeons who perform minimally invasive surgery for THA use more frequently fast-track-concepts for post-operative rehabilitation. According to the interviewees, the application of fast-track-concepts leads to reduced periods of hospital stay after THA.
Conclusion
Our data demonstrate that patterns of practice during THA in Germany are in line with the evidence provided by current literature. This study can be seen as a stimulus to conduct similar surveys in other countries in order to promote minimally invasive surgery for THA.
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.
In this paper we study properties of the Laplace approximation of the posterior distribution arising in nonlinear Bayesian inverse problems. Our work is motivated by Schillings et al. (Numer Math 145:915–971, 2020. https://doi.org/10.1007/s00211-020-01131-1), where it is shown that in such a setting the Laplace approximation error in Hellinger distance converges to zero in the order of the noise level. Here, we prove novel error estimates for a given noise level that also quantify the effect due to the nonlinearity of the forward mapping and the dimension of the problem. In particular, we are interested in settings in which a linear forward mapping is perturbed by a small nonlinear mapping. Our results indicate that in this case, the Laplace approximation error is of the size of the perturbation. The paper provides insight into Bayesian inference in nonlinear inverse problems, where linearization of the forward mapping has suitable approximation properties.
T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression.
NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point.
ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a.
We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells.
Purpose
The present study determined the postoperative phenotypes after unrestricted calipered kinematically aligned (KA) total knee arthroplasty (TKA), whether any phenotypes were associated with reoperation, implant revision, and lower outcome scores at 4 years, and whether the proportion of TKAs within each phenotype was comparable to those of the nonarthritic contralateral limb.
Methods
From 1117 consecutive primary TKAs treated by one surgeon with unrestricted calipered KA, an observer identified all patients (N = 198) that otherwise had normal paired femora and tibiae on a long-leg CT scanogram. In both legs, the distal femur–mechanical axis angle (FMA), proximal tibia–mechanical axis angle (TMA), and the hip–knee–ankle angle (HKA) were measured. Each alignment angle was assigned to one of Hirschmann’s five FMA, five TMA, and seven HKA phenotype categories.
Results
Three TKAs (1.5%) underwent reoperation for anterior knee pain or patellofemoral instability in the subgroup of patients with the more valgus phenotypes. There were no implant revisions for component loosening, wear, or tibiofemoral instability. The median Forgotten Joint Score (FJS) was similar between phenotypes. The median Oxford Knee Score (OKS) was similar between the TMA and HKA phenotypes and greatest in the most varus FMA phenotype. The phenotype proportions after calipered KA TKA were comparable to the contralateral leg.
Conclusion
Unrestricted calipered KA’s restoration of the wide range of phenotypes did not result in implant revision or poor FJS and OKS scores at a mean follow-up of 4 years. The few reoperated patients had a more valgus setting of the prosthetic trochlea than recommended for mechanical alignment. Designing a femoral component specifically for KA that restores patellofemoral kinematics with all phenotypes, especially the more valgus ones, is a strategy for reducing reoperation risk.
In this doctoral thesis we cover the performance evaluation of next generation data plane architectures, comprised of complex software as well as programmable hardware components that allow fine granular configuration. In the scope of the thesis we propose mechanisms to monitor the performance of singular components and model key performance indicators of software based packet processing solutions. We present novel approaches towards network abstraction that allow the integration of heterogeneous data plane technologies into a singular network while maintaining total transparency between control and data plane. Finally, we investigate a full, complex system consisting of multiple software-based solutions and perform a detailed performance analysis. We employ simulative approaches to investigate overload control mechanisms that allow efficient operation under adversary conditions. The contributions of this work build the foundation for future research in the areas of network softwarization and network function virtualization.
Background
It is unknown whether technological advancement of stent-retriever devices influences typical observational indicators of safety or effectiveness.
