1 Summary 1.1 Overview of Presented Topics In this cumulative dissertation the following topics are presented: 1) Interfacial modulation of MoS2 with oxo-functionalized graphene and its derivatives a. Oxo-functionalized graphene/MoS2 and reduced oxo-functionalized graphene/MoS2 heterostructures b. Porous oxo-functionalized graphene/MoS2 2) Interfacial modulation of laser-induced functional graphene/MoS2 heterostructures Oligophenyl-functionalized graphene/MoS2 The research was conducted in collaboration with the groups of 1) Dr. Patryk Kusch from the Department of Physics at Freie Universität Berlin; 2) Prof. Dr. Hyeon S. Shin from the Department of Chemistry at Ulsan National Institute of Science and Technology. The complete results and experimental details are included in the attached publications in Chapter 5 and 6. To keep the explanation of heterostructures concise, we define the vertical stacking order of the heterostructure layers as (substrate/)bottom layer/upper layer. 1.2 Summary of Results 1.2.1 Interfacial modulation of MoS2 with oxo-functionalized graphene and its derivatives a. Oxo-functionalized graphene/MoS2 and reduced oxo-functionalized graphene/MoS2 heterostructures Tuning the electronic and optical properties of monolayer MoS2 and gaining profound insights into the fundamental mechanisms that govern these properties is of utmost significance for the development of efficient optoelectronic devices, such as photodetectors, photodiodes etc. Intrinsic structural defects of monolayer MoS2, such as S vacancies, induce electrons gathering in neighboring Mo atoms, which function as nonradiative traps, thereby impairing the photoluminescence (PL) efficiency. Stacking MoS2 layers on different two-dimensional (2D) materials, such as hexagonal boron nitride (h-BN) and graphene, provides a way to modulate the PL performance. Oxygen-functionalized graphene (oxo-G), a graphene derivative, has a defective graphene network with oxygen species decorating the edges and the plane. Electron-withdrawing groups, such as hydroxyl, epoxy, and organosulfates, make oxo-G a p-doping material. By reducing oxo-G (r-oxo-G), sp2-hybridized graphene domains with a lateral size of up to 10 nm are recovered with the removal of most oxygen groups. During the reduction process, in-plane defects such as vacancies, holes, and non-six-membered carbon rings with sp3 hybridization are formed, which act as structural motifs or active sites and significantly change the electronic and surface properties of roxo-G. Therefore, the use of oxo-G and r-oxo-G is suggested for tuning the carrier concentration of MoS2. Herein, heterostructures of monolayer MoS2 with three types of monolayer graphene are fabricated: mechanically exfoliated pristine graphene, oxo-G (a high amount of oxygen of 60%), and r-oxo-G (a defect density of 0.5%). Raman and PL spectroscopy combined with Kelvin probe force microscopy (KPFM, collaboration with the Shin group, Ulsan National Institute of Science and Technology) measurements are carried out to study optoelectronic properties and mechanism of interface interaction. Oxo-G with a work function (WF) of 5.67 eV and r-oxo-G with a WF of 5.85 eV serving as hole injection layers significantly enhance the PL intensity of MoS2, whereas pristine graphene with a WF of 5.02 eV resulted in PL quenching of MoS2. The electron-withdrawing functional groups of oxo-G and the defects in r-oxo-G layers facilitate the recombination of neutral exciton and result in PL enhancement. Furthermore, the r-oxo-G/MoS2 heterostructure exhibits a higher increase (5-fold) in the overall PL intensity than the oxo-G/MoS2 (3-fold) heterostructure. Our research demonstrates the PL modulation of monolayer MoS2 by monolayer graphene with a varying ability in extracting electrons. The enhancement of PL plays a vital role in high performance optoelectronic devices by improving photovoltaic efficiency, sensitivity, and photoresponse etc. b. Porous oxo-functionalized graphene/MoS2 The in-plane lattice defects in r-oxo-G can affect the electron transfer between graphene and MoS2, leading to an enhancement in the PL of MoS2. To investigate interfacial charge transfer and PL performance, porous graphene with large lattice defects is prepared and stacked with MoS2. Oxo-G with a low density of initial vacancy defects (0.8%) is used as a precursor to etch pores assisted by a Mn-species at 400 °C in Ar atmosphere. By controlling the reaction conditions, it is possible to gain a certain control over the size of pores on porous oxo-G (Pr-oxo-G) with diameters between 100–200 nm. The PL of MoS2 on SiO2, oxo-G, Pr-oxo-G6h (etching time of 6 h), and Pr-oxoG12h (etching time of 12 h) are studied. The amplitudes of the PL are increased for oxo-G/MoS2 (4 times), Pr-oxo-G6h/MoS2 (3 times), and Pr-oxo-G12h/MoS2 (10 times), compared to the PL of the pristine MoS2 monolayer. Overall, Pr-oxoG12h reflects a p-doped material, as indicated by Raman shifts, achieving the highest PL enhancement. The Pr-oxoG12h/MoS2 PL intensity map measured by scanning nearfield optical microscopy (s-SNOM) with nano-scale resolution shows a constant PL intensity over the MoS2 flake, exhibiting no sign of a spatial PL modulation that may arise from free-standing MoS2. (collaboration with Dr. Patryk Kusch, FU Berlin) Furthermore, the Pr-oxo-G6h/MoS2 showed slightly lower PL intensity than oxo-G/MoS2. The Mn-impurities in Pr-oxo-G6h/MoS2 are supposed to limit the increase of the PL of MoS2, and the interaction of Mnspecies with carbonyl groups may be responsible. 