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Solute carrier (SLC) are related to various diseases in human and promising pharmaceutical targets but more structural and functional information on SLCs is required to expand their use for drug design and therapy. The 7-transmembrane segment inverted (7-TMIR) fold was identified for the SLC families 4, 23 and 26 in the last decade thus detailed analysis of the structure function relationship of one of these families might also yield insights for the other two. SVCT1 and SVCT2 from the SLC23 family are sodium dependent ascorbic acid transporters in human but structural analysis of the SLC23 family is exclusively based on two homologs – UraA from E. coli and UapA from A. nidulans – yielding two inward-facing and one occluded conformation. In combination with outward-facing conformations from SLC4 transporters, and additional information from the SLC26 family, an elevator transport mechanism for all 7-TMIR proteins was identified but detailed mechanistic features of the transport remain elusive due to the lack of multiple conformations from individual transporters.
To increase the understanding of 7-TMIR protein structure and function in this study, the transport mechanism of SLC23 transporters was analyzed by two strategies including selection of alpaca derived nanobodies and synthetic nanobodies against UraA as prokaryotic model protein of the SLC23 family. The second strategy involved mutagenesis of UraA at functional relevant positions regarding the conformational change during transport. Therefore, available structures of 7-TMIR proteins and less related elevator transporters were analyzed and a common motif identified – the alpha helical inter-domain linkers. The proposed rigid body movement for transport in combination with the characteristic alpha helical secondary structure of the linkers connecting both rigid bodies led to the hypothesis of functional relevance of the linkers and a conformational hinge being located in close proximity to the linkers. These positions were identified and used to modulate the biophysical properties of the transporter. Mutagenesis at three relevant positions led to loss of transport functionality and these UraA variants could be recombinantly produced and purified to further examine the underlying mechanistic effects. The variants UraAG320P and UraAP330G from the periplasmic inter-domain linker showed increased dimerization and thermal stability as well as substrate binding in solution. The substrate affinity of UraAG320P was identified to be 5-fold higher compared to the wildtype. The solvent accessibility of the substrate binding site in UraAG320P and UraAP330G revealed reduced open probability that indicated an altered conformational space compared to UraAWT. This phenomenon was analyzed in more detail by differential hydrogen-deuterium exchange mass spectrometry and the results supported the hypothesis of a reduced open probability and gave further insights into the impact of the two mutations in the periplasmic inter-domain linker in UraA.
This thesis further presents strategies for phage display selection of nanobodies with epitope bias and a post selection analysis pipeline to identify nanobodies with desired binding characteristics. Thereby, whole cell transport inhibition highlighted periplasmic epitope binders and conformational selectivity. A cytoplasmic epitope could be identified by pulldown with inside-out membrane vesicles for one cytoplasmic side binder. Thermal stabilization analysis of the target protein in differential scanning fluorometry was performed in presence of two different nanobodies to identify simultaneous binding by additional thermal stabilization respectively competition by intermediate melting temperatures. Combination of epitope information with simultaneous DSF could be used to identify the stabilization of different UraA conformations by a set of binders and presents a general nanobody selection strategy for other SLCs. Synthetic nanobodies (sybodies) were also included in the analysis pipeline and Sy45 identified as promising candidate for co-crystallization that gave rise to UraAWT crystals in several conditions in presence or absence of uracil. Similar crystals could be obtained in combination with UraAG320P that were further optimized to gain structural information on this mutant. The structure was solved by molecular replacement and the model refined at 3.1 Å resolution confirming the cytoplasmic epitope of Sy45 as predicted by the selection pipeline. The stabilized conformation was inward-facing similar to the reported UapA structure but significantly different to the previously reported inward-facing structure of UraA. The structure further confirmed the structural integrity of the UraA mutant G320P. Despite the monomeric state of UraA in the structure, the gate domain aligned reasonably well with the gate domain of the previously published dimeric UraA structure in the occluded conformation and allowed detailed analysis of the conformational transition in UraA from inward-facing to occluded by a single rigid body movement. Thereby little movement in the gate domain of UraA was observed in contrast to a previously reported transport mechanism. Core domain rotation around a rotation axis parallel to the substrate barrier was found to explain the major part of conformational transition from inward-facing to occluded and experimentally supported the hypothesized mechanism by Chang et al. (2017). Additionally, the conformational hinge around position G320 in UraA could be identified as well as the impact of the backbone rigidity introduced by the highly conserved proline residue at position 330 in UraA on the conformational transition. This position was found to serve as anchoring point the inter-domain linker and determines the coordinated movement of inter-domain linker and core domain. The functional analysis further highlighted the requirement of alpha helical secondary structure within the inter-domain linker that serves as amphipathic structural entity that can adjust to changed core-gate domain distances and angles during transport by extension/compression or bending while preserving the rigid linkage.
