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Die Rolle des NLRP3 Inflammasoms in Podozyten bei der strukturellen Anpassung an Diabetes-induzierte glomeruläre Hyperfiltration
Get PDFAdvancing single-molecule imaging analysis via deep learning
Get PDFSingle-molecule experiments have revolutionized our understanding of the physical world, offering unparal- leled insights into dynamic processes. However, a bottleneck persists in the time-consuming and potentially biased nature of data analysis. The main goal of this thesis is to address these issues through the devel- opment of deep learning techniques tailored for the analysis of fluorescence data specifically focusing on surface-based single-molecule measurements of Förster resonance energy transfer (FRET). The culmination of this effort is Deep-LASI (Deep-Learning Assisted Single-molecule Imaging analysis), a software suite lever- aging the predictive capabilities of deep neural networks (DNNs). Designed for rapid analysis of single-color, two-color FRET, and three-color FRET data, Deep-LASI fully automates trajectory sorting and FRET correc- tion factor determination, followed by the automated prediction of observed states and state dwell times for each molecule. The pre-trained ensemble of DNNs are able to analyze previously unseen data sets in approxi- mately 20–100 ms per trajectory. In extensive benchmarking, the DNNs demonstrated their efficacy through ground truth simulations and comparisons with manually analyzed experimental data, validated by expert users. Beyond the development of these deep learning techniques, Deep-LASI has evolved into a compre- hensive software suite that provides robust methods for extracting raw intensity data from single-molecule movies across multiple channels. A key feature was the integration of alternative approaches for user in- tervention, applicable to every step that the DNNs undertake automatically. This user-centric framework of Deep-LASI encompasses human evaluation of single-molecule trajectories, offering flexibility to override DNN classifications, and the option to employ hidden Markov models (HMM) for the analysis of kinetic rates, along with various downstream analysis methods post trajectory sorting. Additionally, the evaluation of various software tools for extracting kinetic rate constants from single-molecule FRET trajectories is pre- sented in this thesis. By analyzing specific data sets with different levels of complexity, the comparison of all employed methods shed light on their limitations and revealed important aspects that need to be considered for consistent analysis results. Lastly, established computational methods were coupled with experimen- tal data to elucidate the conformational dynamics of bacterial adhesin SdrG, which can form an extremely mechanostable complex with its target peptide human fibrinogen β (Fgβ). The SdrG:Fgβ complex can with- stand forces greater than 2 nN, representing the strongest non-covalent bond of known to date. Combining molecular dynamics simulations with single-molecule FRET measurements provided new insights into the behavior of the locking strand and ligand-induced structural changes of the SdrG protein
Characterisation of DNA-protein crosslink repair with the Purification of x-linked Proteins technique
Get PDFDNA-Protein Crosslinks (DPCs) have emerged as an important source of endogenous and exogenous DNA damage. DPCs arise when proteins get covalently trapped on DNA, what can happen during the action of enzymes that naturally establish covalent intermediates with DNA -such as topoisomerases- but also by the action of reactive compounds. One of these compounds, formaldehyde, is an environmental toxin and a metabolite produced during one-carbon metabolism and methanol detoxification. Formaldehyde is extremely reactive and generates protein-protein crosslinks, RNA-protein crosslinks and DPCs. DPCs are toxic because they can block essential chromatin transactions such as replication or transcription. Their toxicity has been exploited in chemotherapy with the use of different drugs that induce these genomic lesions either specifically, camptothecin (CPT) traps TOP1 or etoposide traps TOP2, or unspecifically, with compounds like cisplatin commonly used in breast cancer treatment.
DPC-repair generally involves specialized proteases which mediate the destruction of the protein adduct. In higher eukaryotes, the metalloprotease SPRTN is recruited to the lesion in a process that involves the collision of the replication fork with the adduct. SPRTN is activated by the DNA structure generated by polymerase stalling, cleaves the protein and allows peptide bypass by translesion synthesis (TLS). The study of DPC-repair and the identification of DPCs generated by non-specific crosslinkers has remained elusive due to limited methodology to study these lesions. Previous methods to isolate DPCs, rely on DNA precipitation and are prone to technical biases due to the presence of protein aggregates, giving false positives, or changes in DNA precipitation behaviour due to the variable crosslinked protein nature, giving false negatives. Therefore, I established during my master thesis, and optimized during my PhD, a method for the isolation, identification and monitoring of DPCs, the Purification of x-linked Proteins (PxP). Using PxP in combination with mass spectrometry, I uncovered the identity of DPCs generated by physiologically relevant levels of formaldehyde. Strikingly, formaldehyde-induced DPCs are less complex than anticipated, they mostly consist of crosslinked nucleosomes. Then, we decided to apply PxP for the study of 5-azadC-induced DPCs, which consists mostly of the DNA Methyl Transferase 1 (DNMT1). We observed DNMT1-DPC formation in a dose-dependent manner and could monitor their repair kinetics. We discovered that repair of this crosslinks involved the SUMOylation of the adduct and SUMO-targeted ubiquitylation by the StUbL RNF4, which triggered proteasomal degradation of crosslinked DNMT1. In agreement with biochemical data and previous work, RNF4 knock-out cells displayed 5-azadC sensitivity.