1.2.2 Interfacial modulation of laser-induced functional graphene/MoS2 heterostructures Oligophenyl-functionalized graphene/MoS2 The electron-withdrawing effects of the functional groups and lattice defects in graphene have been demonstrated to enhance the PL intensity of monolayer MoS2. In addition, Interlayer van der Waals interactions and interlayer distance are very important factors in studying the PL of graphene/MoS2 heterostructures (G/MoS2) as they are only a few atomic thin. Functionalization of graphene with specific functional groups is of great significance for the further development of covalent modification of graphene and the interface construction in G/MoS2 heterostructures, thereby facilitating the study of interlayer coupling of G/MoS2 heterostructures. Monotopic covalently modified graphene, oligophenyl-functionalized graphene (F-G), are prepared by a laser-induced reaction and stacked with a monolayer MoS2. The functionalization of graphene is regioselective with the assistance of the mapping function of the scanning Raman spectrometer. Through Raman, PL, KPFM and scanning near-field optical microscopy (collaboration with Dr. Patryk Kusch, FU Berlin) measurements, the boundaries and the distinct characteristics of the functionalized and the non-functionalized areas are identified on the heterostructure. More importantly, the layer stacking sequence of F-G and MoS2 brings different interface structures in perpendicular orientation. MoS2 supported by F-G (F-G/MoS2) results in a sandwiched structure consisting of graphene/oligophenyl-groups/MoS2 with an enlarged interlayer distance of 8 nm between the graphene basal plane and MoS2. In the case of MoS2 stacked underneath F-G (MoS2/F-G) a direct interface is formed between the graphene basal plane and MoS2, with the oligophenyl-groups located on the top surface of the heterostructure. The different interfaces in the heterostructures result in a significant difference in the PL enhancement of MoS2. F-G/MoS2 shows a 5-fold PL enhancement, while MoS2/F-G only shows a 1.8-fold PL enhancement compared to pristine G/MoS2. Accordingly, the results indicate that the oligophenyl-groups in F-G/MoS2 not only have a pdoping effect on MoS2 but also largely prevent electron donation from the graphene basal plane with the enlarged interlayer distance. Consequently, the PL enhancement is restored with the thermal de-functionalization of F-G. Thus, we conclude that the functional groups can be considered as separate molecular component with the vertical arrangement in the functionalized heterostructure system. The photoactive graphene acts as a template for perpendicular molecular alignment in the heterointerface construction, thus opening more possibilities for the fabrication of heterointerfaces. In this thesis, the interfaces of G/MoS2 are engineered through the introduction of oxo-functional groups, structural defects, and laser-induced perpendicular functional-groups on graphene. The interfacial modulation via interlayer charge transfers and interlayer distances results in significant changes in the PL properties of G/MoS2 heterostructures. These findings offer novel insights into the design and exploration of optoelectronic devices. Furthermore, the PL enhancement of G/MoS2 opens up numerous possibilities for optoelectronic applications, for instance, wavelength-tunable phototransistors, broadband photodetectors, single-photon emission sites for quantum information science, improved electrical performance for high-speed optoelectronics, and signal enhancement in photodetectors and sensors. Based on the findings in this thesis, there are still ample opportunities for future research to continue this study. First, the development of diverse interlayer functional groups with diverse electron transfer capabilities and spatial effects can be achieved through laser-induced functionalization methods to realize a more versatile interface modulation. Second, further investigation into the interface modulation of different 2D materials beyond graphene and MoS2 is expected. Third, a thorough fundamental understanding of hetero-interfaces is required. A deeper insight into the effects of interface modulation on the electronic structure, band alignment, and van der Waals interactions of heterostructures can be achieved through theoretical studies and techniques such as charge transport measurements, ultrafast optical spectroscopy, and magnetic characterization.
