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Molecular interaction networks lay the foundation for studying how biological functions are controlled by the complex interplay of genes and proteins. Investigating perturbed processes using biological networks has been instrumental in uncovering mechanisms that underlie complex disease phenotypes. Rapid advances in omics technologies have prompted the generation of high-throughput datasets, enabling large-scale, network-based analyses. Consequently, various modeling techniques, including network enrichment, differential network extraction, and network inference, have proven to be useful for gaining new mechanistic insights. We provide an overview of recent network-based methods and their core ideas to facilitate the discovery of disease modules or candidate mechanisms. Knowledge generated from these computational efforts will benefit biomedical research, especially drug development and precision medicine. We further discuss current challenges and provide perspectives in the field, highlighting the need for more integrative and dynamic network approaches to model disease development and progression.

The blood-brain barrier (BBB) is the bottleneck in the development of new drugs to reach the brain. Due to the BBB, toxic substances cannot enter the brain, but promising drug candidates also pass the BBB poorly. Suitable in vitro BBB models are therefore of particular importance during the preclinical development process, as they can not only reduce animal testing but also enable new drugs to be developed more quickly. The aim of this study was to isolate cerebral endothelial cells, pericytes, and astrocytes from the porcine brain to produce a primary model of the BBB. Additionally, as primary cells are well suited by their properties but the isolation is complex and better reproducibility with immortalized cells must be ensured, there is a high demand for immortalized cells with suitable properties for use as a BBB model. Thus, isolated primary cells can also serve as the basis for a suitable immortalization technique to generate new cell lines. In this work, cerebral endothelial cells, pericytes, and astrocytes were successfully isolated and expanded using a mechanical/enzymatic method. Furthermore, in a triple coculture model, the cells showed a significant increase in barrier integrity compared with endothelial cell monoculture, as determined by transendothelial electrical resistance measurement and permeation studies using sodium fluorescein. The results demonstrate the opportunity to obtain all three cell types significantly involved in BBB formation from one species, thus providing a suitable tool for testing the permeation properties of new drug candidates. In addition, the protocols are a promising starting point to generate new cell lines of BBB-forming cells as a novel approach for BBB in vitro models.

Capillary zone electrophoresis (CZE) is an important technique for the analysis of monoclonal antibodies (mAbs). A recently released light-emitting diode (LED)-induced fluorescence (LEDIF) detector equipped with a 275 nm LED for the detection of proteins through their native fluorescence was used in this study and compared to results obtained using the predominant detection mode, the measurement of the absorption of ultraviolet light (UV detection). This was accomplished using an established CZE method for the analysis of three mAbs: NISTmAb, matuzumab, and Intact Mass Check Standard (Waters). For this purpose, the detector’s settings were first optimized using a design of experiments approach. Three factors, rise time, photomultiplier high voltage supply, and acquisition frequency, were optimized by means of a D-optimal design. The optimal settings were then used for the investigation of signal-to-noise ratios (S/Ns), linearity, and precision. LEDIF detection offered a similar separation quality, up to 12 times higher S/Ns, and lower limits of detection compared to UV detection. Repeatability was excellent, with relative standard deviations (RSDs) of approximately 1% for percentage areas. For intermediate precision, RSDs of

IntroductionAlzheimer’s disease (AD), is characterized by a gradual cognitive decline associated with the accumulation of Amyloid beta (Aβ)-oligomers, progressive neuronal degeneration and chronic neuroinflammation. Among the receptors shown to bind and possibly transduce the toxic effects of Aβ-oligomers is the p75 neurotrophin receptor (p75NTR). Interestingly, p75NTR mediates several crucial processes in the nervous system, including neuronal survival and apoptosis, maintenance of the neuronal architecture, and plasticity. Furthermore, p75NTR is also expressed in microglia, the resident immune cells of the brain, where it is markedly increased under pathological conditions. These observations indicate p75NTR as a potential candidate for mediating Aβ-induced toxic effects at the interface between the nervous and the immune system, thereby potentially participating in the crosstalk between these two systems.MethodsHere we used APP/PS1 transgenic mice (APP/PS1tg) and compared the Aβ-induced alterations in neuronal function, chronic inflammation as well as their cognitive consequences between 10 months old APP/PS1tg and APP/PS1tg x p75NTRexonIV knockout mice.ResultsElectrophysiological recordings show that a loss of p75NTR rescues the impairment in long-term potentiation at the Schaffer collaterals in the hippocampus of APP/PS1tg mice. Interestingly, however loss of p75NTR does not influence the severity of neuroinflammation, microglia activation or the decline in spatial learning and memory processes observed in APP/PS1tg mice.ConclusionTogether these results indicate that while a deletion of p75NTR rescues the synaptic defect and the impairment in synaptic plasticity, it does not affect the progression of the neuroinflammation and the cognitive decline in a mouse model for AD.

It is insightful to report an estimator that describes how certain a model is in a prediction, additionally to the prediction alone. For regression tasks, most approaches implement a variation of the ensemble method, apart from few exceptions. Instead of a single estimator, a group of estimators yields several predictions for an input. The uncertainty can then be quantified by measuring the disagreement between the predictions, for example by the standard deviation. In theory, ensembles should not only provide uncertainties, they also boost the predictive performance by reducing errors arising from variance. Despite the development of novel methods, they are still considered the “golden-standard” to quantify the uncertainty of regression models. Subsampling-based methods to obtain ensembles can be applied to all models, regardless whether they are related to deep learning or traditional machine learning. However, little attention has been given to the question whether the ensemble method is applicable to virtually all scenarios occurring in the field of cheminformatics. In a widespread and diversified attempt, ensembles are evaluated for 32 datasets of different sizes and modeling difficulty, ranging from physicochemical properties to biological activities. For increasing ensemble sizes with up to 200 members, the predictive performance as well as the applicability as uncertainty estimator are shown for all combinations of five modeling techniques and four molecular featurizations. Useful recommendations were derived for practitioners regarding the success and minimum size of ensembles, depending on whether predictive performance or uncertainty quantification is of more importance for the task at hand.

IntroductionThe cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive.Methods and resultsWe show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer.DiscussionHence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal’s life.

Die weltweit zunehmende Prävalenz macht den Gestationsdiabetes (GDM) zu einer wachsenden Belastung für die Gesundheitssysteme. Dieser ist als eine erstmals während der Schwangerschaft diagnostizierte Glucosetoleranzstörung (IGT) definiert, wobei die pathophysiologischen Mechanismen der Erkrankung noch nicht vollständig geklärt sind. Um der Heterogenität der Erkrankung gerecht zu werden, wird für den GDM eine Unterteilung in Subtypen hinsichtlich des zeitlichen Auftretens der IGT diskutiert. So kann eine IGT bereits eine präkonzeptionelle Störung sein, die erst während der Schwangerschaft diagnostiziert wird. Das Ziel der vorliegenden Arbeit war es, Veränderungen des hepatischen Stoffwechsels unter prädiabetischen Bedingungen während der Schwangerschaft in einem dafür geeigneten Modell zu untersuchen. Dafür wurde das polygene New Zealand Obese (NZO)-Mausmodell, das durch eine bereits vor der Trächtigkeit bestehende IGT charakterisiert ist, sowohl mit dem C57BL/6N-Stamm, der erst während der Trächtigkeit eine IGT entwickelt, als auch mit dem stoffwechselgesunden Naval Medical Research Institute (NMRI)-Stamm verglichen. Die NZO-Maus zeigte im Vergleich zu den anderen Stämmen während der Trächtigkeit erhöhte freie Fettsäure (FFA)- und Triglycerid (TG)-Plasmaspiegel sowie einen erhöhten atherogenen Index. Darüber hinaus zeigte das NZO-Modell abnehmende hepatische Sphingomyelin (SPM)-Gehalte während der Trächtigkeit bei gleichzeitig ansteigenden Sphingosin-1-Phosphat (S1P)-Plasmaspiegeln. NZO-Mäuse zeigten während der Trächtigkeit eine beeinträchtigte hepatische Gewichtsanpassung und Veränderungen im hepatischen FFA-Metabolismus, einhergehend mit einer verminderten PPARα-Expression und verminderter CD36-Translokation. Damit zeigte das NZO-Modell einen gestörten Lipidstoffwechsel, der sowohl im Plasma als auch in der Leber während der Trächtigkeit messbar war und mit einer verminderten AKT-Aktivität in primären Hepatozyten Merkmale einer Insulinresistenz (IR). In diesen Charakteristika unterschied sich das NZO-Modell vom C57BL/6N-Stamm, der mit einer während der Trächtigkeit erworbenen IGT dem klassischen GDM-Phänotyp entsprach. Die Wirkung der erhöhten FFA-Spiegel sowie steigender 17β-Estradiol (E2)- und Serotonin (5-HT)-Plasmaspiegel während der Trächtigkeit auf den Glucosestoffwechsel wurden in ex vivo-Inkubationsversuchen mit primären Hepatozyten des NZO- und des NMRI-Stamms untersucht. Es konnte gezeigt werden, dass FFA durch eine Steigerung der hepatischen Glucoseproduktion und eine Verminderung der Glycogenspeicherung sowie eine Steigerung der IR, gekennzeichnet durch eine verminderte AKT-Aktivierung, zum prädiabetischen Phänotyp beitragen. Auch 5-HT trug zu einer gesteigerten Glucoseproduktion bei und führte ebenfalls zu einer verminderten Glycogenspeicherung. Für E2 hingegen konnte ein protektiver Effekt auf den hepatischen Glucosemetabolismus im NZO-Modell mit steigender AKT-Aktivität und verminderter Glucoseproduktion nachgewiesen werden. Dieser Kompensationsprozess schützt vermutlich vor einer Verschlechterung des NZO-Stoffwechsels und könnte diesen stabilisieren, sodass das NZO-Modell in der Trächtigkeit keine weitere Verschlechterung der IGT zeigte. Das NZO-Modell bildet mit den beschriebenen Charakteristika einen Subtyp der Erkrankung ab, der bereits vor der Schwangerschaft einen gestörten Stoffwechsel aufweist und aufgrund einer ausbleibenden Verschlechterung des Phänotyps keinem klassischen GDM entspricht. Vor zukünftigen Schwangerschaften kann 5-HT ein geeigneter prädiktiver Indikator für den GDM-Subtyp mit präexistierender IGT sein und sich für die frühzeitige Vorhersage des GDM-Risikos noch vor der Empfängnis eignen, da bereits präkonzeptionell erhöhte 5-HT Plasmaspiegel festgestellt werden konnten. Das NZO-Modell kann folglich einen wichtigen Beitrag zur Beschreibung von Patientenkohorten leisten, die bereits vor der Schwangerschaft an einem Prädiabetes erkrankt sind und während der Schwangerschaft als GDM-Patientinnen diagnostiziert werden., The global increase of gestational diabetes (GDM) prevalence places an increasing burden on the health care systems. The disease is defined as an impaired glucose tolerance (IGT) first diagnosed during pregnancy, although the pathophysiological mechanisms of GDM are not yet completely understood. To reflect the heterogeneity of the disease, a differentiation into several subtypes with regard to the time of onset of IGT is being discussed. Thus, an IGT can already be a preconceptional disorder that is only diagnosed during pregnancy, though it is pre-existing. The aim of the present study was to investigate changes in hepatic metabolism in a suitable mouse model under prediabetic conditions during pregnancy. Therefore, the polygenic New Zealand Obese (NZO) mouse model, which is characterised by an IGT already existing before pregnancy, was compared with both the C57BL/6N strain, which only develops IGT during pregnancy, and with the metabolically healthy Naval Medical Research Institute (NMRI) strain. The NZO mouse displayed increased plasma levels of free fatty acids (FFA) and triglycerides (TG) as well as an increased atherogenic index during gestation compared to the other strains. In addition, the NZO model exhibited decreasing hepatic sphingomyelin (SPM) levels during pregnancy with concomitant increasing sphingosine-1-phosphate (S1P) plasma levels. NZO mice showed impaired hepatic weight adjustment and alterations in hepatic FFA metabolism during gestation, accompanied by decreased PPARα expression and reduced CD36 translocation. Thus, the NZO model showed impaired lipid metabolism detectable in both plasma and liver during gestation and with reduced AKT activity in primary hepatocytes characteristics of insulin resistance (IR). These characteristics differentiated the NZO model from the C57BL/6N strain, which resembled the classic phenotype of the disease with IGT acquired during pregnancy. The effect of increased FFA levels, as well as increasing 17β-estradiol (E2) and serotonin (5-HT) plasma levels during pregnancy on glucose metabolism, was investigated using primary hepatocytes of the NZO and NMRI strains. It was shown, that FFA contributed to the prediabetic phenotype by increasing hepatic glucose production and decreasing glycogen storage, as well as increasing IR, which is characterised by decreased AKT activation. 5-HT also contributed to increased glucose production and similarly resulted in decreased glycogen storage. For E2, on the other hand, a protective effect on hepatic glucose metabolism could be demonstrated in the NZO model with an increase in AKT activity and a decrease in glucose production. This compensatory process presumably stabilises and protects against deterioration of the NZO metabolism, thus the model showed no further deterioration of IGT in pregnancy. With the described characteristics, the NZO model reflects a subtype of the disease with an already altered metabolism before pregnancy and does not resemble a classic GDM due to the lack of a deteriorating phenotype. Prior to future pregnancies, 5-HT could be a predictive indicator for a GDM subtype with pre-existing IGT and could be suitable for early prediction of the GDM risk even before conception, as elevated 5-HT plasma levels could already be detected preconceptionally. Thus, the NZO mouse model can make an important contribution to the description of patient cohorts who already have prediabetes before pregnancy and are later diagnosed as GDM patients during gestation.