Methods
Observational retrospective study of APERIO® (AP) vs. new generation APERIO® Hybrid (APH) (Acandis®, Pforzheim, Germany) stent-retriever device (01/2019–09/2020) for mechanical thrombectomy (MT) in large vessel occlusion (LVO) stroke. Primary effectiveness endpoint was successful recanalization eTICI (expanded Thrombolysis In Cerebral Ischemia) ≥ 2b67, primary safety endpoint was occurrence of hemorrhagic complications after MT. Secondary outcome measures were time from groin puncture to first pass and successful reperfusion, and the total number of passes needed to achieve the final recanalization result.
Results
A total of 298 patients with LVO stroke who were treated by MT matched the inclusion criteria: 148 patients (49.7%) treated with AP vs. 150 patients (50.3%) treated with new generation APH. Successful recanalization was not statistically different between both groups: 75.7% for AP vs. 79.3% for APH; p = 0.450. Postinterventional hemorrhagic complications and particularly subarachnoid hemorrhage as the entity possibly associated with stent-retriever device type was significantly less frequent in the group treated with the APH: 29.7% for AP and 16.0% for APH; p = 0.005; however, rates of symptomatic hemorrhage with clinical deterioration and in domo mortality were not statistically different. Neither the median number of stent-retriever passages needed to achieve final recanalization, time from groin puncture to first pass, time from groin puncture to final recanalization nor the number of cases in which successful recanalization could only be achieved by using a different stent-retriever as bail-out device differed between both groups.
Conclusion
In the specific example of the APERIO® stent-retriever device, we observed that further technological developments of the new generation device were not associated with disadvantages with respect to typical observational indicators of safety or effectiveness.
Central European forests experience a substantial loss of open-forest organisms due to forest management and increasing nitrogen deposition. However, management strategies, removing different levels of nitrogen, have been rarely evaluated simultaneously.
We tested the additive effects of coppicing and topsoil removal on communities of dung-inhabiting beetles compared to closed forests. We sampled 57 021 beetles, using baited pitfall traps exposed on 27 plots.
Experimental treatments resulted in significantly different communities by promoting open-habitat species. While alpha diversity did not differ among treatments, gamma diversity of Geotrupidae and Scarabaeidae and beta diversity of Staphylinidae were higher in coppice than in forest. Functional diversity of rove beetles was higher in both, coppice and topsoil-removed plots, compared to control plots. This was likely driven by higher habitat heterogeneity in established forest openings. Five dung beetle species and four rove beetle species benefitted from coppicing, one red-listed dung beetle and two rove beetle species benefitted from topsoil removal.
Our results demonstrate that dung-inhabiting beetles related to open forest patches can be promoted by both, coppicing and additional topsoil removal. A mosaic of coppice and bare-soil-rich patches can hence promote landscape-level gamma diversity of dung and rove beetles within forests.
Background and Objectives
Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient.
Materials and Methods
In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11).
Results
In average, IA reduced the levels of total IgG, IgG1, IgG2 and IgG4 by approximately 60%, the acetylcholine receptor autoantibody levels by more than 70%. IgG3, IgA or IgM were diminished to a lower extent. In contrast to fibrinogen or other coagulation factors, the column markedly removed vitamin K-dependent coagulation factors II, VII, IX and X by approximately 40%–70%. Accordingly, international normalized ratio and activated partial thromboplastin time were increased after IA by 59.1% and 32.7%, respectively. Coagulation tests almost returned to baseline values within 24 h. Blood cell count, electrolytes, total protein or albumin were not essentially affected. No clinical events occurred.
Conclusion
The single-use, multiple-pass protein A adsorber column is highly efficient to remove IgG1, IgG2 and IgG4 or specific acetylcholine receptor autoantibodies from the plasma. Coagulation parameters should be monitored, since the column has the capacity to largely reduce vitamin K-dependent factors.