View lessAnimals need to switch behavioral strategies to adapt to changing environmental conditions. The ability to choose the most advantageous response to a situation is crucial for survival. These decisions are influenced by environmental factors and internal signals such as physiological needs. During foraging behavior, larval zebrafish approach potential preys and avoid potential predators. The choice is influenced by external factors, such as the size of the object they are interacting with: small visual stimuli are perceived as preys while large objects are considered predators. Moreover, it was previously shown that behavioral choice during feeding behavior is modulated by metabolic state: food-deprived larvae are more likely to take risks during hunting and approach small, prey-like objects compared to fed fish. It was also demonstrated that this modulation is mediated by a differential processing of visual stimuli. In zebrafish the visual information is relayed from the retina to a midbrain structure, the optic tectum, where sensory inputs are integrated. Distinct downstream motor centers are subsequently activated to perform either an approach or an escape. This makes the optic tectum a major center for decision-making. In food-deprived larvae, tectal neurons respond preferentially to small visual stimuli compared to fed siblings, showing a shift of the tuning towards cues important for survival. These results suggest feeding induces a change in the excitability of tectal neurons to modulate behavioral choice, however the molecular mechanisms underlying this phenomenon are still unclear. To fill the gap by investigating the molecular pathways mediating the influence of metabolic state on behavioral choice, a proteomic study was performed to identify proteins differentially abundant in fed versus food-deprived larvae. Among all the hits, an especially interesting candidate was selected: a small peptide known to modulate neuronal excitability, Pcp4a, which was less abundant in fed larvae compared to food-deprived fish. pcp4a mRNA levels were also found to be lower in brain samples from fed larvae, suggesting that feeding modulates its expression through a transcriptional mechanism. PCP4, the mammalian ortholog of Pcp4a is known to bind to calmodulin, a molecule involved in several processes during neuronal activation, and to inhibit its target enzymes such as CaMKII. This results in modulation of neuron excitability in vivo. Since pcp4a is expressed in the optic tectum in zebrafish, we hypothesized that it may play a role in the regulation of tectal neurons excitability by feeding state. To test this hypothesis, I first looked at the role of pcp4a in the modulation of behavioral choice by feeding state using a loss-of-function model. Food-deprived larvae lacking Pcp4a show increased avoidance of small objects in a size discrimination assay, thus phenocopying behavioral choice of fed larvae. To understand if the effect was due to modulation of tectal neurons excitability, I investigated the response to visual stimuli of different size in tectal neurons expressing pcp4a (pcp4a+). Tectal pcp4a+ neurons in fed larvae responded preferentially to large objects compared to starved siblings; the same response profile was observed in larvae lacking Pcp4a, suggesting that Pcp4a mediates the effect of feeding on tectal neurons excitability. I then looked into the neuromodulatory mechanisms mediating the influence of metabolic signals on pcp4a expression. I found that feeding increased activity of dopaminergic neurons in the pretectum and hypothalamus, which could exert an effect on tectal pcp4a+ neurons through their direct and indirect projections to the optic tectum. Indeed, pharmacological activation of dopaminergic signaling through D2 receptors decreased pcp4a expression in food-deprived larvae, mimicking the effect of feeding. Dopamine controls pcp4a transcription through the D2 receptor - cAMP signaling cascade. Pharmacological activation of dopaminergic signaling induced a shift of the response profile of pcp4a+ tectal neurons towards large stimuli by altering the tuning properties of individual neurons. In this study we elucidate a novel molecular mechanism mediating the effect of metabolic state on behavioral choice in zebrafish. In our model, feeding activates dopaminergic neurons in the pretectum and hypothalamus, which project to the optic tectum. Dopaminergic signaling through D2 receptors induces a decrease of pcp4a expression through a transcriptional mechanism, which results in a shift of the response profile of pcp4a+ tectal neurons towards large visual stimuli through a cell-autonomous mechanism. This ultimately leads to increased avoidance of small stimuli during foraging behavior. This study advances our knowledge of the molecular mechanisms mediating neuromodulation of decision-making behavior.
View lessThis dissertation examines the construction and instrumentalisation of Generation Y as a discourse in the US and its translation to Turkey. It is guided by the inquiry to understand the enduring emphasis and popularity of generational thinking, despite the lack of empirical evidence and its theoretical shortcomings.