Interestingly, we observed a cleaved DNMT1-DPC fragment whose appearance was dependent on the same modifications -SUMO and ubiquitin- but not generated by the proteasome. We identified SPRTN as the enzyme responsible for this cleavage and, through structure function analysis, conclude that the UBZ domain is responsible for the recruitment of SPRTN to DNMT1-DPCs. Strikingly, only the loss of the UBZ domain completely phenocopied the SPRTN-ΔC allele, which is causative for Ruijs-Aalfs syndrome. Our data support a replication-independent role for the metalloprotease SPRTN during DPC-repair and highlight the importance of the UBZ domain during lesion recognition.
Given the multiple applications of the PxP for the study of DPC biology, we decided to write an article in which we described the method step by step to facilitate its implementation in other labs. We show that the PxP can be used to isolate also other types of DPCs such as those generated by non-competitive inhibitors (e.g etoposide) and that it can successfully isolate relevant physiological DPCs, like those generated by HMCES after short wavelength ultraviolet light (UVC) exposure. In conclusion, we believe that this manuscript will be a great resource for laboratories which do research on the DNA damage field
Modellierung von mit Lahmheit assoziierten Faktoren bei Milchkühen in drei milchkuhdichten Regionen Deutschlands unter besonderer Berücksichtigung regionsspezifischer Strukturen
Get PDFEinfluss mechanischer und chemischer Oberflächenbehandlung auf die Verbundfestigkeit von verschieden gereinigtem 3D gedrucktem Harz
Get PDFGeschlechtsspezifische Zusammenhänge zwischen Blutdruck, Blutgerinnung und Blutlipiden
Get PDFKardiovaskuläre Erkrankungen stellen weltweit und vor allem in industrialisierten Ländern die Hauptursachen für Morbidität und Mortalität dar und gehören damit zu den global bedeutendsten Erkrankungen.
Das kardiovaskuläre Risiko bezeichnet die Wahrscheinlichkeit, an kardiovaskulären Erkrankungen wie z.B. Herzinfarkt, Schlaganfall oder pAVK zu erkranken. Als Hauptrisikofaktoren für kardiovaskuläre Erkrankungen gelten unter anderem Bluthochdruck und Fettstoffwechselstörungen. Diese erhöhen das Risiko für eine Atherosklerose, wodurch das Risiko u.a. für eine koronare Herzerkrankung steigt.
Arteriosklerotische Wandveränderungen und damit verbundene thrombotische Auflagerungen begünstigen eine systemisch als auch lokal bestehende Aktivierung von Hämostasesystemen und eine Störung der Fibrinolyse. Die vorliegende Arbeit, bestehend aus zwei Publikationen, ging deshalb der Frage nach, ob die kardiovaskulären Risikofaktoren Bluthochdruck und Fettstoffwechselstörungen einen Zusammenhang mit Blutgerinnungsparametern zeigen.
Die Arbeit stützte sich auf einen Datensatz, der im Rahmen der KORA Fit-Studie erhoben wurde. KORA bedeutet Kooperative Gesundheitsforschung in der Region Augsburg. Dabei wurden zwischen 2017 und 2019 Daten bei gut 800 Studienteilnehmern, die zwischen 1945 und 1964 geboren waren, Befragungen durchgeführt sowie Untersuchungen und Labordaten erhoben.
In beiden Publikationen wurden neben deskriptiven Analysen die Zusammenhänge zwischen den Blutdruckexpositionen (systolischer, diastolischer und Pulsdruck) bzw. den Lipidparametern (Gesamtcholesterin, HDL-Cholesterin, LDL-Cholesterin, Non HDL-Cholesterin, Triglyzeride) und den Gerinnungsparametern INR, aPTT, Antithrombin III, Fibrinogen, D-Dimer, Protein C, Protein S und Faktor VIII als Outcomes mittels multivariabler linearer Regressionsmodelle untersucht. Die Modelle wurden für eine Reihe von Confoundern, wie z.B. Alter, Taillenumfang, körperliche Aktivität in der Freizeit, Alkoholkonsum, sozioökonomischen Status, BMI, Diabetes, Raucherstatus etc. adjustiert. Personen, die mit gerinnungshemmenden Medikamenten behandelt wurden, wurden von den Analysen ausgeschlossen. Ein Hauptfokus der Arbeit lag auf geschlechtsspezifischen Auswertungen.
