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Characterization of a P2X7 BAC reporter mouse model and investigation of the P2X4/P2X7 interaction in mouse lung tissue
Get PDFP2X receptors (P2XR) comprise a family of ATP-gated, non-selective cation channels. Out of the seven members of the P2XR family (P2X1R-P2X7R), P2X7R presents important characteristics that make it unique. These are a) the low affinity for the ligand ATP, b) its long, intracellular C-terminus and c) the ability to induce the formation of a macropore under sustained stimulation that allows the passage of large molecules and that may eventually lead to cell death. The best characterized function of the P2X7R is the induction of NLRP3 inflammasome assembly for the activation of caspase-1 and release of pro-inflammatory cytokines IL-1beta and IL-18.
The P2X7R is encoded by the P2rx7 gene and is widely expressed in immune cells, where it has proved relevance as a drug target due to its role in inflammation and its demonstrated modulatory effects in various diseases. For example, P2X7R involvement has been found in infectious diseases, autoimmune disorders, and cancer. In the central nervous system, the P2X7R was shown to modulate neuroinflammation.
However, the cell type-specific localization and functions of the P2X7R in the central nervous system are incompletely understood. While its large intracellular C-terminus has been proposed to serve as a platform for the interaction with mediators of signaling pathways, details about the identity of these interaction partners, the signaling mechanisms and functional relevance of such interactions remain largely unknown. A physical interaction of P2X7R with the P2X4R subtype has been suggested, although proof of this interaction is mostly restricted to heterologous overexpression systems and evidence for this interaction in vivo is insufficient.
In an attempt to address these questions, two transgenic P2X7R reporter mouse models have been generated that express EGFP under the control of the endogenous P2rx7 promoter. One of them expresses a soluble EGFP (sEGFP mouse) and the other one expresses a P2X7-EGFP fusion protein (P2X7-EGFP mouse). However, preliminary data revealed divergent distribution of the reporter proteins in the mouse brain.
In this project, a detailed characterization and comparison of both BAC transgenic mouse models was performed. Biochemical and histochemical approaches have been applied to compare the P2X4 and P2X7 expression levels in both mouse models and determine the cell-specific distribution of the EGFP reporter. In the P2X7-EGFP mouse model, this study confirmed that the endogenous P2X7 and P2X4 expression levels were unaffected and that the overexpressed fusion protein is found in microglia and oligodendrocytes. However, analysis of P2X4 and P2X7 levels in the sEGFP mouse model revealed an unexpected overexpression of both proteins, which is explained by the use of a different BAC clone and the respective recombination strategy. Immunohistochemistry experiments demonstrated divergent expression patterns of P2X7 and the sEGFP reporter and revealed its predominant localization in neurons. In conclusion, these results unveiled and explained inconsistencies in the reporter expression of the sEGFP mouse model.
In a second part of this thesis, I investigated the reported interaction between P2X7R and P2X4R using the transgenic P2X7-EGFP mouse model. In a biochemical approach the interaction of both receptors was evaluated by pull-down via EGFP-tag. Control experiments were performed upon expression of both subunits in Xenopus laevis oo-cytes and HEK293 cells. Our results confirmed a previously shown P2X4R-P2X7R interaction in X. laevis oocytes but revealed no proof of such an interaction in mouse lung and HEK293 cells. The possibility of a functional interrelation between P2X4R and P2X7R was further explored by comparison of the expression levels in both subunits in wildtype, P2X7-EGFP overexpressing mice and P2rx4-/- and P2rx7-/- knockout mouse models. Together, these data did not support an interaction or mutual interrelation of both receptors in the mouse lung.
Lastly, a preliminary comparative proteomic study was performed with wildtype, P2rx7-/- and P2X7-EGFP mice to study P2X7R signaling. To this aim, protocols for sample preparation were tested and samples from mouse hippocampus were submitted to liquid chromatography coupled to mass spectrometry (LC-MS) analysis. However, statistical analysis did not identify significantly enriched proteins. Therefore, a cell-specific approach was considered more promising and a protocol for microglia isolation from adult mouse brain was tested for future proteomic studies.