The applied strategies to modulate the conformational space of UraA by mutagenesis at the hinge positions in the inter-domain linkers is transferrable to other transporters and might facilitate their structural and functional characterization.
Further, this study discusses the conformational thermostabilization of UraA that is based on increased melting temperatures upon restriction of its conformational freedom. The term ‘conformational thermostabilization’ introduced by Serrano-Vega et al. (2007) could be experimentally supported and the direct correlation between the conformational freedom and thermostabilization was qualitatively analyzed for UraA. The concept of conformational thermostabilization might help in characterization of other dynamic transport systems as well.
The overall survival for patients with acute lymphoblastic leukemia (ALL) often is the function of age, in particular in 2019 analysis revealed that 5-year overall survival for patients older than 20 years remains below 35% (American Cancer Society, Cancer Facts &Figures 2019). Importantly, one of the major issues in ALL therapy is the ability of tumor cells to escape the treatment via the establishment of an immunosuppressive environment. The tumor microenvironment has gained tremendous importance in the past decade. This is largely based on the reasoning that, in order to devise better therapeutic strategies for patients, we need to gain better understanding into how malignant cells transform their microenvironment to promote growth, escape immune control and gain therapeutic resistance.
TAM receptors (TAMRs) are engaged in innate immune cells as a feed-back mechanism to terminate the immune response and promote the return to homeostasis (Rothlin et al. 2007). In the context of cancers, aberrant TAMR signaling was mainly explored concerning its pro-oncogenic function (Paolino and Penninger 2016). There are only limited data available suggesting the modulation of cancer immune response via TAMR signaling in highly immunogenic solid tumor models (Paolino et al. 2014; Ubil et al. 2018). So far, however, little is known about their potential indirect immune-modulatory function in hematological malignancies. Taking into account the pronounced importance of TAMR signaling in immune cells combined with the leukemic immune tolerance, the current study focused on the function of TAMR and their ligands in anti-leukemic immunity.
This work uncovers the mechanism of dampening anti-leukemic immune response via TAMR signaling on macrophages using the syngeneic BCR-ABL1 B-ALL mouse model. Using genetic depletion of GAS6 in the host environment or ablation of AXL and/or MERTK receptors in macrophages the bone marrow microenvironment could be rewired in order to achieve an efficient anti-leukemic immune response. In particular, the GAS6/AXL blockade triggers an effective NKand T- cell-dependent anti-leukemic response that results in prolonged survival. This finding specifically tackles the obstacle of inefficient bridging between innate and adaptive immune response typical for hematological malignancies in contrast to solid tumors (E. K. Curran, Godfrey, and Kline 2017).
Besides establishing the vital function of TAMR signaling in anti-leukemic immunity using murine models, the analysis of human blood plasma revealed that age-related immune dysregulation was manifested by significant GAS6 decrease and PROS1 upregulation among elderly donors (>60 y.o.) compared to controls (<25 y.o.). These data are indicative that TAMR signaling likely favors the age-dependent immune system decline, which in turn is associated with a poor survival rate of elderly patients diagnosed with leukemia.