Influenza A virus (IAV) subtypes are a major cause of illness and mortality worldwide and pose a threat to human health. Although IAV infection is considered a self-limiting respiratory syndrome, an expanded spectrum of cerebral manifestations has been reported following IAV infection. Neurotropic IAVs, such as the H7N7 subtype, are capable of invading the central nervous system (CNS) and replicating in brain cells, resulting in microglia-induced neuroinflammation. Microglial cells, the brain’s resident immune cells, are instrumental in the inflammatory response to viral infection. While activation of microglia is important to initially contain the virus, excessive activation of these cells leads to neuronal damage. Previous studies have shown that acute and even long-term IAV-induced neuroinflammation leads to CNS damage. Therefore, the search for possible preventive or therapeutic strategies is of great importance. In this study, we investigated the potential effect of vaccination against acute neuroinflammation induced by H7N7 infection and subsequent neuronal damage in the hippocampus, a particularly vulnerable brain region, comparing young and aged mice. Immunosenescence is one of the striking pathophysiological changes during mammalian aging that leads to “inflammaging” and critically limits the protection by vaccines in the elderly. The results suggest that formalin-inactivated H7N7 vaccine has a preventive effect against the inflammatory responses in the periphery and also in the CNS after H7N7 infection. Cytokine and chemokine levels, increased microglial density, and cell volume after H7N7 infection were all attenuated by vaccination. Further structural analysis of microglial cells also revealed a change in branching complexity after H7N7 infection, most likely reflecting the neuroprotective effect of the vaccination. In addition, synapse loss was prevented in vaccinated mice. Remarkably, engulfment of post-synaptic compartments by microglia can be proposed as the underlying mechanism for spine loss triggered by H7N7 infection, which was partially modulated by vaccination. Although young mice showed better protection against neuroinflammation and the resulting deleterious neuronal effects upon vaccination, a beneficial role of the vaccine was also observed in the brains of older mice. Therefore, vaccination can be proposed as an important strategy to prevent neurological sequelae of H7N7 infection.

Trypanosoma- und Leishmania-Parasiten lösen verheerende Infektionskrankheiten in den Tropen aus, die von der Weltgesundheitsorganisation zu den vernachlässigten Tropenkrankheiten gezählt werden. Die aktuell zugelassenen Chemotherapeutika erfüllen die an Arzneimittel zur Behandlung dieser Krankheiten gestellten Anforderungen zu einem großen Teil nicht. Obwohl die Forschungsaktivitäten in den letzten Jahren deutlich zugenommen haben, ist die Entwicklung neuer Wirkstoffe gegen die Parasiten dringend nötig. Paullon-N5-acetamide waren eine vielversprechende Wirkstoffgruppe gegen die einzelligen Erreger. Entsprechend substituierte Paullone hemmen die Proliferation der Protozoen durch eine Inhibition der Trypanothionsynthetase (TryS), eines essenziellen, an der parasitären Redoxhomöostase beteiligten und parasitenspezifischen Enzyms, das nicht in den Wirtsorganismen vorkommt. Paullone eignen sich jedoch nicht als mögliche neue Wirkstoffe, weil sie keine angemessenen Eigenschaften im Sinne der druglikeness besitzen und beispielsweise nur schlecht in wässrigen Medien löslich sind. Die wässrige Löslichkeit ist eine wichtige Eigenschaft von Arzneistoffen zur Behandlung von Tropenkrankheiten, denn die Arzneistoffe sollten aufgrund der infrastrukturellen Verhältnisse der betroffenen Länder oral appliziert werden können. Das Ziel dieser Dissertation war es, durch molekulare Operationen die Paullone so zu vereinfachen, dass Verbindungen entstehen, die die TryS inhibieren, das Parasitenwachstum unterdrücken und gleichzeitig eine verbesserte druglikeness und Löslichkeit besitzen. Im Rahmen der Arbeit wurden verschiedene Struktur-Aktivitäts- und Struktur-Eigenschafts-Beziehungen rund um ein Strukturelement der Paullone, einen Indolylacetamid-Grundkörper, aufgestellt. So wurden Substituenten identifiziert mit denen die antiparasitäre Wirkung gegenüber Trypanosoma brucei brucei-Blutstromform-Parasiten und/oder die druglikeness-Eigenschaften modifiziert werden können. Viele der identifizierten antiparasitären Verbindungen besitzen jedoch eine hohe Lipophilie, die vermutlich zu deren mangelhafter wässriger Löslichkeit beiträgt. Dennoch wurden auch Verbindungen gefunden, die angemessene Löslichkeitseigenschaften besitzen. Nach vorläufigen Ergebnissen hemmt jedoch keine der untersuchten Verbindungen die ursprünglich als Zielenzym adressierte Trypanothionsynthetase von Leishmania infantum. Es sind also andere Zielstrukturen für die antitrypanosomale Wirkung verantwortlich. Die Ergebnisse der vorgelegten Arbeit können als Basis für die Entwicklung neuer Chemotherapeutika zur Behandlung parasitärer vernachlässigter Tropenerkrankungen dienen., Trypanosoma and Leishmania parasites cause devastating infectious diseases in the tropics, which are categorized by the World Health Organization as neglected tropical diseases. To a large extent, the currently approved chemotherapeutic agents do not meet the requirements placed on drugs for the treatment of these diseases. Although research activities have increased significantly in recent years, the development of new agents against the parasites is urgently needed. Paullone-N5-acetamides have been a promising group of agents against the unicellular pathogens. Appropriately substituted paullones inhibit protozoan proliferation by inhibiting trypanothione synthetase (TryS), an essential enzyme involved in parasitic redox homeostasis that is specific to parasites and does not occur in mammalian hosts. However, paullones are not suitable as potential new drugs because they lack appropriate properties in terms of druglikeness and, for example, are poorly soluble in aqueous media. Aqueous solubility is an important property of drugs for the treatment of tropical diseases, because the drugs should be able to be applied orally due to the infrastructural conditions of the affected countries. The aim of this dissertation was the structural simplification of paullones through molecular operations to find compounds that inhibit TryS, suppress parasite growth, and at the same time have improved druglikeness and solubility. As part of the work, various structure–activity and structure–property relationships were established around a structural element of paullones, an indolylacetamide backbone. Thus, substituents modifying antiparasitic activity against Trypanosoma brucei brucei bloodstream form parasites and/or druglikeness properties were identified. However, many of the identified antiparasitic compounds possess high lipophilicity, which probably contributes to their poor aqueous solubility. Nevertheless, compounds possessing adequate solubility properties were also found. However, according to preliminary results, none of the investigated compounds inhibits the trypanothione synthetase of Leishmania infantum, which was originally addressed as a target enzyme. Thus, other targets are responsible for the antitrypanosomal effect. The results of the presented work may serve as a basis for the development of new chemotherapeutic agents for the treatment of parasitic neglected tropical diseases.

The administration of living microorganisms is of special interest, with regard to probiotic microorganisms providing health benefits to the patient. Effective dosage forms require the preservation of microbial viability until administration. Storage stability can be improved by drying, and the tablet is an especially attractive final solid dosage form due to its ease of administration and its good patient compliance. In this study, drying of the yeast Saccharomyces cerevisiae via fluidized bed spray granulation is investigated, as the probiotic Saccharomyces boulardii is a variety of it. Fluidized bed granulation enables faster drying than lyophilization on the one hand and lower temperatures than spray drying on the other hand, which are the two predominantly used techniques for life-sustaining drying of microorganisms. Yeast cell suspensions enriched with protective additives were sprayed onto the carrier particles of common tableting excipients, namely, dicalcium phosphate (DCP), lactose (LAC) and microcrystalline cellulose (MCC). Different protectants, such as mono-, di-, oligo- and polysaccharides, but also skimmed milk powder and one alditol, were tested; as they themselves, or chemically similar molecules, are known from other drying technologies to stabilize biological structures such as cell membranes, and thus, improve survival during dehydration. With the combined use of trehalose and skimmed milk powder, survival rates were 300 times higher than without the use of protective additives. In addition to these formulation aspects, the influence of process parameters such as inlet temperature and spray rate were considered. The granulated products were characterized regarding their particle size distribution, moisture content and the viability of the yeast cells. It has been shown that thermal stress on the microorganisms is especially critical, which can be reduced, for example, by reducing the inlet temperature or increasing the spray rate; however, formulation parameters such as cell concentration also influenced survival. The results were used to specify the influencing factors on the survival of microorganisms during fluidized bed granulation and to derive their linkages. Granules based on the three different carrier materials were tableted and the survival of the microorganisms was evaluated and linked to the tablet tensile strength achieved. Using LAC enabled the highest survival of the microorganisms throughout the considered process chain.

Climate change and the Covid-19 pandemic are global challenges in which scientists play a crucial role, and immediate political actions are necessary. However, in contrast to climate change, strong governmental actions have been taken during the pandemic. While climate change has been on the public agenda for several decades, the pandemic is a rather new issue. In such cases, social media offer scientists the potential to disseminate scientific results to the public and express calls to action and their personal views towards politics. Thus far, little is known about the extent to which scientists make use of this option. In this study, we investigated the similarities and differences between visible German climate experts and visible German Covid-19 experts regarding advocacy and assessments of policies and political actors on Twitter. We conducted a manual content analysis of tweets (N = 5,915) from 2021 of the most visible climate experts (N = 5) and the most visible Covid-19 experts (N = 5). The results show that climate experts addressed politics more often than Covid-19 experts in their tweets. The selected climate experts more often expressed negative evaluations, the degradation of competence and blaming. The Covid-19 experts, however, made more political calls for action. We assume that an issue’s history and context will affect scientists’ public assessments of politics. Our comparative study provides insight into the interrelations between science and politics in digital communication environments and elucidates visible scientists’ communication behaviours towards different socio-scientific issues.

An innovative polymer-based electro-sensor decorated with Tb nanoparticles has been developed for the first time. The fabricated sensor was utilized for trace determination of favipiravir (FAV), a recently US FDA-approved antiviral drug for the treatment of COVID-19. Different techniques, including ultraviolet-visible spectrophotometry (UV-VIS), cyclic voltammetry (CV), scanning electron microscope (SEM), X-ray Diffraction (XRD) and electrochemical impedance spectroscopy (EIS), were applied for the characterization of the developed electrode TbNPs@ poly m-THB/PGE. Various experimental variables, including pH, potential range, polymer concentration, number of cycles, scan rate and deposition time, were optimized. Moreover, different voltammetric parameters were examined and optimized. The presented SWV method showed linearity over the range of 10–150 × 10−9 M with a good correlation coefficient (R = 0.9994), and the detection limit (LOD) reached 3.1 × 10−9 M. The proposed method was applied for the quantification of FAV in tablet dosage forms and in human plasma without any interference from complex matrices, obtaining good % recovery results (98.58–101.93%).

Background During the last decades, the Open Access paradigm has become an important approach for publishing new scientific knowledge. From 2015 to 2020, the Trans-O-MIM research project was undertaken with the intention to identify and to explore solutions in transforming subscription-based journals into Open Access journals. Trans-O-MIM stands for strategies, models, and evaluation metrics for the goal-oriented, stepwise, sustainable, and fair transformation of established subscription-based scientific journals into Open-Access-based journals with Methods of Information in Medicine as an example. Objectives To present an overview of the outcomes of the Trans-O-MIM research project as a whole and to share our major lessons learned. Methods As an approach for transforming journals, a Tandem Model has been proposed and implemented for Methods of Information in Medicine. For developing a metric to observe and assess journal transformations, scenario analysis has been used. A qualitative and a two-tier quantitative study on drivers and obstacles of Open Access publishing for medical informatics researchers was designed and conducted. A project setup with a research team, a steering committee, and an international advisory board was established. Major international medical informatics events have been used for reporting and for receiving feedback. Results Based on the Tandem Model, the journal Methods of Information in Medicine has been transformed into a journal where, in addition to its subscription-based track, from 2017 onwards a Gold Open Access track has been successfully added. An evaluation metric, composed of 5 scenarios and 65 parameters, has been developed, which can assist respective decision makers in assessing such transformations. The studies on drivers and obstacles of Open Access publishing showed that, while most researchers support the idea of making scientific knowledge freely accessible to everyone, they are hesitant about actually living this practice by choosing Open Access journals to publish their own work. Article-processing charges and quality issues are perceived as the main obstacles in this respect, revealing a two-sided evaluation of Open Access models, reflecting the different viewpoints of researchers as authors or readers. Especially researchers from low-income countries benefit from a barrier-free communication mainly in their role as readers and much less in their role as authors of scientific information. This became also evident at the institutional level, as Open Access policies or financial support through funding bodies are most prevalent in Europe and North America. Conclusions With Trans-O-MIM, an international research project was performed. An existing journal has been transformed. In addition, with the support of the International Medical Informatics Association, as well as of the European Federation for Medical Informatics and of the German Association for Medical Informatics, Biometry and Epidemiology as European and German medical informatics organizations, we did run an international experiment on Open Access incentives. Both together are, as far as the authors know, unique. We therefore expect that this research could add new knowledge on Open Access transformation.