The analysis examines, in sequential order, influential actors shaping the Gen Y discourse in the US, uncovering implicit and explicit claims associated with it. The focus then shifts to Turkey, where the discourse gained prominence during the Gezi Park Protests, exploring local influencers and delving into the evolution and translation of Generation Y discourse within this unique context. The investigation extends to companies in Turkey, examining how employees and companies relate to and instrumentalise the Gen Y discourse in their management practices.
To address these inquiries, the dissertation adopts a constructivist approach, combining thematic analysis of texts on Gen Y from mainstream newspapers, popular business magazines, consultancy reports, and bestselling books in Turkish and English, with the in-depth interviews conducted with white-collar workers in Turkey.
The dissertation highlights the role of management and consultancy practitioners in constructing and disseminating the Generation Y discourse, suggesting that they employ strategies similar to those used for popular management fads and fashions, including the use of an ambiguous and polysemous language. The analysis reveals that, despite theoretical controversies, Generation Y is portrayed as a scientific concept based on birth cohorts while simultaneously drawing strategically on historical connotations of generational identity, aligning itself with alternative understandings of generations (such as kinship structures and youth as a driving force in shaping history). This research suggests that the Gen Y discourse is deeply embedded in the idea of a “new” era marked by digitalisation and widespread internet use. In this context, "generational conflicts" and "becoming outdated" are presented as a single problem, forming a central message within the discourse. Consequently, Gen Y discourse extends beyond addressing generational conflicts or explaining youth movements; it is presented as a panacea offering solutions to all current and foreseeable future problems.
View lessThe development of metal-nitrogen-carbons (M-N-Cs) has gained significant momentum in the 21st century, driven by their potential as sustainable alternatives to precious metals for electrocatalytic applications. However, as the pyrolytic synthesis often produces highly disordered carbonized materials, it can be challenging to accurately characterize the active sites and their involvement in the catalytic reactions. In this regard, quantum chemical methodologies like density functional theory (DFT) could provide crucial atomic-level information to guide the characterization and optimization of M-N-Cs. In this thesis, I present computational studies related to the synthesis of M-N-Cs in the first part, followed by their electrocatalytic applications in the second part. In parallel to these studies, potential factors for the material degradation during the synthesis and applications are also identified, especially for the generally active but unstable Fe-N-Cs.
Inspired by the recent success of the templating-transmetallation strategy in producing M-N-Cs with dense active sites, the first part of the thesis features a computational investigation on the chemical properties of metals at the MN4 sites. By constructing and optimizing structural models for the incorporation of MN4 motifs in conductive carbon matrices, we determined several advantages of using molecular DFT methods for modeling the active sites, especially the structurally complex pyrrolic MN4 motifs. Using these models, we further examined the trends among the first-row transition metals pertaining their binding within the MN4 site as well as their relative affinity to the pyrrolic or pyridinic motifs. It could be shown that metals with more significant ionic binding character, such as Zn and Mg, are more likely to form the pyrrolic MN4 sites than the catalytically active metals like Fe and Co, leading to the successful characterization of the pyrrolic MN4 sites from synthesis strategies which utilized Zn2+ or Mg2+ as the sacrificial templating ions. In addition, the binding of Zn was determined to be the weakest among the transition metals, making it an optimal choice for the subsequent transmetallation.
As the transmetallation step generally retains the original structure of the active site, in addition to its morphological properties and active site densities, it became a crucial starting point for intrinsic activity comparisons among metals in the M-N-Cs. In the second part of this thesis, the electrocatalytic mechanisms for the oxygen reduction reaction (ORR) and the carbon dioxide reduction reaction (CO2RR) on the previously identified pyrrolic MN4 sites are presented along with corresponding side reactions including the Fenton reaction and the hydrogen evolution reaction (HER). The combined efforts of theory and experiment led to the identification of inconspicuous species at the interface, such as hydroxides for alkaline ORR, water for the Fenton reaction, and cations for the CO2RR, which in fact play an active role in the mechanisms of the respective reactions. For the ORR, we determined the involvement of the outer-sphere electron transfer in the reaction mechanism, especially in the alkaline medium, leading to the formation of H2O2 whose subsequent reactions could depend on specific interactions with the MN4 site. For the CO2RR, a theory-experiment contradiction in the activity and selectivity of Ni-N-Cs could be resolved with the inclusion of alkali metal ions in the mechanism, whose adsorption was made favorable by the pyrrolic N atoms at the MN4 site. Overall, these insights derived with DFT provide strong theoretical support for the knowledge-based optimization of the activity, selectivity and stability of M-N-Cs for sustainable energy conversion applications.