In conclusion, using a preclinical ALL model here it was identified in vivo, that Axl significantly increases upon B-ALL challenge in Mph and NK cells. Therefore, AXL targeting, using the orally bioavailable selective inhibitor Bemcentinib, could serve as a powerful approach to revert early immunosuppression created by leukemia.
Taken together these data propose the AXL receptor as a novel immune checkpoint and attractive candidate for the development of a new therapeutic approach via unleashing the patient’s own immune system to combat leukemic cells.
For private investors it is imperative to a) understand and define their own, individual risk preferences, b) assess their financial and demographic circumstances to determine the individual risk-taking potential, and c) form and maintain a well-diversified risky portfolio. The three chapters of my thesis each match one of these three tasks. \\ \noindent The first chapter of my thesis presents novel experimental evidence to test the existence of a potential projection bias in loss aversion, a significant determinant of investor preferences, thus matching task a). The second chapter is devoted to the determination of private investors' risk-taking potential based on their financial and socio-demographic circumstances, matching task b): In a large portfolio experiment, we examine the ability and heterogeneity of lay and professional advisors in matching investor demographics, such as age and income, with risky asset portfolio shares. The third and final chapter addresses the question on how to reach and maintain an efficient risky portfolio, therefore matching task c): It analyzes a decision support system for private investors that allows its users to simulate any arbitrary set of securities, and by reporting aggregated expected return and risk, to optimize their current portfolio.
Droughts impair plant growth, limit global net primary production and are predicted to increase in the course of climate change. Knowledge of the plant drought response on a molecular level can facilitate the selection of drought resistant genotypes and genetic engineering and thereby can help to implement strategies, such as assisted migration projects or crop improvement, in order to preserve natural and agricultural vegetation against droughts.
Studies on gene expression under drought stress were conducted in three species each of the genera Quercus and Panicum, to shed light on the molecular drought response in these species and identify drought responsive genes as a basis for technical applications.
In the genus Quercus, gene expression studies were conducted in the three major European forest trees Q. ilex, Q. pubescens and Q. robur, for which a distributional shift caused by climate change is predicted for the 21st century. RNA-Seq experiments were conducted in the three Quercus species for the first time, ortholog groups were assigned and unregulated genes, as well as drought responsive genes, were identified (Madritsch et al. 2019). For a set of the unregulated genes, a stable expression over the course of long-term drought periods was evaluated in order to enable an application as reference genes for normalizing qRT-PCR experiments (Kotrade 2019a). The reference genes were used in subsequent experiments to generate gene expression profiles over the course of a two-year drought experiment with consecutive drought periods for a set of twelve drought responsive genes and revealed a highly variable gene regulation under long-term drought stress in the Quercus species (Kotrade et al. 2019b).
In the genus Panicum, the gene expression in response to drought was examined in the two wild crop species, P. laetum and P. turgidum, and in the less drought tolerant species P. bisulcatum via RNA-Seq experiments (Kotrade et al. 2020 (in revision). The transcriptomes of the species were sequenced for the first time, ortholog groups were assigned and the gene regulation was compared across the species. The common grounds of the drought response in Panicum were determined by identifying similarities across the species, while the identification of differences between the species led to genes that might contribute to the higher drought tolerance of P. laetum and P. turgidum
A comparison across the two genera showed large differences in the gene regulation upon drought. This might be largely explained by different experimental setups that resulted in different drought conditions in the genera, such as drought intensity, drought duration and velocity of drought development.
The sequence information and the drought responsive genes identified in the Quercus and Panicum species can be used to develop marker assays for marker-assisted selection. The genes that putatively contribute to the higher drought tolerance of the two wild crop Panicum species should be considered as candidate targets in genetic engineering studies. Marker-assisted selection and genetic engineering can be applied, for example, in assisted migration projects to support natural vegetation in the course of climate change or to breed more drought tolerant crop strains to mitigate crop failure rates caused by droughts.