This study aimed to develop valid in vitro models for preclinical evaluation of proliferative and anti-inflammatory effects of human allogeneic serum eye drops for dry eye disease (DED) treatment. A DED wound healing model was developed by analyzing the influence of coating and serum concentrations on human corneal epithelial (HCE-T) wound closure. Further, intralaboratory variance, freeze–thaw cycle effects, donor variability and stability assays were conducted. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were used to induce the gene expression of matrix metalloproteinase 9 (MMP9), cyclooxygenase 2 (COX2), transforming growth factor-β (TGFβ) and IL-1β. MMP9 induction was optimized using a design-of-experiments (DoE) approach and applied to examine serum under static and dynamic conditions. MMP9 protein expression was analyzed by ELISA. The DED wound healing model detected proliferative effects of serum down to 1% with a small intralaboratory variance. Serum stability was shown over six months, donor variance could be detected, and freeze–thaw cycle effects did not affect wound closure. Serum decreased MMP9 expression on the gene and protein levels. The induction method was successfully optimized using DoE modeling and transferred to a dynamic setting mimicking tear film fluidics. The DED wound healing and inflammatory DED model present useful in vitro models for the preclinical evaluation of allogeneic serum eye drops without the use of animal experiments.

Die Kapillarelektrophorese ist eine etablierte Technik in der Pharmazeutischen Analytik, die besonders in den Bereichen Anwendung findet, in denen die Hochleistungs-Flüssigchromatographie nicht optimal geeignet ist. Hierzu gehört u. a. die Analytik von Proteinen und von Arzneistoffen in Anwesenheit von Proteinen. Dabei sind die Wechselwirkungen der Proteine mit der Kapillarwand eine ständige Herausforderung, da sie zu Veränderungen in der Trennleistung, schlechter Reproduzierbarkeit und schließlich zum Verstopfen der Kapillare führen können. Weiterhin sind Arzneistoffe häufig an Proteine gebunden und so der analytischen Erfassung entzogen. In der vorliegenden Arbeit wurden beide Aspekte jeweils anhand eines kapillarelektrophoretischen Verfahrens beleuchtet und entsprechende Problemlösungen entwickelt. Zuerst sollte ein Zellmodell der Blut-Hirn-Schranke auf seine Eigenschaften hinsichtlich der Penetration von Arzneistoffen untersucht werden. Dazu wurden sechs Arzneistoffe mit bekannten Penetrationseigenschaften (Carbamazepin, Cimetidin, Coffein, Indometacin, Paracetamol und Propranolol) einzeln im komplex aufgebauten Nährmedium des Modells gelöst und auf das Modell aufgegeben. Nach sechs Stunden wurde jeweils der penetrierte und zurückgehaltene Anteil bestimmt, außerdem wurde die Stabilität der Arzneistoffe in dem Medium untersucht. Die hierzu verwendete mizellare elektrokinetische Chromatographie (MEKC) erlaubt die direkte Injektion des Mediums ohne vorherige Abtrennung der Matrixbestandteile. Eine entsprechende Methode wurde für dieses Trennproblem adaptiert und hinsichtlich der zeitlichen und analytischen Anforderungen optimiert. Angepasst wurden die Wellenlänge, Druck und Dauer der Injektion, die Konzentration an Natriumlaurylsulfat (SDS), die angelegte Spannung und schließlich die Spülung der Kapillare. Dabei konnte gezeigt werden, dass die MEKC gut an unterschiedliche Matrices angepasst werden kann und dass das untersuchte Modell hinsichtlich der Arzneistoffpenetration ausreichend realistische Eigenschaften aufweist. Bei der kapillar-isoelektrischen Fokussierung (CIEF), mit der sich der zweite Teil der Arbeit beschäftigt, liegen Proteine während der Analyse an ihrem isoelektrischen Punkt (pI) netto ungeladen und in relativ hoher Konzentration vor. Es kommt daher zu besonders starken Wechselwirkungen der Proteine untereinander und mit der Kapillare. Untersucht wurden verschiedene Möglichkeiten, die Adsorption zu verringern oder adsorbierte Proteine von der Kapillarwand zu entfernen. Dazu wurden drei gut bekannte Modellproteine (Myoglobin, Ovalbumin und β-Lactoglobulin) verwendet. Erreicht werden sollten möglichst geringe Variabilitäten der Migrationszeit und der relativen Peakfläche. Untersucht wurden zunächst verschiedene Innendurchmesser der Kapillare, wobei sich 75 µm als optimal erwiesen. Weiterhin wurden die nichtionischen Tenside Brij® 35 (1% und 2%), Tween® 20 (1%) und Tween® 80 (1%) sowie Acetonitril (10%) dem Hintergrundelektrolyten zugesetzt. Dabei traten die beiden letzten deutlich hinter Brij® und Tween 20 zurück, außerdem wurde deutlich, dass die Alterung des verwendeten Trägerampholyten das Ergebnis deutlich beeinflusst. Bei weiteren Versuchen wurden verschiedene Spülreagenzien eingesetzt (Brij® allein und in Kombination mit Propan-2-ol sowie Palmitylsulfobetain), wobei für die Migrationszeit relative Standardabweichungen im Bereich von 1,7% erreicht werden konnten. Die Verwendung eines Internen Standards verbessert die Präzision nicht, gibt aber Aufschluss über den pH-Gradienten. Schließlich konnte gezeigt werden, dass ein gewisser Spüldruck erforderlich ist, um Aggregate mechanisch von der Kapillare zu entfernen., Capillary electrophoresis is a well-established technique in pharmaceutical analysis. It is especially applied whenever high performance liquid chromatography (HPLC) is not ideally suited, e. g., for the analytics of proteins, or of drugs in presence of proteins. Interaction between proteins and the capillary wall is a permanent challenge, as it leads to changes in separation performance, low reproducibility, and eventually capillary clogging. In this thesis, both aspects are examined and suggestions for appropriate solutions are made. First, the drug penetration properties of cell-based blood-brain-barrier model were studied. Six drugs (acetaminophen, carbamazepine, cimetidine, caffeine, indomethacin, and propranolol) with known penetration properties were separately dissolved in the growth medium and given onto the model. After six hours, the penetrated and retained amounts of drug were determined. In addition, drug stability in the growth medium was examined. Micellar electrokinetic chromatography (MEKC) allowed direct sample injection without previous separation steps. A method has been adapted to this analytical problem and optimized to fulfil the analytical requirements and time restrictions. The following parameters were adjusted: wavelength, injection pressure and time, sodium dodecyl sulphate (SDS) concentration, voltage, and capillary rinsing. The adaptability of MEKC could be demonstrated, and the model showed sufficiently realistic properties for drug penetration. In capillary isoelectric focusing (CIEF), being the subject of the second part of this thesis, proteins are net uncharged and in higher concentrations at their isoelectric points during analysis. This leads to particularly strong interactions between the proteins and between proteins and the capillary. Various ways of reducing adsorption and of removing adsorbed proteins from the capillary wall were investigated. Three well-known proteins (myoglobin, β-lactoglobulin, and ovalbumin) were used for this purpose. The aim should be to achieve the lowest possible variability in migration time and relative peak area. Initially, various inner diameters of the capillary were examined, with 75 µm proving to be optimal. Furthermore, the non-ionic surfactants Brij® 35 (1% and 2%), Tween® 20 (1%) and Tween® 80 (1%) and acetonitrile (10%) were added to the background electrolyte, the first two being clearly superior to the latter. It also became clear that the aging of the carrier ampholyte used significantly influences the result. Various rinsing reagents were used in further tests (Brij® alone and in combination with propan-2-ol and palmitylsulfobetaine), and relative standard deviations (RSD%) in the range of 1.7% could be achieved for the migration time. The use of an internal standard does not improve the precision. Nevertheless, it provides information about the pH gradient. Finally, it could be shown that a certain rinsing pressure is required to mechanically remove aggregates from the capillary.

The poor bioavailability of many newly developed active pharmaceutical ingredients (APIs) poses a major challenge in formulation development. To overcome this issue, strategies such as the preparation of amorphous solid dispersions (ASDs), and the application of the APIs in lipid nanocarriers or the wet-milling of the substances into nanoparticles have been introduced. In addition to an efficient formulation strategy, a dosage form that is accepted by all patients is also of great importance. To enable a simple application of the oral dosage form for all patients, orodispersible films (ODFs) are a very promising delivery platform for the APIs because the films directly disintegrate in the mouth. In this study, two poorly water-soluble APIs, fenofibrate and naproxen, were formulated using five different formulation strategies and then embedded in ODFs. It was found that the deliverable amount of API with one ODF highly depends on the formulation strategy as well as the physicochemical properties of the formulated API. The most promising film formulations were ASD-ODFs as well as films with API-loaded lipid nanoemulsions. Both showed a reduction of the dissolution time of the APIs from the ODF compared to an ODF with unformulated API micro particles. In addition, short disintegration times were achieved, although the mechanical film properties were slightly worse compared to the API-free film formulation.

In our continuous study for some African plants as a source for antitrypanosomally and cytotoxic active drugs, nine different plants belonging to the Crassulaceae family have been selected for the present study. Sedum sieboldii leaves extract showed an antitrypanosomal activity against Trypanosoma brucei with an IC50 value of 8.5 µg/mL. In addition, they have cytotoxic activities against (HCT-116), (HEPG-2) and (MCF-7), with IC50 values of 28.18 ± 0.24, 22.05 ± 0.66, and 26.47 ± 0.85 µg/mL, respectively. Furthermore, the extract displayed inhibition against Topoisomerase-1 with an IC50 value of 1.31 µg/mL. It showed the highest phenolics and flavonoids content among the other plants’ extracts. In order to identify the secondary metabolites which may be responsible for such activities, profiling of the polar secondary metabolites of S. sieboldii extract via Ultra-Performance Liquid Chromatography coupled to High-Resolution QTOF-MS operated in negative and positive ionization modes, which revealed the presence of 46 metabolites, including flavonoids, phenolic acids, anthocyanidins, coumarin, and other metabolites.

Im Rahmen der vorliegenden Arbeit wurden verschiedene NHC-Gold-Komplexverbindungen in der Weiterentwicklung bereits etablierter Strukturen (Substanzgruppe A) und NHC-Gold(I)-Metronidazol-Hybridverbindungen mit ihren Vorstufen (Substanzgruppe B) erfolgreich synthetisiert, chemisch und biologisch charakterisiert. Die analysierten Verbindungen der Gruppe A zeigten analoge Aktivitäten zu bereits etablierten NHC-Gold(I)-Komplexen: eine moderate bis gute antiproliferative Aktivität gegen verschiedene Zelllinien, gute antibiotische Eigenschaften gegenüber grampositiven Bakterien und eine gute Hemmung der bakteriellen Thioredoxin-Reduktase (TrxR). Eine relevante Differenz basierend auf der Oxidationsstufe des Goldes konnte nicht gezeigt werden. Es konnten röntgenkristallografische Strukturen von vier Verbindungen der Gruppe A und zwei weiteren auf früheren Arbeiten basierenden Komplexen erhalten und bestimmt werden. Die beobachteten Trends folgen bekannten Entwicklungen in der Literatur. Weiterhin wurden Anstrengungen unternommen hybride NHC-Gold(I)-Antibiotika- und NHC-Gold(I)-Siderophor-Konjugate herzustellen. Nach mehreren erfolglosen Kombinationen konnten zwei Metronidazolesterkonjugate synthetisiert und charakterisiert werden. Während die Beurteilung eines dieser Konjugate durch Löslichkeitsprobleme nicht gesichert vollständig möglich war und die Konjugate keine messbaren Aktivitäten in Antiproliferationsstudien an Zelllinien oder gegenüber verschiedenen aeroben Bakterienarten zeigten, waren sie gegenüber isolierter bakterieller TrxR und gegenüber verschiedenen Stämmen von C. difficile aktiv. Ein Hybrid konnte darüber hinaus einen gegen Metronidazol resistenten Stamm (CD-22-00115) in weiten Konzentrationsbereichen hemmen., In this work, several NHC-gold complexes based on established compounds (group A) and two NHC-gold(I)-metronidazole conjugates and their precursors (group B) were successfully synthesised and biologically and chemically analysed. The examined compounds of group A exhibited moderate to good antiproliferative effects against different tumorigenic and non-tumorigenic cell lines, good antibiotic activity against gram-positive bacteria and were good inhibitors for the bacterial thioredoxin reductase. These results were overwhelmingly in accordance with previously published data, but no relevant difference in activity was found based on the oxidation state of the gold. While working on this thesis, four x-ray crystallographic structures of compounds in group A were found and two more based on previous publications in the group. The structures were solved and followed trends in good alignment with published literature. Additionally, synthesis of hybrid NHC-gold(I)-antibiotic and NHC-gold(I)-siderophore compounds was attempted. After several attempts two NHC-gold(I)-metronidazole conjugates were successfully synthesised and characterised. While solubility issues prevented an optimal biological characterisation, the conjugates showed no relevant antiproliferative properties and were inactive against aerobic bacteria. They performed well against bacterial thioredoxin reductase and multiple strains of anaerobic C. difficile. Furthermore, one conjugate was able to inhibit the growth of a strain resistant to metronidazole (CD-22-00115) over a wide range of concentration.

