Analysen zum Kernimport von Notch1

Der Notch Signalweg ist ein wichtiges Regulationssystem für die Entwicklung und Selbsterneuerung verschiedener Gewebe. Eine Besonderheit dieser Signalkaskade stellt die Funktion von Notch als Oberflächen-Rezeptor und direkter Regulator der Genexpression dar. Die Aktivierung und Signalübertragung von Notch erfordert daher die proteolytische Freisetzung der intrazellulären Domäne (NICD), welche nach dem Transport in den Zellkern die Transkription zellspezifischer Gene anschaltet. Bis heute existieren nur wenige Kenntnisse über die Mechanismen, die den Kernimport von NICD vermitteln. Frühere Studien weisen zwar auf den klassischen Importin α/β1-Transportweg in diesem Zusammenhang hin, dennoch fehlten bisher eine Charakterisierung der potentiellen Transportsignale sowie die Aufklärung des genauen Kerntransportmechanismus. Daher war es das Ziel der vorliegenden Arbeit, den Kernimport von NICD im Detail zu analysieren. Der erste Abschnitt zielte auf die Identifizierung des funktionellen Kernlokalisierungssignals (NLS) sowie der Importrezeptoren, welche dieses Transportsignal erkennen. Anhand vorausgegangener Studien war bereits bekannt, dass die intrazelluläre Domäne von Notch1 vier potentielle klassische Kernlokalisierungssignale (NLS1 - NLS4) enthält. Durch die Anwendung von Mutationsanalysen konnte nachgewiesen werden, dass ausschließlich das NLS3 für den Transport von NICD in den Zellkern essentiell ist. In Abhängigkeit von dieser Sequenz interagiert NICD mit den Importinen α3, α4 und α7, welche als Adapterproteine für den klassischen Importin α/β1-Transportweg agieren. Zudem konnten für die verschiedenen Isoformen unterschiedliche Affinitäten gegenüber NICD dokumentiert werden. So zeigte Importin α4 die stärkste und Importin α7 die schwächste Bindung an NICD. Weiterhin wurde mittels Reportergen- und immunzytochemischer Analysen demonstriert, dass die Importine α3, α4 und α7 den Kernimport von NICD in vivo vermitteln. Allerdings scheinen hierbei vor allem die Importine α3 und α4 von Bedeutung zu sein. Importin α7 spielt in diesem Zusammenhang wahrscheinlich eine untergeordnete Rolle

Performance analysis on percentage of wheel slip for a passenger car using GPS and wheel speed sensor

This thesis deals with the analysis on percentage of wheel slip for a passenger car using GPS and wheel speed sensor. The objective of this thesis is to analyze the percentage of wheel slip for a passenger car in a various velocity, road condition and driving mode. The thesis describes the post-processing method to analyze the percentage of wheel slip and identify the effective rolling radius and the longitudinal tire stiffness for maximum tire life and performance. Driving and braking behaviour of vehicle were both studied in this thesis for paved and unpaved sandy road condition which commonly the contributing factors to the wheel slip to occur. The data used for the analysis is obtained through experimental test using UMP Test Car which has been installed with Wheel Pulse Transducer, Global Positioning System and DEWESOFT software for data acquisition purpose. The post-processing method was performed using Flexpro and Microsoft Office Excel. The post-processing method to analyze the percentage of wheel slip was performed using the SAE definition of wheel slip and the percent error in the distance travel by the car between free rolling and actual condition. Finally, the longitudinal force, the effective rolling radius and the longitudinal tire stiffness was determined for both driving and braking maneuver of vehicle on paved and unpaved sandy road condition. From the results, it is observed that the percentage of wheel slip during driving maneuver is higher for unpaved sandy road condition compares to that the paved road. It is also observed that the longitudinal force of the tire is lower for unpaved sandy road compare to the paved road condition. The effective rolling radius of the tire during driving maneuver was determined to be lower compare to the free rolling radius of the tire. During braking manuever, the results show that the percentage of wheel slip is higher for unpaved sandy road compare to that for paved road condition. The longitudinal force and tire stiffness also observed lower for unpaved sandy road condition. The effective rolling radius of the tire during braking determined higher compared to that in the free rolling radius. The results concluded that the percentage of wheel slip is strongly dependent to the longitudinal force and the tire road friction. Therefore, effective rolling radius and longitudinal tire stiffness obtained can significantly use to improve tire design and construction. The results also can be use to improve the energy usage efficiency and fuel consumption of vehicle

Changes of Proteases, Antiproteases, and Pathogens in Cystic Fibrosis Patients' Upper and Lower Airways after IV-Antibiotic Therapy