This work describes development of a comprehensive methodology for analyzing vibro-acoustic and wear mechanisms in transmission systems. The thesis addresses certain gaps present in the fields of structure dynamics and abrasion mechanism and opens new areas for further research.
The paper attempts to understand new and relatively unexplored challenges like influences of wear on the dynamics of drive train. It also focuses on developing new techniques for analyzing the vibration and acoustic behavior of the drive unit structures and surrounding fluids respectively.
The developed methodology meets the requirements of both the complete system and component level modeling by using specially identified combination of different simulation techniques. Based on the created template model, a three-stage spur plus helical gearbox is constructed and simulated as an application example. In addition to the internal mechanical excitation mechanisms, the transmission model also includes the rotational and translational dynamics of the gears, shafts and bearings. It is followed by illustration of wear among the rotating components.
Different kinds of static and dynamic analyses are performed and coupled at various levels depending on the mechanical complexities involved. Furthermore, the structure dynamic vibration of the housing and the associated sound particle radiations are mapped into the surrounding fluid. Additionally, the approach for selection of the potential parameters for optimization is depicted. Final part focuses on the measurements of different system states used for validation of the model. In the end, results obtained from both simulations and experiments are analyzed and assessed for there respective performances.
Machine Learning (ML) is so pervasive in our todays life that we don't even realise that, more often than expected, we are using systems based on it. It is also evolving faster than ever before. When deploying ML systems that make decisions on their own, we need to think about their ignorance of our uncertain world. The uncertainty might arise due to scarcity of the data, the bias of the data or even a mismatch between the real world and the ML-model. Given all these uncertainties, we need to think about how to build systems that are not totally ignorant thereof. Bayesian ML can to some extent deal with these problems. The specification of the model using probabilities provides a convenient way to quantify uncertainties, which can then be included in the decision making process.
In this thesis, we introduce the Bayesian ansatz to modeling and apply Bayesian ML models in finance and economics. Especially, we will dig deeper into Gaussian processes (GP) and Gaussian process latent variable model (GPLVM). Applied to the returns of several assets, GPLVM provides the covariance structure and also a latent space embedding thereof. Several financial applications can be build upon the output of the GPLVM. To demonstrate this, we build an automated asset allocation system, a predictor for missing asset prices and identify other structure in financial data.
It turns out that the GPLVM exhibits a rotational symmetry in the latent space, which makes it harder to fit. Our second publication reports, how to deal with that symmetry. We propose another parameterization of the model using Householder transformations, by which the symmetry is broken. Bayesian models are changed by reparameterization, if the prior is not changed accordingly. We provide the correct prior distribution of the new parameters, such that the model, i.e. the data density, is not changed under the reparameterization. After applying the reparametrization on Bayesian PCA, we show that the symmetry of nonlinear models can also be broken in the same way.
In our last project, we propose a new method for matching quantile observations, which uses order statistics. The use of order statistics as the likelihood, instead of a Gaussian likelihood, has several advantages. We compare these two models and highlight their advantages and disadvantages. To demonstrate our method, we fit quantiled salary data of several European countries. Given several candidate models for the fit, our method also provides a metric to choose the best option.
We hope that this thesis illustrates some benefits of Bayesian modeling (especially Gaussian processes) in finance and economics and its usage when uncertainties are to be quantified.