Patterns of resource use by animals can clarify how ecological communities have assembled in the past, how they currently function and how they are likely to respond to future perturbations. Bumble bees (Hymentoptera: Bombus spp.) and their floral hosts provide a diverse yet tractable system in which to explore resource selection in the context of plant–pollinator networks. Under conditions of resource limitation, the ability of bumble bees species to coexist should depend on dietary niche overlap. In this study, we report patterns and dynamics of floral morphotype preferences in a mountain bumble bee community based on ~13 000 observations of bumble bee floral visits recorded along a 1400 m elevation gradient. We found that bumble bees are highly selective generalists, rarely visiting floral morphotypes at the rates predicted by their relative abundances. Preferences also differed markedly across bumble bee species, and these differences were well-explained by variation in bumble bee tongue length, generating patterns of preference similarity that should be expected to predict competition under conditions of resource limitation. Within species, though, morphotype preferences varied by elevation and season, possibly representing adaptive flexibility in response to the high elevational and seasonal turnover of mountain floral communities. Patterns of resource partitioning among bumble bee communities may determine which species can coexist under the altered distributions of bumble bees and their floral hosts caused by climate and land use change.
Objective
At high altitude (HA), acute mountain sickness (AMS) is accompanied by neurologic and upper gastrointestinal symptoms (UGS). The primary aim of this study was to test the hypothesis that delayed gastric emptying (GE), assessed by \(^{13}\)C-octanoate breath testing (OBT), causes UGS in AMS. The secondary aim was to assess post-gastric mechanisms of OBT, which could confound results under these conditions, by determination of intermediary metabolites, gastrointestinal peptides, and basal metabolic rate.
Methods
A prospective trial was performed in 25 healthy participants (15 male) at 4559 m (HA) and at 490 m (Zurich). GE was assessed by OBT (428 kcal solid meal) and UGS by visual analogue scales (VAS). Blood sampling of metabolites (glucose, free fatty acids (FFA), triglycerides (TG), beta-hydroxyl butyrate (BHB), L-lactate) and gastrointestinal peptides (insulin, amylin, PYY, etc.) was performed as well as blood gas analysis and spirometry. Statistical analysis: variance analyses, bivariate correlation, and multilinear regression analysis.
Results
After 24 h under hypoxic conditions at HA, participants developed AMS (p < 0.001). \(^{13}\)CO\(_{2}\) exhalation kinetics increased (p < 0.05) resulting in reduced estimates of gastric half-emptying times (p < 0.01). However, median resting respiratory quotients and plasma profiles of TG indicated that augmented beta-oxidation was the main predictor of accelerated \(^{13}\)CO\(_{2}\)-generation under these conditions.
Conclusion
Quantification of \(^{13}\)C-octanoate oxidation by a breath test is sensitive to variation in metabolic (liver) function under hypoxic conditions. \(^{13}\)C-breath testing using short-chain fatty acids is not reliable for measurement of gastric function at HA and should be considered critically in other severe hypoxic conditions, like sepsis or chronic lung disease.
Background
Student performance is a mirror of teaching quality. The pre-/post-test design allows a pragmatic approach to comparing the effects of interventions. However, the calculation of current knowledge gain scores introduces varying degrees of distortion. Here we present a new metric employing a linear weighting coefficient to reduce skewness on outcome interpretation.
Methods
We compared and contrasted a number of common scores (raw and relative gain scores) with our new method on two datasets, one simulated and the other empirical from a previous intervention study (n = 180) employing a pre-/post-test design.
Results
The outcomes of the common scores were clearly different, demonstrating a significant dependency on pre-test scores. Only the new metric revealed a linear relationship to the knowledge baseline, was less skewed on the upper or lower extremes, and proved well suited to allow the calculation of negative learning gains. Employing the empirical dataset, the new method also confirmed the interaction effect of teaching formats with specific subgroups of learner characteristics.
Conclusion
This work introduces a new weighted metric enabling meaningful comparisons between interventions based on a linear transformation. This method will form the basis to intertwine the calculation of test performance closely with the outcome of learning as an important factor reflecting teaching quality and efficacy. Its regular use can improve the transparency of teaching activities and outcomes, contribute to forming rounded judgements of students' acquisition of knowledge and skills and enable valuable feedforward to develop and enhance curricular concepts.