Background. In cystic fibrosis (CF) the upper (UAW) and lower airways (LAW) are reservoirs for pathogens like Pseudomonas aeruginosa. The consecutive hosts' release of proteolytic enzymes contributes to inflammation and progressive pulmonary destruction. Objectives were to assess dynamics of protease : antiprotease ratios and pathogens in CF-UAW and LAW sampled by nasal lavage (NL) and sputum before and after intravenous-(IV-) antibiotic therapy. Methods. From 19 IV-antibiotic courses of 17 CF patients NL (10 mL/nostril) and sputum were collected before and after treatment. Microbiological colonization and concentrations of NE/SLPI/CTSS (ELISA) and MMP-9/TIMP-1 (multiplex bead array) were determined. Additionally, changes of sinonasal symptoms were assessed (SNOT-20). Results. IV-antibiotic treatment had more pronounced effects on inflammatory markers in LAW, whereas trends to decrease were also found in UAW. Ratios of MMP-9/TIMP-1 were higher in sputum, and ratios of NE/SLPI were higher in NL. Remarkably, NE/SLPI ratio was 10-fold higher in NL compared to healthy controls. SNOT-20 scores decreased significantly during therapy ( = 0.001). Conclusion. For the first time, changes in microbiological patterns in UAW and LAW after IV-antibiotic treatments were assessed, together with changes of protease/antiprotease imbalances. Delayed responses of proteases and antiproteases to IV-antibiotic therapy were found in UAW compared to LAW

COVID-19 patients share common, corticosteroid-independent features of impaired host immunity to pathogenic molds

Patients suffering from coronavirus disease-2019 (COVID-19) are susceptible to deadly secondary fungal infections such as COVID-19-associated pulmonary aspergillosis and COVID-19-associated mucormycosis. Despite this clinical observation, direct experimental evidence for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)-driven alterations of antifungal immunity is scarce. Using an ex-vivo whole blood stimulation assay, we challenged blood from twelve COVID-19 patients with Aspergillus fumigatus and Rhizopus arrhizus antigens and studied the expression of activation, maturation, and exhaustion markers, as well as cytokine secretion. Compared to healthy controls, T-helper cells from COVID-19 patients displayed increased expression levels of the exhaustion marker PD-1 and weakened A. fumigatus - and R. arrhizus -induced activation. While baseline secretion of proinflammatory cytokines was massively elevated, whole blood from COVID-19 patients elicited diminished release of T-cellular (e.g., IFN-γ, IL-2) and innate immune cell-derived (e.g., CXCL9, CXCL10) cytokines in response to A. fumigatus and R. arrhizus antigens. Additionally, samples from COVID-19 patients showed deficient granulocyte activation by mold antigens and reduced fungal killing capacity of neutrophils. These features of weakened anti-mold immune responses were largely decoupled from COVID-19 severity, the time elapsed since diagnosis of COVID-19, and recent corticosteroid uptake, suggesting that impaired anti-mold defense is a common denominator of the underlying SARS-CoV-2 infection. Taken together, these results expand our understanding of the immune predisposition to post-viral mold infections and could inform future studies of immunotherapeutic strategies to prevent and treat fungal superinfections in COVID-19 patients

Host response to Candida albicans bloodstream infection and sepsis

Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management

First Insights in NK—DC Cross-Talk and the Importance of Soluble Factors During Infection With Aspergillus fumigatus

Invasive aspergillosis (IA) is an infectious disease caused by the fungal pathogen Aspergillus fumigatus that mainly affects immunocompromised hosts. To investigate immune cell cross-talk during infection with A. fumigatus, we co-cultured natural killer (NK) cells and dendritic cells (DC) after stimulation with whole fungal structures, components of the fungal cell wall, fungal lysate or ligands for distinct fungal receptors. Both cell types showed activation after stimulation with fungal components and were able to transfer activation signals to the counterpart not stimulated cell type. Interestingly, DCs recognized a broader spectrum of fungal components and thereby initiated NK cell activation when those did not recognize fungal structures. These experiments highlighted the supportive function of DCs in NK cell activation. Furthermore, we focused on soluble DC mediated NK cell activation and showed that DCs stimulated with the TLR2/Dectin-1 ligand zymosan could maximally stimulate the expression of CD69 on NK cells. Thus, we investigated the influence of both receptors for zymosan, Dectin-1 and TLR2, which are highly expressed on DCs but show only minimal expression on NK cells. Specific focus was laid on the question whether Dectin-1 or TLR2 signaling in DCs is important for the secretion of soluble factors leading to NK cell activation. Our results show that Dectin-1 and TLR2 are negligible for NK cell activation. We conclude that besides Dectin-1 and TLR2 other receptors on DCs are able to compensate for the missing signal

Comparative assessment of immune evasion mechanisms in human whole-blood infection assays by a systems biology approach.

Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms

Quantitative Simulations Predict Treatment Strategies Against Fungal Infections in Virtual Neutropenic Patients

The condition of neutropenia, i.e., a reduced absolute neutrophil count in blood, constitutes a major risk factor for severe infections in the affected patients. Candida albicans and Candida glabrata are opportunistic pathogens and the most prevalent fungal species in the human microbiota. In immunocompromised patients, they can become pathogenic and cause infections with high mortality rates. In this study, we use a previously established approach that combines experiments and computational models to investigate the innate immune response during blood stream infections with the two fungal pathogens C. albicans and C. glabrata. First, we determine immune-reaction rates and migration parameters under healthy conditions. Based on these findings, we simulate virtual patients and investigate the impact of neutropenic conditions on the infection outcome with the respective pathogen. Furthermore, we perform in silico treatments of these virtual patients by simulating a medical treatment that enhances neutrophil activity in terms of phagocytosis and migration. We quantify the infection outcome by comparing the response to the two fungal pathogens relative to non-neutropenic individuals. The analysis reveals that these fungal infections in neutropenic patients can be successfully cleared by cytokine treatment of the remaining neutrophils; and that this treatment is more effective for C. glabrata than for C. albicans