In summary, this study provided the first detailed characterization of the sEGFP P2X7 reporter mouse model and revealed important caveats for its use in basic research. It further showed evidence against the proposed interaction between P2X7R and P2X4R and finally describes a comparative proteomics approach to investigate P2X7R signaling cascades
Establishing multiplex in vivo assays in a xenograft mouse model of acute leukaemia to correlate preclinical treatment trials with molecular screens
Get PDFAcute leukaemia is a rare disease, but the number of cases reported each year is steadily increasing. Standard therapy includes classical chemotherapy, which most patients respond to at the beginning. However, a major problem is relapse, which is associated with a poorer prognosis, so better treatment options are urgently needed.
With advances in sequencing technology, more and more molecular features can be identified. As a result, patients benefit from more specific diagnostics and can be matched to potential specific treatment options. The number of targeted inhibitors has been increasing for years, and while there are many drugs existing, the biomarker to link the needed drug to the patient is not yet available.
In the present study, the results of molecular dropout screens were used as a basis. The aim of the present study was to test the effect of selected targeted therapies against five PDX ALL models with different molecular alterations. Specific inhibitors were used to determine whether the molecular screen could predict a response to an inhibitor and results were correlated with the existing data from molecular screens.
In order to preclinically test novel therapies, this work aimed to increase the efficiency of preclinical therapy trials. I was able to establish a multiplex protocol for in vivo treatment trials, allowing up to five ALL-PDX samples to be tested simultaneously in competitive in vivo trials. Flow cytometry was used to detect individual samples and investigate response towards targeting drugs. With this method, I was able to test three targeted therapies in vivo, while reducing the required number of mice by a factor of five, in line with the 3R concept.
Detected dependencies in vivo were BCL2, MCL1 and XPO1. The correlation of in vivo therapy trials with the molecular functional results was high, with a positive correlation in three of five samples for all tested target / drug pairs, one sample with two positive correlations, and only one sample with no correlation between the two independent approaches. However, the PDX samples responded differently to the specific inhibitors, which highlights the concept of personalised medicine. Furthermore, the results state that the screen overestimates the efficacy of the drug
Machine learning and data-driven techniques for verification and synthesis of cyber-physical systems
Get PDFSafety and performance are the most important requirements for designing and manufacturing complex life-critical systems. Consider a self-driving car which is not equipped with certain safety functionalities. It can cause fatal accidents, severe injuries, or serious damages to the environment. Hence, rigorous analysis required to ensure the correctness of functionalities in many safety-critical applications.
Model-based approaches for satisfying such requirements have been studied extensively in the literature. Unfortunately, a precise model of the system is not always available in many practical scenarios. Hence, in this thesis we focus on data-driven methods and machine learning techniques to tackle this challenge.
First, we assume that only an incomplete parameterized model of the system is available. The main goal is to study formal verification of linear time-invariant systems with respect to a fragment of temporal logic specifications when only a partial knowledge of the model is available, i.e., a parameterized model of the system is known but the exact values of the parameters are unknown. We provide a probabilistic measure for the satisfaction of the specification by trajectories of the system under the influence of uncertainty. We assume that these specifications are expressed as signal temporal logic formulae and provide an approach that relies on gathering input-output data from the system. We employ Bayesian inference on the collected data to associate a notion of confidence with the satisfaction of the specification.
Second, we assume that we do not have any knowledge about the model of the system and just have access to input-output data from the system. We study verification and synthesis problems for safety specifications over unknown discrete-time stochastic systems. When a model of the system is available, notion of barrier certificates have been successfully applied for ensuring the satisfaction of safety specifications. Here, we formulate the computation of barrier certificates as a robust convex program (RCP). Solving the acquired RCP is difficult in general because the model of the system that appears in one of the constraints of the RCP is unknown. We propose a data-driven approach that replaces the uncountable number of constraints in the RCP with a finite number of constraints by taking finitely many random samples from the trajectories of the system. We thus replace the original RCP with a scenario convex program (SCP) and show how to relate their optimizers. We guarantee that the solution of the SCP is a solution of the RCP with a priori guaranteed confidence when the number of samples is larger than a specific value. This provides a lower bound on the safety probability of the original unknown system together with a controller in the case of synthesis.