Glucosinolates, specialized metabolites of the Brassicales including Brassica crops and Arabidopsis thaliana, have attracted considerable interest as chemical defenses and health-promoting compounds. Their biological activities are mostly due to breakdown products formed upon mixing with co-occurring myrosinases and specifier proteins, which can result in multiple products with differing properties, even from a single glucosinolate. Whereas product profiles of aliphatic glucosinolates have frequently been reported, indole glucosinolate breakdown may result in complex mixtures, the analysis of which challenging. The aim of this study was to assess the breakdown of indole glucosinolates in A. thaliana root and rosette homogenates and to test the impact of nitrile-specifier proteins (NSPs) on product profiles. To develop a GC-MS-method for quantification of carbinols and nitriles derived from three prominent indole glucosinolates, we synthesized standards, established derivatization conditions, determined relative response factors and evaluated applicability of the method to plant homogenates. We show that carbinols are more dominant among the detected products in rosette than in root homogenates of wild-type and NSP1- or NSP3-deficient mutants. NSP1 is solely responsible for nitrile formation in rosette homogenates and is the major NSP for indolic nitrile formation in root homogenates, with no contribution from NSP3. These results will contribute to the understanding of the roles of NSPs in plants.

A growing number of complex neurostimulation strategies promise symptom relief and functional recovery for several neurological, psychiatric, and even multi-organ disorders. Although pharmacological interventions are currently the mainstay of treatment, neurostimulation offers a potentially effective and safe alternative, capable of providing rapid adjustment to short-term variation and long-term decline of physiological functions. However, rapid advances made by clinical studies have often preceded the fundamental understanding of mechanisms underlying the interactions between stimulation and the nervous system. In turn, therapy design and verification are largely driven by clinical-empirical evidence. Even with titanic efforts and budgets, it is infeasible to comprehensively explore the multi-dimensional optimization space of neurostimulation through empirical research alone, especially since anatomical structures and thus outcomes vary dramatically between patients. Instead, we believe that the future of neurostimulation strongly depends on personalizable computational tools, i.e. Digital Neuro Twins (DNTs) to efficiently identify effective and safe stimulation parameters. DNTs have the potential to accelerate scientific discovery and hypothesis-driven engineering, and aid as a critical regulatory and clinical decision support tool. We outline here how DNTs will pave the way toward effective, cost-, time-, and risk-limited electronic drugs with a broad application bandwidth.

Oral tadalafil (TD) proved promising in treating pediatric pulmonary arterial hypertension (PAH). However, to ensure higher efficacy and reduce the systemic side effects, targeted delivery to the lungs through nebulization was proposed as an alternative approach. This poorly soluble drug was previously dissolved in nanoemulsions (NEs). However, the formulations could not resist aqueous dilution, which precluded its dilution with saline for nebulization. Thus, the current study aimed to modify the previous systems into dilutable TD-NEs and assess their suitability for a pulmonary application. In this regard, screening of various excipients was conducted to optimize the former systems; different formulations were selected and characterized in terms of physicochemical properties, nebulization performance, stability following sterilization, and biocompatibility. Results showed that the optimal system comprised of Capmul-MCM-EP:Labrafac-lipophile (1:1) (w/w) as oil, Labrasol:Poloxamer-407 (2:1) (w/w) as surfactant mixture (Smix) and water. The optimum formulation P2TD resisted aqueous dilution, exhibited reasonable drug loading (2.45 mg/mL) and globule size (25.04 nm), acceptable pH and viscosity for pulmonary administration, and could be aerosolized using a jet nebulizer. Moreover, P2TD demonstrated stability following sterilization and a favorable safety profile confirmed by both in-vitro and in-vivo toxicity studies. These favorable findings make P2TD promising for the treatment of pediatric PAH.

Embedded porous membranes are a key element of various organ-on-chip systems. The widely used commercial polymer membranes impose limits with regard to chip integration and thinness. We report a microfluidic chip platform with the key element of a monolithically integrated, ultra-thin (700 nm) nanoporous membrane made of ultra-low-stress (<35 MPa) SixNy for culturing and testing reconstructed tissue. The membrane is designed to support various in vitro tissues including co-cultures and to allow passage of molecules but not of cells. A digital laser write method was used to produce nanopores with deterministic but highly flexible positioning within the membrane. A thin layer of photoresist was exposed by accumulation of femtosecond pulses for local two-photon polymerization, which allowed nanopores as small as 350 nm in diameter to be generated through the membranes in a subsequent plasma etch process. The fabricated membranes were used to separate a microfluidic chip into two compartments, which are connected to the outside by microchannel structures. With unique side inlets for fluids, all cells are exposed to identical flow velocities and shear stresses. With the hydrophilic nature of chip materials the self-loading seeding is controlled bottom-up by capillary forces, which makes the seeding procedure homogeneous and less dependent on the operator. The chip is designed to allow fabrication by wafer-level MEMS manufacturing technologies without critical assembly steps, thereby promoting reproducibility and scale-up of fabrication. In order to establish a fully functional test system to be used in a lab incubator, a holder for the bare chip was designed and 3D-printed with additional elements for gravity driven pumping. In order to mimic physiological conditions, the holder was designed to provide not only media delivery but also appropriate shear stress to the tissue. To prove usability, murine microvascular endothelial cells (muMEC) were seeded on the membrane within the chip. Cell compatibility was confirmed after 3 days of dynamic cultivation using fluorescence live/dead assays. Cultivation proved to be reproducible and led to confluent layers with cells preferentially grown on nanoporous areas. The system can in future be cost effectively manufactured in larger quantities in MEMS foundries and can be used for a wide variety of in vitro tissues and test scenarios including pumpless operation within cell incubator cabinets.

Background The prevalence of gestational diabetes mellitus (GDM) is increasing worldwide. There is increasing evidence that GDM is a heterogeneous disease with different subtypes. An important question in this context is whether impaired glucose tolerance (IGT), which is a typical feature of the disease, may already be present before pregnancy and manifestation of the disease. The latter type resembles in its clinical manifestation prediabetes that has not yet manifested as type 2 diabetes (T2DM). Altered lipid metabolism plays a crucial role in the disorder's pathophysiology. The aim was to investigate the role of lipids which are relevant in diabetes-like phenotypes in these both models with different time of initial onset of IGT. Methods Two rodent models reflecting different characteristics of human GDM were used to characterize changes in lipid metabolism occurring during gestation. Since the New Zealand obese (NZO)-mice already exhibit IGT before and during gestation, they served as a subtype model for GDM with preexisting IGT (preIGT) and were compared with C57BL/6 N mice with transient IGT acquired during gestation (aqIGT). While the latter model does not develop manifest diabetes even under metabolic stress conditions, the NZO mouse is prone to severe disease progression later in life. Metabolically healthy Naval Medical Research Institute (NMRI) mice served as controls. Results In contrast to the aqIGT model, preIGT mice showed hyperlipidemia during gestation with elevated free fatty acids (FFA), triglycerides (TG), and increased atherogenic index. Interestingly, sphingomyelin (SM) concentrations in the liver decreased during gestation concomitantly with an increase in the sphingosine-1-phosphate (S1P) concentration in plasma. Further, preIGT mice showed impaired hepatic weight adjustment and alterations in hepatic FFA metabolism during gestation. This was accompanied by decreased expression of peroxisome proliferator-activated receptor alpha (PPARα) and lack of translocation of fatty acid translocase (FAT/CD36) to the hepatocellular plasma membrane. Conclusion The preIGT model showed impaired lipid metabolism both in plasma and liver, as well as features of insulin resistance consistent with increased S1P concentrations, and in these characteristics, the preIGT model differs from the common GDM subtype with aqIGT. Thus, concomitantly elevated plasma FFA and S1P concentrations, in addition to general shifts in sphingolipid fractions, could be an interesting signal that the metabolic disorder existed before gestation and that future pregnancies require more intensive monitoring to avoid complications. Graphical Abstract This graphical abstract was created with BioRender.com.

Ausgangspunkt der Arbeit war die Frage, welche intrazellulären Kriterien über die Fusions-kompetenz der Insulingranula entscheiden. Zu diesem Zweck wurde überprüft, ob das Mobi-litätsverhalten der submembranären Granula sowohl nach Desensibilisierung und Ruhestatus als auch während der Modulation der GLP-1-vermittelten Signalwege und Inhibition der oxi-dativen Phosphorylierung Rückschlüsse auf die Sekretionsleistung der MIN6-Zellen zu lässt. Die Insulingranula zeigten sowohl nach Desensibilisierung als auch Ruhestatus eine Reduktion des Austauschs und der lateralen Mobilität im Submembranärraum. Jedoch war nach Desen-sibilisierung unter anderem die Sekretionsrate sowie die Effizienz des Ca2+-Signals vermindert und nach Ruhestatus erhöht. Ferner zeigte sich auch eine Beeinflussung des Granulaalters. Hier-aus ergibt sich, dass die Anzahl, das Alter und die Mobilität der Insulingranula den Funktions-zustand der Zellen widerspiegeln und möglicherweise zum metabolischen Gedächtnis beitra-gen. Die Aktivierung der GLP-1-vermittelten Signalwege ging mit einer Steigerung der Exozytose- und Insulinsekretionsrate einher. Nichtsdestotrotz ließ sich kein einzelner Mobilitätsparamter ausmachen, welcher mit der Sekretionsleistung korrelierte, wofür wahrscheinlich der indivi-duelle Grundzustand der Zellen verantwortlich war. Ferner wirken sich diese Signalwege auch direkt auf die Fusionsporenbildung aus. Dieser Interaktion kommt möglicherweise ein höherer Stellenwert als der Beeinflussung der Granulamobilität bei. Hingegen führte die Inhibition der oxidativen Phosphorylierung zu einer starken Beeinflussung des Granulaverhaltens, wobei die Effekte vom verwendeten Inhibitor abhängig waren. Grund hierfür scheint eine unterschiedliche Selektivität zu sein. Die selektivste Minderung des ATP-Gehalts ging vom Oligomycin aus, welches zu einer Abnahme der Verweildauer sowie zu einer Zunahme der lateralen Mobilität im Submembranärraum führte. Hieraus ist zu schließen, dass das ATP maßgeblich an der Regulation der Granulamobilität beteiligt ist. Die hier vorgestellte Arbeit zeigt auf, dass der Granulamobilität eine Schlüsselrolle für die Se-kretionsleistung der insulinsezernierenden Zellen beikommt. Zunächst müssen die energe-tischen Voraussetzungen geklärt werden, die für die Rezirkulation der Insulingranula verant-wortlich sind, damit ein spezifisches Muster erkannt werden kann, welches die Beweglichkeit der Granula mit der Insulinsekretionsleistung in Verbindung setzt. Als wegweisendes Target stellte sich das ATP heraus, welches scheinbar nicht nur einen permissiven Effekt hat, sondern wahrscheinlich eine regulatorische Funktion erfüllt., The starting point of this work was the question which intracellular criteria determine the fusion competence of insulin granules. Therefore, we examined whether the mobility of submembrane granules, both after desensitization and resting state as well as during modulation of GLP-1-mediated signaling pathways and inhibition of oxidative phosphorylation, allows conclusions about the secretion rate of MIN6 cells. Insulin granules showed a decrease in granule turnover and lateral mobility at the plasma membrane after both desensitization and resting state. However, secretion rate and efficiency of Ca2+-signaling, among others, were decreased after desensitization and increased after resting state. Furthermore, granule age was also found to be affected. This suggests that the number, age, and mobility of insulin granules reflect the functional state of cells and may contribute to metabolic memory. Activation of GLP-1-mediated signaling pathways was associated with an increase in the rate of exocytosis and insulin secretion. Nevertheless, no single mobility parameter could be identified that correlated with secretion performance, for which the individual basal state of the cells was probably responsible. Furthermore, these signaling pathways also directly affect fusion pore formation. This interaction may be more important than influencing granule mobility. In contrast, inhibition of oxidative phosphorylation strongly affected granule behavior, although the effects were dependent on the inhibitor used. The reason for this appears to be a difference in selectivity. The most selective reduction in ATP content was from oligomycin, which led to a decrease in residence time as well as an increase in lateral mobility in the submembrane space. So it can be concluded that ATP is crucial involved in the regulation of granule mobility. The work presented here demonstrates that granule mobility plays a key role in secretory response. First, the energetic requirements of mobility responsible for insulin granule recirculation must be clarified in order to identify a specific pattern linking granule mobility to insulin secretion. ATP turned out to be a relevant target, which appears to have not only a permissive effect but probably a regulatory function.