This paper shows that labor demand plays an important role in the labor market reactions to a pension reform in Germany. Employers with a high share of older worker inflow compared with their younger worker inflow, employers in sectors with few investments in research and development, and employers in sectors with a high share of collective bargaining agreements allow their employees to stay employed longer after the reform. These employers offer their older employees partial retirement instead of forcing them into unemployment before early retirement because the older employees incur low substitution costs and high dismissal costs.
Gifted underachievers perform worse in school than would be expected based on their high intelligence. Possible causes for underachievement are low motivational dispositions (need for cognition) and metacognitive competences. This study tested the interplay of these variables longitudinally with gifted and non-gifted students from Germany (N = 341, 137 females) in Grades 6 (M = 12.02 years at t1) and 8 (M = 14.07 years). Declarative and procedural metacognitive competences were assessed in the domain of reading comprehension. Path analyses showed incremental effects of procedural metacognition over and above intelligence on the development of school achievement in gifted students (β = .139). Moreover, declarative metacognition and need for cognition interactively predicted procedural metacognition (β = .169), which mediated their effect on school achievement.
Quantifying tree defoliation by insects over large areas is a major challenge in forest management, but it is essential in ecosystem assessments of disturbance and resistance against herbivory. However, the trajectory from leaf-flush to insect defoliation to refoliation in broadleaf trees is highly variable. Its tracking requires high temporal- and spatial-resolution data, particularly in fragmented forests.
In a unique replicated field experiment manipulating gypsy moth Lymantria dispar densities in mixed-oak forests, we examined the utility of publicly accessible satellite-borne radar (Sentinel-1) to track the fine-scale temporal trajectory of defoliation. The ratio of backscatter intensity between two polarizations from radar data of the growing season constituted a canopy development index (CDI) and a normalized CDI (NCDI), which were validated by optical (Sentinel-2) and terrestrial laser scanning (TLS) data as well by intensive caterpillar sampling from canopy fogging.
The CDI and NCDI strongly correlated with optical and TLS data (Spearman's ρ = 0.79 and 0.84, respectively). The ΔNCDII\(_{Defoliation(A−C)}\) significantly explained caterpillar abundance (R\(^{2}\) = 0.52). The NCDI at critical timesteps and ΔNCDI related to defoliation and refoliation well discriminated between heavily and lightly defoliated forests.
We demonstrate that the high spatial and temporal resolution and the cloud independence of Sentinel-1 radar potentially enable spatially unrestricted measurements of the highly dynamic canopy herbivory. This can help monitor insect pests, improve the prediction of outbreaks and facilitate the monitoring of forest disturbance, one of the high priority Essential Biodiversity Variables, in the near future.
Within the Spessart low mountain range in central Germany, numerous castle ruins of the 13th century ce exist. Their construction and destruction were often determined by the struggle for political and economic supremacy in the region and for control over the Spessart's natural resources. Wahlmich Castle is located in a relatively uncommon strategic and geomorphological position, characterized by a fairly remote position and atypical rough relief. In order to reconstruct the local relief development and possible human impact, a multi-method approach was applied combining two-dimensional geoelectrical measurements, geomorphological mapping and stratigraphic-sedimentological investigations. This provides new insights into the influence of landscape characteristics on choices of castle locations.
The combined geoelectrical, geomorphological and stratigraphic-sedimentological data show that the rough relief is of natural origin and influenced by regional faulting, which triggered sliding and slumping as well as weathering and dissection of the surface deposits. The rough relief and the lithology permitted intensive land use and building activities. However, the location of the castle offered access to and possibly control over important medieval traffic routes and also represented certain ownership claims in the Aschaff River valley.
The economic situation combined with rivalry between different elites led to the castle being built in a geomorphological challenging and strategically less valuable location. Focusing on castles located in rare and challenging geomorphological positions may therefore lead to a better understanding of castle siting in the future.