Lastly, to address the high demand for data in our data-driven barrier-based approach, we propose three remedies. First, the wait-and-judge approach that checks a condition over the optimal value of the SCP using a fixed number of samples, ensuring a lower bound probability and the desired confidence for satisfying safety specifications. Second, the repetition-based scenario framework that iteratively solves the SCP with samples, checking feasibility and achieving the desired violation error. A safety condition is verified, enabling the computation of a lower bound for safety satisfaction. Third, the wait, judge, and repeat framework that solves the SCP iteratively until a feasibility condition, based on computed support constraints, is met. If the safety condition is satisfied, the system is considered safe with a lower bound probability determined using the optimizer of the successful iteration
Rolle der Makrophagendifferenzierung und -interaktion mit regulatorischen T-Zellen bei der Progression und Regression der HPV-induzierten Zervixdysplasie
Get PDFDie Roller des Wilms Tumor 1 Gens in der akuten myeolischen Leukämie
Get PDFDie akut myeloische Leukämie (AML) ist die häufigste Form akuter Leukämien im Erwachsenenalter und wird durch genetische Aberrationen verursacht. Im Rezidiv kann bei 10% der Patienten eine Akquirierung von Wilms Tumor 1 Gen (WT1) Mutationen festgestellt werden. WT1 ist beim Nephroblastom als Tumorsuppressor bekannt. Loss-of-Function-Mutationen im Sinne eines Tumorsuppressorgens können zur Resistenzbildung in der AML beitragen. WT1 Mutationen bei AML-Patienten zeigen eine ungünstigere Prognose in Form eines schlechteren klinischen Outcomes bedingt durch ein vermehrtes Auftreten von Rezidiven.
Eine Zunahme von WT1 Mutationen im Rezidiv weist daraufhin, dass WT1 eine Rolle bei der Induktion von Resistenzen in der AML spielt. Wir wollen in der vorliegenden Arbeit den Einfluss von WT1 auf Therapieresistenzen in der AML in Modellsystemen untersuchen.
Zur Untersuchung der Rolle von WT1 in der AML haben wir zum einen ein WT1 Knockout Zell-Modell und zum anderen ein Re-expressions-Zell-Modell entwickelt. Mit Hilfe von CRISPR/Cas9 konnte zunächst ein Gen-Knockout von WT1 in K562 Zellen eingeführt werden. Anschließend wurde mittels PiggyBac (PB) Vektorsystems WT1 Wildtyp oder WT1 Mutanten (R430X, D464G) in K562 WT1 Knockout-Zellen re-exprimiert. Anhand von Viabilitäts-Assays wurden Dosis-Wirkungs-Kurven erstellt. Die Interaktionsfähigkeit von WT1 und WT1 Mutanten mit wichtigen Proteinpartnern (TP53, TET2 und STAT3) wurde in einer Immunpräzipitation (IP) untersucht.
Im Knockout-Modell konnte kein Unterschied in der Viabilität von WT1 Wildtyp versus WT1 Knockout unter Behandlung mit den Chemotherapeutika Cytarabin (Ara-C), Daunorubicin und Decitabin gezeigt werden. Ebenso führte die Re-expression von WT1 Wildtyp im Vergleich zu WT1 Knockout zu keinem signifikanten Unterschied der Dosis-Wirkungs-Kurven. Bei der Re-expression von WT1 Mutanten versus WT1 Knockout wurde ein signifikanter Unterschied in der Viabilität unter Ara-C und Decitabin Behandlung festgestellt. Eine Interaktion von WT1 Wildtyp und den WT1 Mutationen mit den Interaktionspartnern TP53, TET2 und STAT3 konnte in der IP nachgewiesen werden.