Across the entire animal kingdom, sociality, i.e. the tendency of individual animals to form a group with conspecifics, is a common trait. Environmental changes have to be met with corresponding, quick adaptations. For social species, the presence of conspecifics is important for survival and if social animals are deprived of access to conspecifics, this can lead to strong and lasting changes on a physiological level as well as behaviour. Gene expression changes responsible for these adaptations have so far not been understood in detail. As social isolation leads to changes on a neuronal level, it is important to investigate the gene expression changes that are induced in the brain. In this thesis, next-generation RNA-sequencing was applied to zebrafish, a well-established model organism characterized by its high degree of companionship. Within the entire brain, gene expression was analysed in zebrafish that were raised either with conspecifis or in isolation, ranging from 5 to 21 days post fertilization. Using this approach, several genes were identified that were downregulated by social isolation. In this thesis, I focused on one of these consistently downregulated genes, parathyroid hormone 2 (pth2). The expression of pth2 was demonstrated to be bidirectionally regulated by the number of conspecifics present and to be responsive to changes in the social environment within 30 minutes. Regulation of pth2 does not occur by visual or chemosensory access to conspecifcs, but is mediated by mechanosensory perception of other fish via the lateral line. In an experiment using an artificial mechanical stimulation paradigm, it was shown that the features necessary to elicit pth2 transcription closely mimick the locomotion of actual zebrafish. Other, similar stimulation paradigms are not capable to induce this transcriptional response.
BH3 mimetics are novel anticancer therapeutics that induce apoptosis by targeting anti‐apoptotic BCL‐2 proteins. Highly specific inhibitors of the main anti-apoptotic proteins BCL-2, BCL‐XL and MCL‐1 promise new opportunities for the treatment of AML. However, it is currently unclear which of these anti-apoptotic BCL-2 proteins represents the most promising target in AML. Therefore, we investigated the effect of BH3 mimetics targeting either BCL-2 (ABT-199, S55746), BCL-XL (A-1331852) or MCL-1 (S63845) on eleven AML cell lines. Drug sensitivity screening revealed heterogeneous sensitivity towards the different BH3 mimetics, with the best responses observed upon targeting of MCL-1. Selected cell lines that displayed sensitivity towards the specific BH3 mimetics underwent intrinsic apoptosis, which was characterized by loss of mitochondrial membrane potential, exposure of phosphatidylserine and activation of caspases. Furthermore, S63845 turned out to displace BIMS and NOXA from MCL-1 to induce apoptotic cell death. Importantly, the translational relevance of this study was demonstrated by experiments in primary AML blasts, which displayed similar sensitivity towards BH3 mimetics as the cell lines did. Additionally, experiments with nonmalignant cells could confirm the clinical relevance of the MCL-1 inhibitor. There we could show, that S63845 does not cause cytotoxicity on HPCs at efficacious doses.
In conclusion, our findings reveal that the inhibition of BCL-2 proteins, especially MCL-1, by BH3 mimetics can be a promising strategy in AML treatment.
The present study approached two related but conceptually different questions of EV biology in cancer. In both approaches, tailored variants of the Cre LoxP system were utilized. First, in the context of intradermal and intracranial tumours, it was examined which cells in the tumour microenvironment (TME) take up tumour derived
EVs and what effects EV uptake has on recipient cells. Secondly, in the context of glioma, peripheral macrophages (MF) were directly traced to the brain and
separated from brain resident microglia (MG). Furthermore, EV signalling between these entities was analysed.
Regarding the first approach, multidirectional transfer of functional Cre recombinase RNA in intradermal and intracranial mouse tumour models was observed. In spite of robust recombination rates in all tumour models, the total number of EV-uptaking cells is around three times higher than the total number of recombined cells, suggesting that interactions of cells and EVs which contain CremRNA does not necessarily lead to marker gene expression. Subsequent studies can build up on this established system and isolate and characterise EV-uptaking cells to identify geno- and phenotypical changes induced by EV uptake.
The second, conceptionally different aspect that was investigated in this study is the distinction and tracing of peripheral MF to the brain and their distinction from
brain resident MG in glioma. Glioblastoma multiforme (GBM) is the most common and the most malignant brain tumour. The average patient survival of 15 months
past diagnosis did not change much during the last decades, which stresses the need for new therapies. GBM location in the immune privileged brain, its characteristically highly immune suppressive TME and its highly invasive growth
makes this disease so difficult to treat. Immune therapies, which in general show good results in other types of cancer, are not effective in GBM. To a great extent, this can be ascribed to the lack of understanding of MG and MF function in GBM and their roles in tumour progression.