The capacity to develop immunological memory is a hallmark of the adaptive immune system. To investigate the role of Samd3 for cellular immune responses and memory development, we generated a conditional knock-out mouse including a fluorescent reporter and a huDTR cassette for conditional depletion of Samd3-expressing cells. Samd3 expression was observed in NK cells and CD8 T cells, which are known for their specific function against intracellular pathogens like viruses. After acute viral infections, Samd3 expression was enriched within memory precursor cells and the frequency of Samd3-expressing cells increased during the progression into the memory phase. Similarly, during chronic viral infections, Samd3 expression was predominantly detected within precursors of exhausted CD8 T cells that are critical for viral control. At the functional level however, Samd3-deficient CD8 T cells were not compromised in the context of acute infection with Vaccinia virus or chronic infection with Lymphocytic choriomeningitis virus. Taken together, we describe a novel multifunctional mouse model to study the role of Samd3 and Samd3-expressing cells. We found that Samd3 is specifically expressed in NK cells, memory CD8 T cells, and precursor exhausted T cells during viral infections, while the molecular function of this enigmatic gene remains further unresolved.
High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed–Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.
The US National Research Council (NRC) report "Toxicity Testing in the 21st Century: A Vision and a strategy (Tox21)", published in 2007, calls for a complete paradigm shift in tox-icity testing. A central aspect of the proposed strategy includes the transition from apical end-points in in vivo studies to more mechanistically based in vitro tests. To support and facilitate the transition and paradigm shift in toxicity testing, the Adverse Outcome Pathway (AOP) concept is widely recognized as a pragmatic tool. As case studies, the AOP concept was ap-plied in this work to develop AOPs for proximal tubule injuries initiated by Receptor-mediated endocytosis and lysosomal overload and Inhibition of mtDNA polymerase-. These AOPs were used as a mechanistic basis for the development of in vitro assays for each key event (KE). To experimentally support the developed in vitro assays, proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) were treated with model compounds. To measure the dis-turbance of lysosomal function in the AOP – Receptor-mediated endocytosis and lysosomal overload, polymyxin antibiotics (polymyxin B, colistin, polymyxin B nonapeptide) were used as model compounds. Altered expression of lysosomal associated membrane protein 1/2 (LAMP-1/2) (KE1) and cathepsin D release from lysosomes (KE2) were determined by im-munofluorescence, while cytotoxicity (KE3) was measured using the CellTiter-Glo® cell via-bility assay. Importantly, significant differences in polymyxin uptake and susceptibility be-tween cell lines were observed, underlining the importance of in vitro biokinetics to determine an appropriate in vitro point of departure (PoD) for risk assessment. Compared to the in vivo situation, distinct expression of relevant transporters such as megalin and cubilin on mRNA and protein level in the used cell lines (RPTEC/TERT1 and NRK-52E) could not be con-firmed, making integration of quantitative in vitro to in vivo extrapolations (QIVIVE) neces-sary. Integration of QIVIVE by project partners of the University of Utrecht showed an im-provement in the modelled biokinetic data for polymyxin B. To assess the first key event, (KE1) Depletion of mitochondrial DNA, in the AOP – Inhibition of mtDNA polymerase-, a RT-qPCR method was used to determine the mtDNA copy number in cells treated with mod-el compounds (adefovir, cidofovir, tenofovir, adefovir dipivoxil, tenofovir disoproxil fumarate). Mitochondrial toxicity (KE2) was measured by project partners using the high-content imaging technique and MitoTracker® whereas cytotoxicity (KE3) was determined by CellTiter-Glo® cell viability assay. In contrast to the mechanistic hypothesis underlying the AOP – Inhibition of mtDNA polymerase-, treatment with model compounds for 24 h resulted in an increase rather than a decrease in mtDNA copy number (KE1). Only minor effects on mitochondrial toxicity (KE2) and cytotoxicity (KE3) were observed. Treatment of RPT-EC/TERT1 cells for 14 days showed only a slight decrease in mtDNA copy number after treatment with adefovir dipivoxil and tenofovir disoproxil fumarate, underscoring some of the limitations of short-term in vitro systems. To obtain a first estimation for risk assessment based on in vitro data, potential points of departure (PoD) for each KE were calculated from the obtained in vitro data. The most common PoDs were calculated such as the effect concentra-tion at which 10 % or 20_% effect was measured (EC10, EC20), the highest no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), the benchmark 10 % (lower / upper) concentrations (BMC10, BMCL10, BMCU10) and a modelled non-toxic con-centration (NtC). These PoDs were then compared with serum and tissue concentrations de-termined from in vivo studies after treatment with therapeutic / supratherapeutic doses of the respective drugs in order to obtain a first estimate of risk based on in vitro data. In addition, AOPs were used to test whether the quantitative key event relationships between key events allow prediction of downstream effects and effects on the adverse outcome (AO) based on measurements of an early key event. Predictions of cytotoxicity from the mathematical rela-tionships showed good concordance with measured cytotoxicity after treatment with colistin and polymyxin b nonapeptide. The work also revealed uncertainties and limitations of the ap-plied strategy, which have a significant impact on the prediction and on a risk assessment based on in vitro results.