Im Zellmodell konnten wir unter bestimmten Dosierungen von Ara-C und Decitabin ein besseres Überleben von Zellen mit WT1 Mutationen im Vergleich zum Knockout von WT1 zeigen. Diese Daten weisen darauf hin, dass WT1 ein Resistenzfaktor in der klonalen Evolution der AML darstellt und als therapeutische Zielstruktur bei Patienten mit resistenter Erkrankung geeignet sein könnte
A cognitive and computational model of the sound change from pre- to post-aspiration in Andalusian Spanish
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Biokompatibilität und Zytotoxizität von 3D-gedruckten zahnärztlichen Werkstoffen
Get PDFDurch die Nutzung des dreidimensionalen Drucks in der Zahnmedizin, wurden die Methoden zur Herstellung zahnärztlicher Werkstücke erweitert und gleichzeitig eine Vielzahl neuer Materialien auf den Markt gebracht. Ziel der Studie war es, Materialien für den 3D Druck von provisorischen zahnärztlichen Versorgungen hinsichtlich ihrer Wirkung auf gingivale Keratinozyten zu untersuchen. Dabei standen vor allem Einflüsse auf die Vitalität der Zellen, sowie auf die Produktion proinflammatorischer Zytokine im Zentrum.
Untersucht wurden vier verschiedene 3D Druck Materialien auf Harzbasis (Nextdent C&B MFH, GC Temp PRINT, Freeprint Temp UV, Deltamed 3Delta Temp) im Vergleich mit einem subtraktiv (fräsend) zu bearbeitenden CAD/CAM Material und einem konventionellen Provisorienkunststoff. Die vorkultivierten Keratinozyten wurden durch Eluate in Kontakt mit den Materialien gebracht. Veränderungen der Zellvitalität wurden mittels XTT ermittelt, während die proinflammatorischen Zytokine IL6 und IL8 mit Hilfe eines ELISAs gemessen wurden.
Dabei zeigten die gedruckten Materialproben im Vergleich zur Kontrolle und den CAD/CAM Proben im XTT-Test signifikant geringere Vitalitätswerte, sowie eine Hemmung der Expression der proinflammatorischen Interleukine 6 und 8. Die untersuchten 3D-Druck Materialien mit dem geringsten Füllkörperanteil, sowie dem Inhaltsstoff HEMA (MFH und Freeprint Temp UV) ließen, verglichen mit den anderen Materialien für die additive Fertigung durch 3D Druck, die geringsten Effekte auf die biologischen Zellfunktionen erkennen. Materialien mit höheren Anteilen an Füllkörpern zeigten demgegenüber eine raschere Reduktion der Zellvitalität und Expression von Interleukin 6 und 8, wobei das Material mit den meisten Füllkörpern die Zellfunktion am schnellsten beeinträchtigt. Somit scheint die Höhe des Füllkörperanteils, sowie die Monomerzusammensetzung maßgeblich an der Beeinflussung der Zellvitalität und Interleukinproduktion beteiligt zu sein. Ein geringer Füllkörperanteil in Kombination mit der Abwesenheit der beiden Monomere TEGDMA und UDMA scheint die geringste Beeinträchtigung der biologischen Funktionen auszulösen.The use of three-dimensional printing in dentistry has expanded the methods for producing dental workpieces and, at the same time, introduced a variety of new materials to the market. The aim of the study was to investigate materials for 3D printing of temporary dental restorations with regard to their effect on gingival keratinocytes. The main focus was on influences on the vitality of the cells and on the production of proinflammatory cytokines. Four different resin-based 3D printing materials (Nextdent C&B MFH, GC Temp PRINT, Freeprint Temp UV, Deltamed 3Delta Temp) were investigated in comparison with a subtractive CAD/CAM material and a conventional temporary resin. Pre-cultured keratinocytes were brought into contact with the materials by eluates. Changes in cell viability were determined by XTT, while proinflammatory cytokines IL6 and IL8 were measured by ELISA. Thereby, the printed material samples showed significantly lower vitality values, as well as inhibition of the expression of the proinflammatory interleukins 6 and 8, compared to the control and the CAD/CAM samples in the XTT assay. The investigated 3D printed materials with the lowest filler content, as well as the ingredient HEMA (MFH and Freeprint Temp UV) suggested the lowest effects on the biological cell functions, compared to the other materials for additive manufacturing by 3D printing. In contrast, materials with higher levels of filler showed a more rapid reduction in cell viability and expression of interleukin 6 and 8. The material with the highest filler content was affecting cell function the most rapidly. Thus, the level of filler content, as well as the monomer composition, appears to be significantly involved in affecting cell viability and interleukin production. A low filler content in combination with the absence of the two monomers TEGDMA and UDMA seems to trigger the least impairment of biological functions