Anthranoids are a group of natural products with a broad spectrum of applications. Many anthranoids exhibit crucial pharmaceutical uses, as exemplified by laxative anthraquinones and antitumoral hypericin. Hypericin is a potent drug in the photodynamic therapy (PDT) and diagnosis (PDD) of cancer and it contributes to the anti-depressant efficacy of pharmaceutical preparations from Hypericum perforatum. However, the continuous supply of many anthranoid drugs remains challenging due to their low yield in plants, difficult extraction, and complicated chemical synthesis. The core of the anthranoid scaffold is biosynthesized in plants via the polyketide pathway which is not fully clarified. For these reasons, elucidation of anthranoid biosynthesis is a demand. Cassia bicapsularis, which is rich in anthranoids, provided a model system for the present studies. A comparative transcriptomic analysis between non-elicited and yeast-extract-elicited C. bicapsularis cell cultures was performed. An octaketide-producing type III polyketide synthase from C. bicapsularis (CbOKS) was successfully identified and functionally characterized. Subcellular localization of reporter fusions revealed the presence of CbOKS in the cytosol. Another octaketide-producing type III polyketide synthase cloned from Polygonum cuspidatum, likewise an anthranoid-rich plant, was also functionally characterized. Assay-guided fractionation of C. bicapsularis crude protein extract suggested the contribution of a tailoring factor/enzyme to the biosynthesis of the anthranoid scaffold. However, the subsequent proteomic analysis was not specific enough to identify a putative polyketide cyclase. Additionally, the endogenous cyclization capability of Nicotiana benthamiana leaves as plant-based heterologous expression system was insufficient to support the correct cyclization of the linear octaketide intermediate upon transient expression of the OKSs. Moreover, a group of putative polyketide cyclases from C. bicapsularis and H. perforatum were identified using combined bioinformatic analyses. The interaction of polyketide synthase and polyketide cyclase may lead to the formation of the anthranoid scaffold, thereby paving the way for heterologous production of anthranoid drugs, including hypericin., Anthranoide sind eine Gruppe von Naturstoffen mit einem breiten Anwendungsspektrum. Viele Anthranoide zeigen bedeutende pharmazeutische Anwendungen wie beispielsweise abführende Anthrachinone und antitumorales Hypericin. Das Hypericin ist ein effizienter Wirkstoff in der photodynamischen Therapie (PDT) und Diagnose (PDD) von Krebs und trägt auch zur antidepressiven Wirksamkeit von pharmazeutischen Präparaten aus Hypericum perforatum bei. Die kontinuierliche Bereitstellung vieler Anthranoid-Wirkstoffe bleibt jedoch aufgrund ihrer geringen Ausbeute in Pflanzen, der schwierigen Extraktion und der komplizierten chemischen Synthese eine Herausforderung. Der Kern des Anthranoid Gerüsts wird in Pflanzen über den noch nicht vollständig geklärten Polyketidweg aufgebaut. Aus diesen Gründen ist die Aufklärung der Anthranoid Biosynthese von großer Bedeutung. Als Modellsystem für die vorliegenden Untersuchungen diente die anthranoidreiche Pflanze Cassia bicapsularis. Es wurde eine vergleichende transkriptomische Analyse zwischen nicht-behandelten und Hefeextrakt-behandelten Zellkulturen von C. bicapsularis durchgeführt. Eine Octaketid-produzierende Polyketid-Synthase vom Typ III aus C. bicapsularis (CbOKS) wurde erfolgreich identifiziert und funktionell charakterisiert. Die subzelluläre Lokalisation von Reporterfusionen zeigte das Vorkommen von CbOKS im Zytosol. Eine weitere Octaketid-produzierende Typ-III-Polyketid-Synthase, die aus der ebenfalls anthranoidreichen Pflanze Polygonum cuspidatum, kloniert wurde, erfuhr gleichfalls eine funktionelle Charakterisierung. Eine Enzymtest-gesteuerte Fraktionierung von C. bicapsularis-Rohproteinextrakt deutete auf den Beitrag eines Hilfsfaktors/Enzyms in der Anthranoid-Gerüstbiosynthese hin. Die anschließende proteomische Analyse war jedoch nicht spezifisch genug, um eine mutmaßliche Polyketid Zyklase zu identifizieren. Außerdem war die endogene Zyklisierungsfähigkeit von Nicotiana benthamiana-Blättern als ein pflanzenbasiertes heterologes Expressionssystem nicht ausreichend, um die korrekte Zyklisierung des linearen Octaketid-Zwischenprodukts bei vorübergehender Expression der OKS-Enzyme zu unterstützen. Darüber hinaus wurde eine Gruppe mutmaßlicher Polyketid Zyklasen aus C. bicapsularis und H. perforatum mittels kombinierter bioinformatischer Analysen identifiziert. Die Wechselwirkung zwischen Polyketid-Synthase und Polyketid-Zyklase könnte zur Bildung des Anthranoid-Gerüsts führen und damit den Weg für eine heterologe Produktion von Anthranoid-Wirkstoffen einschließlich Hypericin ebnen.

Application of Sustainable analytical chemistry concepts has become crucial in order to remove the environmentally harmful impacts originating from the routine use of analytical techniques. Here, a new LC method is developed and its parameters are analyzed, depending on a mixed micellar mobile phase. This was primarily aimed at getting rid of the use of organic solvents in conventional routine analyses. Combinations of tazobactam (TZB) with piperacillin (PPC) or cefepime (CFM) are commonly used as effective antimicrobial therapies, especially for resistant strains. Therefore, the three drugs were separated and quantified using an organic solvent-free mobile phase. The mixed micellar mobile phase was comprised of 15 mM Brij-35 with 38 mM SDS, adjusted to pH 3.5. Separation was performed by HPLC on monolithic RP-C18 column Chromolith® Performance RP-18e (100 mm × 4.6 mm) at a rate of 1 mL per minute of flow in conjunction with a measurement wavelength 210 nm. The method was found valid and applicable in accordance of precision, and accuracy within ranges of 5–100 µg mL−1 for PPC and CFM and of 0.625–12.5 µg mL−1 for TZB. The quality-by-design technique was used to analyze the effect of modifying the mixed micellar ratios on separation efficiency and conclude their behavior. Finally, the suggested approach was assessed applying the green analytical procedure index against the greenest published methodology to show superiority.

Clonazepam (CLZ), an antipsychotic drug reported for its efficiency in managing anxiety-related disorders, is being marketed only as conventional tablets. Some patients have abstention to swallow the conventional tablets; therefore, the proposed study was aimed at developing a buccal lozenge tablet by direct compression of two types of optimized granules. Conazepam’s water solubility was first enhanced by a solid dispersion technique for a fast and better dissolution of type 1 granules, while the impact of gelling polymers was investigated on controlled-release type 2 granules. The optimized formulae met the acceptable pharmacopeial limits for tablets’ evaluation. A differential scanning calorimetry study revealed the compatibility between the drug and used excipients. All formulae gave a burst release of CLZ in the first hour of investigation, followed by a sustained release over 24 h. The formula that showed the highest prolonged in vitro release (99.0 + 0.1%), following the Higuchi diffusion model (R2 = 0.99), was then selected for further study. The formula succeeded in controlling the induced stress in a rat model with a significant impact on the behavioral tests throughout the experiment. The results were further confirmed by a pharmacokinetic study that showed a significant increase in Cmax, Tmax, and AUC (1.5, 2, and 3.9 folds), respectively, compared to oral suspension. The newly proposed delivery system has proven a better efficacy with a reduced dosing frequency.

Die Legionärskrankheit ist eine schwere Pneumonie, die durch Inhalation des humanen Lungenpathogens Legionella pneumophila ausgelöst wird. Durch eine umfassende Charakterisierung auf verschiedenen Organisationsebenen konnte in dieser Arbeit eine vielseitige Rolle der bakteriellen Zink-Metalloprotease ProA als Virulenzfaktor aufgezeigt und spezifiziert werden. Infektionsstudien mit proA-Mutagenesestämmen offenbarten, dass die Protease maßgeblich zur Vermehrung von L. pneumophila in humanen Lungengewebsexplantaten (HLTEs) beiträgt. Die intrazelluläre Replikation in Makrophagen und Epithelzelllinien ließ dabei vornehmlich auf extrazelluläre Effekte schließen. Erstmals konnte in histologischen Untersuchungen eine Verdickung der Alveolarsepten infolge ProA-vermittelter Degradation von Kollagen IV quantifiziert und eine verbesserte bakterielle Ausbreitung durch die Gewebeschädigung beobachtet werden. Darüber hinaus legte eine erhöhte Sensitivität der proA-defizienten Mutante gegenüber humanem Serum eine zentrale Rolle der Protease im Schutz vor der humoralen Immunantwort nahe. Auch das Zusammenspiel von ProA und bakteriellem FlaA ermöglicht dem Pathogen eine verbesserte Evasion. So wurde in einem HEK-Blue hTLR5-Modellsystem deutlich, dass die Flagellin-vermittelte Stimulation des TLR5-NF-κB-Signalwegs im Wirt durch einen Abbau der FlaA-Monomere über ProA reduziert wird. Eine Mutagenese und phänotypische Charakterisierung weiterer lasB-annotierter Proteasen von L. pneumophila Corby enthüllte einzelne synergistische Funktionen hinsichtlich der Vermehrung in HLTEs, der Tensidfilmsezernierung sowie der Serumresistenz des Pathogens. Eine Kristallisation und Strukturaufklärung von ProA bei 1,48 Å zeigte zudem Übereinstimmung mit homologen M4-Proteasen anderer Pathogene wie Pseudolysin von Pseudomonas aeruginosa. Unterschiede wurden insbesondere in äußeren Loopstrukturen wie der Substratbindestelle S1' deutlich. Die Substratdeterminierung verschiedener Vertreter der Enzymfamilie scheint daher vorwiegend von spezifischen Aminosäuren in definierten Bindungsmotiven und weniger von allgemeinen strukturellen Ähnlichkeiten abhängig zu sein. Insgesamt demonstriert die vorliegende Arbeit eine elementare Bedeutung von ProA für die Pathogenese der Legionärskrankheit durch Zerstörung pulmonalen Gewebes, Hemmung der Wirtsabwehrmechanismen und Unterstützung der Vermehrung und Ausbreitung von L. pneumophila in der menschlichen Lunge., Legionnaires' disease is a severe pneumonia caused by inhalation of the human lung pathogen Legionella pneumophila. By a comprehensive characterization at different organizational levels, this thesis showed and specified a versatile role of the bacterial zinc metalloprotease ProA as an important virulence factor. Infection studies with proA mutagenesis strains revealed that the protease contributes significantly to the proliferation of L. pneumophila in human lung tissue explants (HLTEs). The intracellular replication in macrophages and epithelial cell lines particularly indicated extracellular effects. For the first time, thickening of alveolar septa as a consequence of ProA-mediated degradation of collagen IV was quantified in histopathological analyses, and improved bacterial dissemination due to tissue damage was observed. Furthermore, an increased susceptibility of the proA-deficient mutant to human serum suggested a central role of the protease in antagonizing the humoral immune response. The interplay of ProA and bacterial FlaA also improves evasion for the pathogen. Hence, in a HEK-Blue hTLR5 model system, it became evident that flagellin-mediated stimulation of the TLR5-NF-κB signaling pathway in the host is reduced by degradation of FlaA monomers via ProA. Mutagenesis and phenotypical characterization of other lasB-annotated proteases from L. pneumophila Corby revealed individual synergistic functions regarding the replication in HLTEs, secretion of surfactants and serum resistance of the pathogen. Crystallization and structure determination of ProA at 1.48 Å also showed high conformity with homologous M4 proteases of other pathogens such as pseudolysin from Pseudomonas aeruginosa. Differences were especially evident in outer loop structures like the substrate binding pocket S1'. Substrate determination of different members of the enzyme family therefore seems to depend primarily on specific amino acids in defined binding motifs and less on general structural similarities. Overall, the present thesis demonstrates crucial importance of ProA for the pathogenesis of Legionnaires' disease by destroying pulmonary tissue, inhibiting host defense mechanisms and promoting proliferation and dissemination of L. pneumophila in the human lung.