Paclitaxel (PTX) is one of the leading drugs against breast and ovarian cancer. Due to its low solubility, treatment of the patients with this drug requires a very well-suited combination with a soluble pharmaceutical excipient to increase the bioavailability and reduce the strong side ef-fects. One efficient way to achieve this in the future could be the incorporation of PTX into pol-ymeric micelles composed of poly(2-oxazoline) based triblock copolymers (POL) which ena-bles PTX loadings of up to 50 wt.%. However, structural information at an atomic level and thus the knowledge of interaction sites within these promising but complex PTX-POL formula-tions were not yet available. Such results could support the future development of improved excipients for PTX and suitable excipients for other pharmaceutical drugs. Therefore, a solid-state MAS NMR investigation of these amorphous formulations with different POL-PTX com-positions was performed in this thesis as this gives insights of the local structure at an atomic level in its solid state. NMR in solution showed very broad 13C signals of PTX for this system due to the reduced mobility of the incorporated drug which exclude this as an analytical meth-od.
In a first study, crystalline PTX was structurally characterized by solid-state NMR as no com-plete 13C spectrum assignment and no 1H NMR data existed for the solid state. In addition, the asymmetric unit of the PTX crystal structure consists of two molecules (Z'=2) that can only be investigated in its solid state. As crystalline PTX in total has about 100 different 13C and 1H chemical shifts with very small differences due to Z’=2, and furthermore, its unit cell consisting of more than 900 atoms, accompanying GIPAW (CASTEP) calculations were required for NMR signal assignments. These calculations were performed using the first three available purely hydrous and anhydrous PTX structures, which were determined by XRD and published by Vel-la-Zarb et al. in 2013. Within this thesis, is was discovered that two investigated batches of commercially available PTX from the same supplier both contained an identical and so far un-known PTX phase that was elucidated by PXRD as well as solid-state NMR data. One of the two batches consists of an additional phase that was shown to be very similar to a known hy-drated phase published in 2013.[1] By heating the batch with the mixture of the two phases un-der vacuum, it is transformed completely to the new dry phase occurring in both PTX batches. Since the drying conditions to obtain anhydrous PTX in-situ on the PXRD setup described by Vella-Zarb et. al.[1] were much softer than ours, we identify our dry phase as a relaxed version of their published anhydrate structure. The PXRD data of the new anhydrate phase was trans-ferred into a new structural model, which currently undergoes geometry optimization. Based on solid-state NMR data at MAS spinning frequencies up to 100 kHz, a 13C and a partial 1H signal assignment for the new anhydrous structure were achieved. These results provided sufficient structural information for further investigations of the micellar POL-PTX system.