Im Rahmen der ersten Arbeit ergaben sich Unterschiede bei Männern mit bzw. ohne Hypertonie in Bezug auf die Gerinnungsparameter Fibrinogen und Faktor VIII-Aktivität. Bei den Frauen zeigten sich entsprechend statistisch signifikante Unterschiede bei den Parametern Fibrinogen, D-Dimere, Protein S-Aktivität und Faktor VIII-Aktivität. Die Analyse mittels linearer Regression zeigte bei den Männern keine signifikanten Assoziationen zwischen Blutdruck- und Blutgerinnungsparametern. Bei Frauen zeigte sich eine signifikant positive Assoziation zwischen systolischem Blutdruck und D-Dimer-Leveln.
In der zweiten Arbeit konnten ebenfalls signifikante Unterschiede zwischen Männern und Frauen festgestellt werden: Bei Männern war Gesamt-Cholesterin invers mit aPTT, aber positiv mit der Protein C-Aktivität assoziiert. HDL-Cholesterin war bezogen auf aPTT und Fibrinogen invers assoziiert. LDL-Cholesterin, Non HDL-Cholesterin und Triglyzeride zeigten eine positive Assoziation mit Protein C- und Protein S-Aktivitäten. Bei Frauen zeigten LDL-Cholesterin, Gesamtcholesterin und Non HDL-Cholesterin einen positiven Zusammenhang mit AT III-Konzentrationen und Protein C- und S-Aktivitäten. Zusätzlich war Non-HDL-Cholesterin positiv assoziiert mit der Faktor VIII-Aktivität, HDL-Cholesterin und invers mit Fibrinogen. Triglyzeride zeigten einen positiven Zusammenhang mit der Protein C-Aktivität.
Zusammenfassend lässt sich sagen, dass sich in beiden Arbeiten deutliche geschlechtsspezifische Assoziationen zeigten. Die weitere Erforschung und Ausarbeitung dieser Unterschiede muss eine wichtige Rolle bei zukünftigen Studien spielen, um sowohl kardiovaskuläre Risiken als auch die Einflüsse von Serumlipiden in Bezug auf die Blutgerinnung bei Männern und Frauen aus der Allgemeinbevölkerung besser verstehen zu können.Cardiovascular diseases are the main causes of morbidity and mortality worldwide, especially in industrialized countries, and are therefore among the most important diseases worldwide.
Cardiovascular risk refers to the probability of developing cardiovascular diseases such as myocardial infarction, stroke, or peripheral arterial disease. The main risk factors for cardiovascular disease include high blood pressure and dyslipidemia. These increase the risk of atherosclerosis, which increases the risk of coronary heart disease, among other things.
Arteriosclerotic wall changes and associated thrombotic deposits favor a systemic as well as local activation of hemostasis systems and a disturbance of fibrinolysis. The present study, consisting of two publications, therefore investigated whether the cardiovascular risk factors hypertension and dyslipidemia are related to blood coagulation parameters.
The work was based on data collected as part of the KORA Fit study. KORA stands for Cooperative Health Research in the Augsburg Region. Between 2017 and 2019, data were collected from about 800 study participants born between 1945 and 1964, surveys were conducted, and examinations and laboratory data were collected.
In both publications, in addition to descriptive analyses, the associations between blood pressure exposures (systolic, diastolic and pulse pressure) as well as lipid parameters (total cholesterol, HDL cholesterol, LDL cholesterol, non-HDL cholesterol, triglycerides) and the coagulation parameters INR, aPTT, antithrombin III, fibrinogen, D-dimer, protein C, protein S and factor VIII as outcomes were investigated using multivariable linear regression models. The models were adjusted for a number of confounders, such as age, waist circumference, leisure-time physical activity, alcohol consumption, socioeconomic status, BMI, diabetes, smoking status, etc. Individuals treated with anticoagulant medications were excluded from the analyses. A main focus of the work was on gender-specific evaluations.
In the first study, differences were found in men with and without hypertension with regard to the coagulation parameters fibrinogen and factor VIII activity. In women, statistically significant differences were found for the parameters fibrinogen, D-dimer, protein S activity, and factor VIII activity. Linear regression analysis showed no significant associations between blood pressure and coagulation parameters in men. In women, there was a significant positive association between systolic blood pressure and D-dimer levels.
In the second work, significant differences were also found between men and women: In men, total cholesterol was inversely associated with aPTT but posi-tively associated with protein C activity. HDL cholesterol was inversely associated related to aPTT and fibrinogen. LDL cholesterol, non HDL-cholesterol, and triglycerides showed a positive association with protein C and protein S activities. In women, LDL cholesterol, total cholesterol, and non HDL-cholesterol showed a positive association with AT III concentrations and protein C and S activities. In addition, non HDL cholesterol was positively associated with factor VIII activity, HDL cholesterol and inversely with fibrinogen. Triglycerides showed a positive association with protein C activity.
In conclusion, significant gender differences were evident in both investigations. Further exploration and elaboration of these differences must play an important role in future studies to better understand both cardiovascular risks and the influences of serum lipids in relation to blood coagulation in men and women from the general population