Background Adherence and motivation are key factors for successful treatment of patients with chronic diseases, especially in long-term care processes like rehabilitation. However, only a few patients achieve good treatment adherence. The causes are manifold. Adherence-influencing factors vary depending on indications, therapies, and individuals. Positive and negative effects are rarely confirmed or even contradictory. An ontology seems to be convenient to represent existing knowledge in this domain and to make it available for information retrieval. Methods First, a manual data extraction of current knowledge in the domain of treatment adherence in rehabilitation was conducted. Data was retrieved from various sources, including basic literature, scientific publications, and health behavior models. Second, all adherence and motivation factors identified were formalized according to the ontology development methodology METHONTOLOGY. This comprises the specification, conceptualization, formalization, and implementation of the ontology “Ontology for factors influencing therapy adherence to rehabilitation” (OnTARi) in Protégé. A taxonomy-oriented evaluation was conducted by two domain experts. Results OnTARi includes 281 classes implemented in ontology web language, ten object properties, 22 data properties, 1440 logical axioms, 244 individuals, and 1023 annotations. Six higher-level classes are differentiated: (1) Adherence, (2) AdherenceFactors, (3) AdherenceFactorCategory, (4) Rehabilitation, (5) RehabilitationForm, and (6) RehabilitationType. By means of the class AdherenceFactors 227 adherence factors, thereof 49 hard factors, are represented. Each factor involves a proper description, synonyms, possibly existing acronyms, and a German translation. OnTARi illustrates links between adherence factors through 160 influences-relations. Description logic queries implemented in Protégé allow multiple targeted requests, e.g., for the extraction of adherence factors in a specific rehabilitation area. Conclusions With OnTARi, a generic reference model was built to represent potential adherence and motivation factors and their interrelations in rehabilitation of patients with chronic diseases. In terms of information retrieval, this formalization can serve as a basis for implementation and adaptation of conventional rehabilitative measures, taking into account (patient-specific) adherence factors. OnTARi also enables the development of medical assistance systems to increase motivation and adherence in rehabilitation processes.

Der Diabetes mellitus ist trotz intensiver Forschung und stetig verbesserter Therapieverfahren weltweit eine der wichtigsten Stoffwechselerkrankung. Da neben dem Mangel an Insulin auch der Überschuss von Glucagon zur Entstehung des Krankheitsbildes beiträgt, liegt in der Reduktion der Glucagonsekretion ein aussichtsreicher therapeutischer Ansatzpunkt. Die vorliegende Arbeit soll dazu dienen, die der Insulin- und Glucagonsekretion zugrunde liegenden Mechanismen und ihre Wechselwirkungen weiter aufzuklären, um daraus bessert therapeutische Interventionen entwickeln zu können. Während der auslösende Weg (triggering pathway), der die Insulinsekretion einleitet, relativ gut aufgeklärt ist, ist der verstärkende Weg (amplifying pathway) der die tatsächliche Sekretionsstärke zu regeln scheint, noch in großen Teilen unverstanden. Um diesen zu untersuchen wurde ein etabliertes Protokoll verwendet, in dem Glucose selbst in maximal effektiver Konzentration vollständig ihre insulinsekretionssteigernde Wirkung verliert, während die Ketosäure alpha-Ketoisocapronsäure (KIC) weiterhin in der Lage ist, die Sekretion zu stimulieren, wahrscheinlich, weil sie Alpha-Ketoglutarat für den Citracyclus bereitstellt. Es ließ sich nun zeigen, dass die Ketosäure alpha-Ketoisovaleriansäure (KIV), die allein nicht insulinotrop ist, die verloren gegangene insulinotrope Wirkung der Glucose wiederherstellen kann und diese dann auch bei alleiniger Anwesenheit von Glucose weiter bestehen bleibt. Daraus kann geschlussfolgert werden, dass das Wiederauffüllen des Citratcyclus durch KIV dessen dauerhafte Funktion und damit den Export von Metaboliten (Kataplerose) ermöglicht, die für die Amplifikation der Insulinsekretion notwendig sind. Die Identifikation der Metabolite sowie ihrer Zielstrukturen bedarf jedoch weiterer Aufklärung. Im Gegensatz zur Insulinsekretion ist der Mechanismus der Glucagonsekretion auch in seinen Grundlagen weitgehend kontrovers. Um die Bedeutung der KATP-Kanäle und der Depolarisation des Plasmamembran für die Glucagonsekretion weiter zu klären, wurden simultane Messungen der Insulin- und Glucagonsekretion, der intrazellulären Calciumkonzentration und von elektrophysiologischen Parametern durchgeführt. Dadurch konnten wir zeigen, dass auch in Alpha-Zellen der Schluss des KATP-Kanals zu einer Depolarisation der Zellmembran führt, der ein Calciumeinstrom und eine Sekretionsauslösung folgt, diese Ereignisse jedoch in ihrem Ausmaß deutlich geringer sind als die entsprechenden Ereignisse in Beta-Zellen. Messungen des verbleibenden Stromflusses nach Blockade des KATP-Kanals durch ATP legen zudem eine weniger dominante Rolle für das Membranpotential der Alpha-Zellen als für dasjenige der Beta-Zellen nahe. Um die Konsequenzen für die Glucagonsekretion von intakten Inseln darstellen zu können, bedurfte es der Aufhebung der parakrinen Hemmung durch die Beta-Zellen. Der Alpha-2-Agonist Clonidin erwies sich dabei als geeignetes Werkzeug, da es die Insulinsekretion effektiv hemmte, ohne die durch Depolarisation stimulierte Glucagonsekretion relevant zu beeinflussen. Dadurch konnte die Wirkung depolarisierender Stimuli auf die Glucagonsekretion untersucht werden, ohne dass sie von der parakrinen Hemmung beeinflußt wurden. Eine bemerkenswerte Ausnahme bildete die durch 40 mM Kalium ausgelöste Stimulation, die trotz gesteigerter Insulinsekretion auch eine gesteigerte Glucagonsekretion erbrachte. Die direkte glucagonotrope Wirkung der Sulfonylharnstoffe Tolbutamid und Gliclazid war gering und vorübergehend. Ob die parakrine Hemmung durch Beta-Zellen direkt oder indirekt über Somatostatin aus Delta-Zellen erfolgt, bedarf weiterer Untersuchung. Insgesamt konnten wir zeigen, dass sowohl autonome als auch parakrine Mechanismen die Glucagonsekretion regulieren. Da in der Signalkaskade der Alpha-Zellen ab dem Schluss des KATP-Kanals kein grundlegender Unterschied zu den Beta-Zellen festgestellt werden konnte, der die inverse Glucoseabhängigkeit der Glucagonsekretion erklären würde, scheint eine Untersuchung des Glucosemetabolismus der Alpha-Zellen der logische Ansatzpunkt für künftige Untersuchungen zu sein., Despite intensive research and continuously improved therapy, diabetes mellitus is still one of the most significant metabolic diseases in the world. In addition to the deficient insulin secretion the (relative) excess of glucagon secretion contributes to the pathophysiology development of the disease. Thus, the control of glucagon secretion may be of therapeutic value. The present study aims to further clarify the mechanisms underlying insulin and glucagon secretion and their interactions in order to develop better therapeutic interventions. While the triggering pathway that initiates insulin secretion is comparatively well understood, the amplifying pathway, which appears to be responsible for setting the actual level of secretion, is still poorly understood. This pathway was investigated by using an established protocol where glucose, even at a maximally effective concentration, loses its ability to amplify insulin secretion, while the keto acid alpha-ketoisocaproic acid (KIC) is still able to do so. A possible explanation for this ability of KIC is its anaplerotic effect on the citrate cycle by providing alpha-ketoglutarate. It could now be shown that the keto acid alpha-ketoisovaleric acid (KIV), which is not insulinotropic on its own, can restore the lost amplifying effect of glucose, which persists even in the presence of glucose alone. This role as a “starter” likely reflects the anaplerosis by KIV of the citrate cycle which had previously become depleted and lends support to the notion that the export of metabolites (cataplerosis) is necessary for the amplification of insulin secretion. In contrast to insulin secretion, even the basic features of glucagon stimulus-secretion coupling are controversial. To clarify the role of KATP channels and plasma membrane depolarization for glucagon secretion, simultaneous measurements of insulin and glucagon secretion were performed as well as measurements of the intracellular calcium concentration and electrophysiological parameters. It could be shown that the sulfonylurea-mediated closure of the KATP channel occurs less reliably than in beta-cells, but once established also leads to depolarisation followed by calcium influx and increase of the cytosolic Ca2+ concentration. Measurements of the residual currents after blockade of the KATP channel by ATP also suggest a less dominant role than in beta-cells of the KATP channel for the metabolism-dependent control of the membrane potential. To check the direct effects of KATP channel closure and of potassium depolarization on glucagon secretion by perifused islets, it was necessary to abolish the paracrine inhibition by the beta cells. The alpha-2 agonist clonidine proved to be a suitable tool for this purpose, as it effectively inhibited insulin secretion without having a relevant effect on stimulated glucagon secretion. The direct glucagonotropic effect of sulfonylureas thus characterized was small and transient. Both sulfonylureas tested, tolbutamide and gliclazide, displayed the same characteristics. In contrast to the depolarization by 15 mM KCl the depolarization by 40mM KCl moderately but significantly increased insulin secretion in the presence of clonidine. In spite of the elevated insulin secretion glucagon secretion was also increased, even more so than by 15 mM KCl. This points to the supraphysiological strength of depolarization by 40 mM KCl. To which degree the paracrine inhibition by beta cells is direct or exerted via somatostatin release of the delta cells needs further investigation. To conclude, we could show that both alpha cell-autonomous and paracrine mechanisms regulate glucagon secretion. Since no fundamental differences could be found between the KATP channel-dependent signaling cascade of alpha cells and those of beta cells, a detailed investigation of the glucose metabolism of alpha cells may be needed to explain the inverse glucose dependence of glucagon secretion.

Gestational diabetes (GDM) is characterized by a glucose tolerance disorder. This may first appear during pregnancy or pre-exist before conception as a form of prediabetes, but there are few data on the pathogenesis of the latter subtype. Female New Zealand obese (NZO) mice serve as a model for this subpopulation of GDM. It was recently shown that GDM is associated with elevated urinary serotonin (5-hydroxytryptamine, 5-HT) levels, but the role of the biogenic amine in subpopulations with prediabetes remains unclear. 5-HT is synthesized in different tissues, including the islets of Langerhans during pregnancy. Furthermore, 5-HT receptors (HTRs) are expressed in tissues important for the regulation of glucose homeostasis, such as liver and pancreas. Interestingly, NZO mice showed elevated plasma and islet 5-HT concentrations as well as impaired glucose-stimulated 5-HT secretion. Incubation of isolated primary NZO islets with 5-HT revealed an inhibitory effect on insulin and glucagon secretion. In primary NZO hepatocytes, 5-HT aggravated hepatic glucose production (HGP), decreased glucose uptake (HGU), glycogen content, and modulated AKT activation as well as cyclic adenosine monophosphate (cAMP) increase, indicating 5-HT downstream modulation. Treatment with an HTR2B antagonist reduced this 5-HT-mediated deterioration of the metabolic state. With its strong effect on glucose metabolism, these data indicate that 5-HT is already a potential indicator of GDM before conception in mice.