In a second study, the applicability and benefit of two-dimensional solid-state 14N-1H HMQC MAS NMR spectra for the characterization of amorphous POL-PTX formulations was investi-gated. The mentioned technique has never been applied to a system of similar complexity be-fore and was chosen because around 84% of the small-molecule drugs contain at least one nitrogen atom. In addition, the number of nitrogen atoms in both POL and PTX is much smaller than the number of carbons or hydrogens, which significantly reduces the spectral complexity. 14N has a natural abundance of 99.6% but leads to quadrupolar broadening due to its nuclear spin quantum number I = 1. While this is usually undesirable due to broadening in the resulting 1D 14N NMR spectra, this effect is explicitly used in the 2D 14N-1H HMQC MAS experiment. The indirect 14N measurement can avoid the broadening while maintaining the advantage of the high natural abundance and making use of the much more dispersed signals due to the additional quadrupolar shifts as compared to 15N.
This measurement method could be successfully applied to the complex amorphous POL-PTX mixtures. With increasing PTX loading of the formulations, additional peaks arise as spatial proximities of the amide nitrogens of POL to NH or OH groups of PTX. In addition, the 14N quadrupolar shift of these amide nitrogens decreases with increasing PTX content indicating a more symmetric nitrogen environment. The latter can be explained by a transformation of the trigonal planar coordination of the tertiary amide nitrogen atoms in pure POL towards a more tetrahedral environment upon PTX loading induced by the formation of hydrogen bonds with NH/OH groups of PTX.
In the third and last project, the results of the two abovementioned studies were used and ex-tended by solid state 13C and two-dimensional 1H-13C as well as 1H-1H MAS NMR data with the aim to derive a structural model of the POL-PTX formulations at an atomic level. The knowledge of the NMR signal assignments for crystalline PTX was transferred to amorphous PTX (present in the micelles of the formulations). The 13C solid-state NMR signals were evalu-ated concerning changes in chemical shifts and full widths of half maximum (FWHM) for the different PTX loadings. In this way, the required information about possible interaction sites at an atomic level becomes available. Due to the complexity of these systems, such proximities often cannot be assigned to special atoms, but more to groups of atoms, as the individual de-velopments of line widths and line shifts are mutually dependent. An advantageous aspect for this analysis was that pure POL already forms unloaded micelles. The evaluation of the data showed that the terminal phenyl groups of PTX seem to be most involved in the interaction by the establishment of the micelle for lowest drug loading and that they are likely to react to the change in the amount of PTX molecules as well. For the incorporation of PTX in the micelles, the following model could be obtained: For lowest drug loading, PTX is mainly located in the inner part of the micelles. Upon further increasing of the loading, it progressively extends to-ward the micellar shell. This could be well shown by the increasing interactions of the hydro-phobic butyl chain of POL and PTX, proceeding in the direction of the polymer backbone with rising drug load. Furthermore, due to the size of PTX and the hydrodynamic radius of the mi-celles, even at the lowest loading, the PTX molecules partially reach the core-shell interface of the micelle. Upon increasing the drug loading, the surface coverage with PTX clusters increas-es based on the obtained model approach. The latter result is supported by DLS and SANS data of this system. The abovementioned results of the 14N-1H HMQC MAS investigation of the POL-PTX formulations support the outlined model.
As an outlook, the currently running geometry optimization and subsequently scheduled calcu-lation of the chemical shieldings of the newly obtained anhydrous PTX crystal structure can further improve the solid-state NMR characterization through determination of further spatial proximities among protons using the existing 2D 1H(DQ)-1H(SQ) solid-state MAS NMR spec-trum at 100 kHz rotor spinning frequency. The 2D 14N-1H HMQC MAS NMR experiments were shown to have great potential as a technique for the analysis of other disordered and amor-phous drug delivery systems as well. The results of this thesis should be subsequently applied to other micellar systems with varying pharmaceutical excipients or active ingredients with the goal of systematically achieving higher drug loadings (e.g., for the investigated PTX, the similar drug docetaxel or even different natural products). Additionally, it is planned to transfer the knowledge to another complex polymer system containing poly(amino acids) which offers hy-drogen bonding donor sites for additional intermolecular interactions. Currently, the POL-PTX system is investigated by further SANS studies that may provide another puzzle piece to the model as complementary measurement method in the future. In addition, the use of MD simu-lations might be considered in the future. This would allow a computerized linking of the differ-ent pieces of information with the aim to determine the most likely model.