"Forschung fördern, Lehre entwickeln, Versorgung stärken - für die Zukunft der Physiotherapie" war das Motto des 5. Forschungssymposiums Physiotherapie, welches durch die Brandenburgische Technische Universität Cottbus-Senftenberg (BTU) und die Deutsche Gesellschaft für Physiotherapiewissenschaft e. V. (DGPTW) am 26. und 27.11.2021 veranstaltet wurde. Im Abstractband des 5. Forschungssymposiums Physiotherapie sind die Abstracts der eingereichten und beim 5. Forschungssymposiums Physiotherapie präsentierten Beiträge veröffentlicht. Zudem sind die veranstalteten Workshops zusammenfassend dargestellt., All abstracts from accepted workshops and presentations from the 5. Research Symposium Physical Therapy., Schriftenreihe des Instituts für Gesundheit; 5

In 2018, the discovery of carcinogenic nitrosamine process related impurities (PRIs) in a group of widely used drugs led to the recall and complete withdrawal of several medications that were consumed for a long time, unaware of the presence of these genotoxic PRIs. Since then, PRIs that arise during the manufacturing process of the active pharmaceutical ingredients (APIs), together with their degradation impurities, have gained the attention of analytical chemistry researchers. In 2020, favipiravir (FVR) was found to have an effective antiviral activity against the SARS-COVID-19 virus. Therefore, it was included in the COVID-19 treatment protocols and was consequently globally manufactured at large-scales during the pandemic. There is information indigence about FVR impurity profiling, and until now, no method has been reported for the simultaneous determination of FVR together with its PRIs. In this study, five advanced multi-level design models were developed and validated for the simultaneous determination of FVR and two PRIs, namely; (6-chloro-3-hydroxypyrazine-2-carboxamide) and (3,6-dichloro-pyrazine-2-carbonitrile). The five developed models were classical least square (CLS), principal component regression (PCR), partial least squares (PLS), genetic algorithm-partial least squares (GA-PLS), and artificial neural networks (ANN). Five concentration levels of each compound, chosen according to the linearity range of the target analytes, were used to construct a five-level, three-factor chemometric design, giving rise to twenty-five mixtures. The models resolved the strong spectral overlap in the UV-spectra of the FVR and its PRIs. The PCR and PLS models exhibited the best performances, while PLS proved the highest sensitivity relative to the other models.

Parkinson’s disease (PD) is the second-most abundant neurodegenerative disease, and PD patients have been medicated with levodopa (L-DOPA) for decades. Between 1990 and 2000, a lot of in vitro studies reported neurotoxic effects of the drug, but in vivo studies could never confirm these neurotoxic effects. As a result, L-DOPA remained the gold standard for PD medication. In this study, I investigated the effects of L-DOPA on the central energy metabolism of the LUHMES cell line. For this purpose, I first treated the cells with L-DOPA under normoxic conditions to compare the results with previous studies, which were almost always performed with other in vitro systems. I could confirm the neurotoxic effect of L-DOPA as well as the common mode of action, excessive reactive oxygen species (ROS) formation due to L-DOPA auto-oxidation and therefore several alterations of the central energy metabolism. Next, I minimized the impact of the auto-oxidation of L-DOPA by lowering the oxygen levels from atmospheric pressure to hypoxic conditions (2% O2), which also better reflected the low oxygen levels within the brain. Under these low oxygen tension conditions, L-DOPA auto-oxidation was heavily decelerated resulting in greatly decreased neurotoxicity. Further experiments revealed a double-edged effect of L-DOPA on intracellular ROS levels. Auto-oxidation of the drug result in increased ROS tension, but L-DOPA itself acts as a ROS scavenger. I further observed several alterations of the central energy metabolism induced by L-DOPA such as reduced respiration and impaired mitochondrial membrane potential. The use of stable-isotope labeling confirmed altered mitochondrial metabolism. A transcriptome analyses further revealed an altered gene expression of the calcium signaling pathway induced by L-DOPA. As a result, this study potentially represents an important part of LID research if these results can be confirmed in vivo., Die Parkinson-Krankheit (PD) ist die zweithäufigste neurodegenerative Erkrankung, und PD-Patienten werden seit Jahrzehnten mit Levodopa (L-DOPA) behandelt. Zwischen 1990 und 2000 berichteten viele in vitro Studien über neurotoxische Wirkungen des Arzneimittels, welche jedoch nie durch in vivo Studien bestätigt werden konnten. Infolgedessen blieb L-DOPA der Goldstandard für PD-Medikamente. In meiner Thesis untersuchte ich die Auswirkungen von L-DOPA auf den zentralen Energiestoffwechsel der LUHMES-Zelllinie. Zu diesem Zweck habe ich die Zellen zunächst mit L-DOPA unter normoxischen Bedingungen inkubiert, um die Ergebnisse mit früheren Studien zu vergleichen, die fast immer mit anderen in vitro Systemen durchgeführt wurden. Ich konnte die neurotoxische Wirkung von L-DOPA sowie die gemeinsame Wirkungsweise, die übermäßige Bildung reaktiver Sauerstoffspezies (ROS) durch die Autooxidation von L-DOPA, und damit verbunden mehrere Veränderungen des zentralen Energiestoffwechsels bestätigen. Im Anschluss minimierte ich die Auswirkungen der Autooxidation von L-DOPA, indem ich den Sauerstoffgehalt vom atmosphärischen Druck auf hypoxische Bedingungen (2% O2) senkte, was auch den niedrigen Sauerstoffgehalt im Gehirn besser widerspiegelt. Unter diesen niedrigen Sauerstoffbelastung wurde die Autooxidation von L-DOPA stark abgebremst, was zu einer stark verringerten Neurotoxizität führte. Weitere Experimente zeigten eine zweischneidige Wirkung von L-DOPA auf intrazelluläre ROS-Spiegel. Die Autooxidation von L-DOPA führt zu einer erhöhten ROS-Spannung, aber L-DOPA selbst wirkt als ROS-lindernd. Ich beobachtete weiterhin mehrere Veränderungen des zentralen Energiestoffwechsels, die direkt durch L-DOPA induziert wurden, wie etwa reduzierte Atmung und beeinträchtigtes mitochondriales Membranpotential. Die Verwendung von stabiler Isotopenmarkierung bestätigte einen veränderten mitochondrialen Metabolismus. Eine Transkriptomanalyse offenbarte zudem eine veränderte Genexpression des Calcium-Signalwegs, die durch L-DOPA induziert wurde. Infolgedessen stellt diese Studie möglicherweise einen wichtigen Teil der LID-Forschung dar, wenn diese Ergebnisse in vivo bestätigt werden können.

Following the spread of the COVID-19 pandemic crisis, a race was initiated to find a successful regimen for postinfections. Among those trials, a recent study declared the efficacy of an antiviral combination of favipiravir (FAV) and molnupiravir (MLP). The combined regimen helped in a successful 60% eradication of the SARS-CoV-2 virus from the lungs of studied hamster models. Moreover, it prevented viral transmission to cohosted sentinels. Because both medications are orally bioavailable, the coformulation of FAV and MLP can be predicted. The developed study is aimed at developing new green and simple methods for the simultaneous determination of FAV and MLP and then at their application in the study of their dissolution behavior if coformulated together. A green micellar HPLC method was validated using an RP-C18 core-shell column (5 μm, 150 × 4.6 mm) and an isocratic mixed micellar mobile phase composed of 0.1 M SDS, 0.01 M Brij-35, and 0.02 M monobasic potassium phosphate mixture and adjusted to pH 3.1 at 1.0 mL min−1 flow rate. The analytes were detected at 230 nm. The run time was less than five minutes under the optimized chromatographic conditions. Four other multivariate chemometric model methods were developed and validated, namely, classical least square (CLS), principal component regression (PCR), partial least squares (PLS-1), and genetic algorithm–partial least squares (GA–PLS-1). The developed models succeeded in resolving the great similarity and overlapping in the FAV and MLP UV spectra unlike the traditional univariate methods. All methods were organic solvent-free, did not require extraction or derivatization steps, and were applied for the construction of the simultaneous dissolution profile for FAV tablets and MLP capsules. The methods revealed that the amount of the simultaneously released cited drugs increases up until reaching a plateau after 15 and 20 min for FAV and MLP, respectively. The greenness was assessed on GAPI and found to be in harmony with green analytical chemistry concepts.

The tendency of using weight loss herbal preparations is continuously increasing, especially for the widespread consumption of junk food that is characterized by high calories. Weight loss herbal preparations are considered a type of food supplement product, and, as such, the regulations governing their quality control might be minimal. These products could be locally formulated in any country or internationally imported. Being non-controlled products, the herbal weight-loss products may contain high levels of elemental impurities that might exceed the permissible ranges. Moreover, these products contribute to the total daily intake (TDI) of such elements, which might represent concerns about their potential toxicological danger. In this research, the elemental contents in such products were investigated. The inductively coupled plasma with optical emission spectrometer (ICP-OES) was used to determine the levels of 15 elemental contents, namely, Na, K, Ca, Mg, Al, Cu, Fe, Li, Mn, As, Co, Cr, Cd, Ni and Pb. The results showed that seven micro-elements, namely Cd, Co, Ni, Cr, Pb, Li and Cu, were either not detectable or at a concentration much lower than their tolerable limits. However, all studied macro-elements (Na, K, Ca and Mg), together with Fe, were found at considerable, yet safe levels. On the other hand, Mn, Al and As contents showed perturbing levels in some of the studied products. Finally, a conclusion was highlighted for the necessity for stricter surveillance of such herbal products.

Four eco-friendly, cost-effective, and fast stability-indicating UV-VIS spectrophotometric methods were validated for cefotaxime sodium (CFX) determination either in the presence of its acidic or alkaline degradation products. The applied methods used multivariate chemometry, namely, classical least square (CLS), principal component regression (PCR), partial least square (PLS), and genetic algorithm-partial least square (GA-PLS), to resolve the analytes’ spectral overlap. The spectral zone for the studied mixtures was within the range from 220 to 320 nm at a 1 nm interval. The selected region showed severe overlap in the UV spectra of cefotaxime sodium and its acidic or alkaline degradation products. Seventeen mixtures were used for the models’ construction, and eight were used as an external validation set. For the PLS and GA-PLS models, a number of latent factors were determined as a pre-step before the modelsʹ construction and found to be three for the (CFX/acidic degradants) mixture and two for the (CFX/alkaline degradants) mixture. For GA-PLS, spectral points were minimized to around 45% of the PLS models. The root mean square errors of prediction were found to be (0.19, 0.29, 0.47, and 0.20) for the (CFX/acidic degradants) mixture and (0.21, 0.21, 0.21, and 0.22) for the (CFX/alkaline degradants) mixture for CLS, PCR, PLS, and GA-PLS, respectively, indicating the excellent accuracy and precision of the developed models. The linear concentration range was studied within 12–20 μg mL–1 for CFX in both mixtures. The validity of the developed models was also judged using other different calculated tools such as root mean square error of cross validation, percentage recoveries, standard deviations, and correlation coefficients, which indicated excellent results. The developed methods were also applied to the determination of cefotaxime sodium in marketed vials, with satisfactory results. The results were statistically compared to the reported method, revealing no significant differences. Furthermore, the greenness profiles of the proposed methods were assessed using the GAPI and AGREE metrics.

Virtual screening of the potential lead chemotherapeutic phytochemicals from medicinal plants has useful application in the field of in-silico modelling and computer-based drug design by orienting and scoring ligands in the active binding site of a target protein. The phytochemical investigation of the Pterocephalus frutescens extract in n-butanol resulted in the isolation and structure elucidation of three iridoids and four flavonoids which were identified as Geniposide (1), Geniposidic acid (2), Nepetanudoside C (3), Isovitexin (4), Luteolin-7-O-glucoside (5) Isoorientin (6) and Orientin (7), respectively. Molecular docking studies were used to compare the binding energies of the isolated phytochemicals at four biological cancer-relevant targets; namely, aromatase, carbonic anhydrase IX, fatty acid synthase, and topoisomerase II-DNA complex. The docking study concluded that the isolated compounds have promising cytotoxic activities, in particular, Luteolin-7-O-glucoside (5) and Orientin (7) which exhibited high binding affinities among the isolated compounds at the active sites of the target enzymes; Aromatase (−8.73 Kcal/mol), and Carbonic anhydrase IX (−8.92 Kcal/mol), respectively, surpassing the corresponding binding scores of the co-crystallized ligands and the reference drugs at these target enzymes. Additionally, among the isolated compounds, Luteolin-7-O-glucoside (5) showed the most outstanding binding affinities at the active sites of the target enzymes; Fatty acid synthase, and Topisomerase II-DNA complex with binding scores of −6.82, and −7.99 Kcal/mol, respectively. Finally, the SwissADME online web tool predicted that most of these compounds possessed acceptable oral bioavailability and drug likeness characteristics.

Continuous health monitoring in a vehicle enables the earlier detection of symptoms of cardiovascular diseases. In this work, we designed flexible and thin electrodes made of polyurethane for long-term electrocardiogram (ECG) monitoring while driving. We determined the time for reliable ECG recording to evaluate the effectiveness of the electrodes. We recorded data from 19 subjects under four scenarios: rest, city, highway, and rural. The recording time was five min for rest and 15 min for the other scenarios. The total recording (950 min) is publicly available under a CC BY-ND 4.0 license. We used the simultaneous truth and performance level estimation (STAPLE) algorithm to detect the position of R-waves. Then, we derived the RR intervals to compare the estimated heart rate with the ground truth, which we obtained from ECG electrodes on the chest. We calculated the signal-to-noise ratio (SNR) and averaged it for the different scenarios. Highway had the lowest SNR (−6.69 dB) and rural had the highest (−6.80 dB). The usable time of the steering wheel was 42.46% (city), 46.67% (highway), and 47.72% (rural). This indicates that steering-wheel-based ECG recording is feasible and delivers reliable recordings from about 45.62% of the driving time. In summary, the developed electrodes allow continuous in-vehicle heart rate monitoring, and our publicly available recordings provide the opportunity to apply more sophisticated data analytics.

Artificial intelligence (AI) technologies enable Conversational Agents (CAs) to perform highly complex tasks in a human-like manner and may help people cope with anxiety to improve their mental health and well-being. To support patients with their mental well-being in an authentic way, CAs need to be imbued with human-like behavior, such as personality. In this paper we cover an innovative form of CA, so-called Personality-Adaptive Conversational Agents (PACAs) that automatically infer users’ personality traits and adapt accordingly to their personality. We empirically investigate their benefits and caveats in mental health care. The results of our study show that PACAs can be beneficial for mental health support, but they also raise concerns about trust and privacy issues. We present a set of relevant requirements for designing PACAs and provide solution approaches that can be followed when designing and implementing PACAs for mental health care. Publishers version

To improve the predictive value of in vitro experimentation, the use of 3D cell culture models, or organoids, is becoming increasingly popular. However, the current equipment of life science laboratories has been developed to deal with cell monolayers or cell suspensions. To handle 3D cell aggregates and organoids in a well-controlled manner, without causing structural damage or disturbing the function of interest, new instrumentation is needed. In particular, the precise and stable positioning in a cell bath with flow rates sufficient to characterize the kinetic responses to physiological or pharmacological stimuli can be a demanding task. Here, we present data that demonstrate that microgrippers are well suited to this task. The current version is able to work in aqueous solutions and was shown to position isolated pancreatic islets and 3D aggregates of insulin-secreting MIN6-cells. A stable hold required a gripping force of less than 30 μN and did not affect the cellular integrity. It was maintained even with high flow rates of the bath perfusion, and it was precise enough to permit the simultaneous microfluorimetric measurements and membrane potential measurements of the single cells within the islet through the use of patch-clamp electrodes.

Hepatitis B and C are viruses causing liver infections and resulting in grave secondary diseases. While there are different treatments for chronic liver infections, the process of evolving chronic diseases is still not fully understood. This paper presents an economic-inspired model for the overall health of an infected organism. The health model is based on the results of a reaction diffusion model for describing the space-dependent dynamics of virus and T cells during a liver infection. The different treatments affect the parameters of the reaction diffusion model and influence therefore the well-being of the infected person during an infection. The health model is selected in a detailed process out of a class of possible models. The presented work provides a foundation for an optimal control problem for finding the best treatment strategy.

A capillary zone electrophoresis method was developed for the determination of NiII(3-OMe-salophene), a substance with anticancer activity in vitro. A fused silica capillary (56 cm × 100 µm) was used for this purpose. The method was optimized in terms of parameters affecting the electrophoretic conditions in order to optimize separation efficiency and total time of migration. The analysis was best performed using an operating buffer of 50 mM borate, adjusted to pH 9.3, mixed with acetonitrile (50%, v/v) as organic modifier. Injections were performed hydrodynamically by applying a pressure of 50 mbar for 8 s, and a 30 kV separation voltage was selected at 25 °C. Detection was carried out at 250 nm using diode array detector (DAD). The method allowed the separation of NiII(3-OMe-salophene) from four other structurally related impurities in a total migration time (tm) of 8 min. Peak identification was achieved using the standard reference of individual impurities. The purity of the migrated NiII(3-OMe-salophene) was confirmed by Ultra-violet (UV) scan overlay depending on DAD. The linear ranges for the determination of NiII(3-OMe-salophene) was 400–20,000 ng mL−1 with limit of detection (LOD) of 120 ng mL−1. Acceptable intra-day and inter-day precisions were achieved (%relative standard deviation (RSD) results were less than 0.76% and 0.30%, respectively). The proposed method was assessed for greenness and compared to reported methodologies to prove superiority.

This study covers chiral analysis for the enantioseparation and binding affinity determination of calcium antagonist drugs using polysaccharide-based and protein-based chiral selectors by chromatographic and electrophoretic techniques. An in-house generated polysaccharide-based column was employed in chiral chromatography, while chiral background electrolyte additives were utilized in capillary electrophoresis. Three experimental studies and computational modeling were conducted using amlodipine and verapamil as drug models. A chondroitin sulfate-based column was successfully generated through reductive amination on a monolithic epoxy silica support and employed as a chiral stationary phase in liquid chromatography. The separation of enantiomers of selected drugs as pure compounds, as well from tablet matrices, was successfully carried out. Systematic optimization with a minimum number of experiments in maltodextrin-based electrokinetic chromatography was set up by applying a design of experiments approach to investigate factors affecting separation. The D-optimal design enabled a strategy to obtain the desired resolution of enantiomers by identifying the most important separation factors. Enantioseparation and simultaneous binding affinity determination of the selected chiral drugs toward human serum albumin were performed using mobility shift-affinity capillary electrophoresis. Association constants were determined through nonlinear regression of effective mobilities as a function of total ligand concentrations. Enantiomers of the employed drug models showed acceptable resolutions, but a sufficient description of the binding curves was necessary to obtain a reliable binding constant. Once a sufficient binding description has been obtained, both enantioseparation and simultaneous binding affinity determination can be performed. Computational modeling was utilized to complement the experimental findings by revealing relevant molecular interactions for enantiorecognition. The selector-selectand complexes with the lowest binding energies were visualized in a qualitative manner. This docking approach is relevant to have an early hint to the interaction of racemic drugs toward their binding partners, which might lead to the development of efficient chiral analysis in the future., Diese Arbeit befasst sich mit der chiralen Analyse zur Enantiomerenerkennung und der Bestimmung der Bindungsaffinität von Calciumantagonisten unter Verwendung von chiralen Selektoren auf Polysaccharid- und Proteinbasis durch chromatographische und elektrophoretische Techniken. Für die chirale Chromatographie wurde eine selbst entwickelte Säule auf Polysaccharidbasis verwendet, während für die Kapillarelektrophorese chirale Hintergrundelektrolytzusätze eingesetzt wurden. Mit den Arzneistoffmodelle Amlodipin und Verapamil wurden drei experimentelle Studien sowie Computermodellierungen mit als durchgeführt. Die selbst entwickelte Säule auf Chondroitinsulfatbasis wurde erfolgreich durch reduktive Aminierung auf einem monolithischen Epoxysilikat-Träger hergestellt und als chirale stationäre Phase in der Flüssigchromatographie eingesetzt. Die Trennung von Enantiomeren gelang hierbei sowohl in ihrer reinen Form als auch aus den Tablettenmatrices der Arzneimittel. Durch eine Versuchsplanung wurde eine systematische Optimierung der die Trennung beeinflussenden Faktoren, mit einer minimalen Anzahl von Experimenten in der elektrokinetischen Chromatographie auf Maltodextrinbasis erreicht. Das D-optimale Design ermöglichte eine Strategie zur Erzielung der gewünschten Enantiomerentrennung durch Identifizierung der wichtigsten Trennfaktoren. Die Enantiomerenerkennung und die simultane Bindungsaffinitätsbestimmung der ausgewählten chiralen Arzneistoffe gegenüber Humanalbumin wurden mit Hilfe der mobility shift-affinity capillary electrophoresis durchgeführt. Die Assoziationskonstanten wurden durch nichtlineare Regression aus den effektiven Mobilitäten als Funktion der gesamten Ligandenkonzentrationen bestimmt. Die Enantiomere der verwendeten Arzneistoffmodelle zeigten akzeptable Auflösungen, jedoch war eine ausreichende Beschreibung der Bindungskurven erforderlich, um eine zuverlässige Bindungskonstante zu erhalten. Sobald eine ausreichende Beschreibung der Bindung vorlag, könnten sowohl die Enantiomerenerkennung als auch die gleichzeitige Bindungsaffinität durchgeführt werden. Computergestützte Modellierung diente der Ergänzung der experimentellen Ergebnisse, durch aufzeigen der relevanten molekularen Wechselwirkungen für die Enantiomerenerkennung. Die Selektor-Selektand-Komplexe mit den niedrigsten Bindungsenergien wurden auf diese Weise qualitativ visualisiert. Dieser Docking-Ansatz ist relevant, um einen frühen Hinweis auf die Wechselwirkungen von racemischen Arzneistoffen mit ihren Bindungspartnern zu erhalten, was in Zukunft zur Entwicklung einer effizienten chiralen Analyse führen könnte.

The discovery of antibiotics revolutionized modern medicine. Their discovery, however, was accompanied by the finding, that bacteria are able to develop mechanisms escaping the treatment with antibiotic compounds. Today, the development of antimicrobial resistance poses one of the most significant threads to public health world-wide. The gram-negative, rod-shaped opportunistic pathogen Pseudomonas aeruginosa is well known for its ability to thrive in numerous environments. This includes soil, water, and nearly every human tissue. It is the cause of severe nosocomial infections in immune compromised individuals such as for example patients suffering from the genetic disorder cystic fibrosis. In the latter, P. aeruginosa often develops life-long chronic infections. P. aeruginosa constantly evolves to adapt to its human habitat causing recurring exacerbations of lung function. Consequently, it is the major cause of morbidity and mortality in these patients P. aeruginosa infected patients receive an extensive antibiotic treatment, that is, however, often resulting in high-level antibiotic resistance in the pathogen. In this thesis, I investigated two major patho-adaptative mechanisms in the context of P. aeruginosa infections: Firstly, the mutational-induced overexpression of the efflux pump MexXY, that is known to transport a variety of antimicrobial classes from the bacterial cytosol into the extra-cytoplasmic space. I discovered a so far undetected dependency of the important resistance factor of aminoglycoside modifying enzymes on the activity of the efflux pump MexXY to reach high-level aminoglycoside resistance. Secondly, I investigated the transcriptional imprint of inactivating mutations in the gene sequence of the global regulator LasR that are regularly found in clinical P. aeruginosa isolates. I hypothesize that the inactivation of this global regulator is not abolishing the expression of certain genes per se but alters their expression dynamics. This shift in the timing of gene expression was dependent on the activity of the global regulator RhlR. I combined state of the art sequencing data with classical microbiological methods in order to gain global insights into the adaptation mechanisms of this important pathogen., Die Entdeckung der Antibiotika hat die moderne Medizin revolutioniert. Ihre Entdeckung wurde jedoch von der Erkenntnis begleitet, dass Bakterien in der Lage sind, Mechanismen zu entwickeln, um der Behandlung mit antibiotischen Wirkstoffen entgehen. Heute stellt die Entwicklung der Antibiotikaresistenzen eines der größten Probleme für die öffentliche Gesundheit weltweit dar. Das gramnegative, stäbchenförmige opportunistische Pathogen Pseudomonas aeruginosa ist für seine Fähigkeit bekannt, in zahlreichen Umgebungen, wie z.B. Boden, Wasser und nahezu jedes menschliche Gewebe zu überleben. Es ist die Ursache schwerer nosokomialer Infektionen bei immungeschwächten Personen wie z.B. Patienten mit der Erbkrankheit Mukoviszidose. Hier entwickelt P. aeruginosa oft lebenslange chronische Infektionen, während derer es sich stetig an das menschliche Habitat anpasst. Dies führt zu wiederkehrenden Exazerbationen der Lungenfunktion. Folglich ist es die Hauptursache für Morbidität und Mortalität bei diesen Patienten. P. aeruginosa-infizierte Personen erhalten eine intensive Antibiotikatherapie, die jedoch häufig zu hochgradigen Antibiotikaresistenzen des Erregers führt. In dieser Arbeit habe ich zwei wesentliche patho-adaptive Mechanismen von P. aeruginosa-Infektionen untersucht: Zum einen die mutationsbedingte Überexpression der Effluxpumpe MexXY, die eine Vielzahl von Antibiotika-Klassen aus dem bakteriellen Zytosol in den extrazytoplasmatischen Raum transportieren kann. Dabei identifizierte ich eine bisher unentdeckte Abhängigkeit des wichtigen Resistenzfaktors der Aminoglykosid-modifizierenden Enzyme von der Aktivität der Effluxpumpe MexXY, um hochgradige Aminoglykosidresistenz hervorzurufen. Zum anderen untersuchte ich den transkriptionellen Fußabdruck von inaktivierenden Mutationen in der Gensequenz des globalen Regulators LasR, die regelmäßig in klinischen P. aeruginosa Isolaten detektiert werden. Ich stelle die Hypothese auf, dass die Inaktivierung dieses globalen Regulators nicht die Expression bestimmter Gene per se aufhebt, sondern deren Expressionsdynamik verändert. Diese Verschiebung im Timing der Genexpression war abhängig von der Aktivität des globalen Regulators RhlR. Ich habe dazu modernste DNA- und RNA-Sequenzierungsdaten mit klassischen mikrobiologischen Methoden kombiniert, um umfassende Einblicke in die Anpassungsmechanismen dieses wichtigen Pathogens zu gewinnen.

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