View lessIn dieser Habilitationsarbeit wurden hochspezifische pathophysiologische Konzepte im Kontext verschiedener klinischer Präsentationen einiger ausgewählter neuromuskulärer Erkrankungen untersucht. Dabei kam ein Spektrum von experimentellen, morphologischen und klinischen Techniken zum Einsatz, um verschiedene Aspekte der jeweiligen Erkrankung zu untersuchen. Im Kontext inflammatorischer (IBM, SSc) und hereditärer (MADD, Danon-Erkrankung) Myopathien konnten morphologische und molekulare Muster herausgearbeitet werden, die mit der klinischen Präsentation korrelieren. Darüber hinaus wurde das bisherige pathophysiologische Verständnis der IBM erweitert und das darauf basierende Konzept der IBM-Spektrumserkrankung eingeführt. Im Rahmen dieser Arbeiten wurden auch mögliche Kandidaten für einen neuen IBM-Biomarker identifiziert. Zudem wurden sowohl im Kontext hereditärer (Danon-Erkrankung), wie auch erworbener Myopathien (IBM/PM-Mito) relevante mitochondriale Veränderungen identifiziert, denen ein bislang nicht ausreichend verstandener Anteil an der Pathophysiologie und dem Phänotyp der Erkrankungen zukommt. Schließlich wurde das klinische Spektrum der TTR-Amyloidose erweitert und die ATTRwt neben der ATTRv als behandelbare Ursache für Polyneuropathien in höherem Lebensalter identifiziert. Zusammengefasst stellen die hier präsentierten klinischen und experimentellen Arbeiten die Grundlage für weitere Forschungsprojekte dar, die, gleichberechtigt zum wissenschaftlichen Erkenntnisgewinn, die Verbesserung der Versorgung von Patient*innen mit neuromuskulären Erkrankungen zum Ziel haben werden.
View lessDuring lactation, the gut mucosa undergoes expansion to accommodate increased food consumption and absorption, in order to meet the increased energy demands associated with nurturing neonates. Previous studies have investigated the impact of litter size, hormones, and diet on lactation-induced gut hyperplasia, yet the role of immune cells remained unexplored. The recently discovered innate lymphocytes type 3 (ILC3s) have emerged as key players not only in organizing lymphoid tissues but also in protecting the intestinal epithelium against tissue damage and genotoxic stress. Thus, we explored the contribution of ILC3s to lactation-induced gut hyperplasia. Our immunofluorescence analysis revealed that mice lacking RORγt, the transcription factor crucial for ILC3 development and function, exhibited shorter villi and reduced proliferation in the crypts, where the intestinal stem cells reside. Subsequent analysis using IL-22-deficient mice, a cytokine primarily expressed by ILC3s, suggests that ILC3s promote lactation-induced gut hyperplasia in an IL-22-dependent manner. Furthermore, our findings demonstrated that STAT3 in the intestinal epithelium, known to be stimulated by IL-22 and EGF, also contributes to increased proliferation and villus growth in lactating mice.
Flow cytometry experiments and histological analyses confirmed increased ILC3 proliferation during lactation and enlargement of the clusters ILC3s form proximal to the crypts, namely cryptopatches and isolated lymphoid follicles. Bulk RNA sequencing and leukocyte stimulation experiments between non-pregnant (NP) and lactating mice, confirmed the upregulation of IL-22 in ILC3s during lactation, a finding further validated through the analysis of IL-22 tdTomato reporter mice. Single-cell RNA sequencing of intestinal epithelial cells from both ILC3-sufficient and deficient mice unveiled downregulation of the EGF signaling in the gut, which was validated by quantification of EGFR phosphorylation in epithelial cells. The phosphorylation of EGFR, linked to ligand binding, was confirmed to be associated with EGF upregulation during lactation, originating from mesenchymal PDPN+ cells. Notably, RORγt-dependent cells were found to regulate EGF production in mesenchymal cells in an IL-22-dependent manner upon lactation.
Moreover, our study demonstrated that IL-22 modulates genes associated with nutrient absorption in gut epithelium in vitro, differentially to the IL-20 and IL-24 cytokines, which also bind to the IL-10RB receptor. Lastly, we observed that lactating RORγt and IL22-deficient mice consumed more food than the WT control mice, presumably to compensate for reduced food absorption. However, due to their reduced absorptive epithelial surface, the body weight of IL-22-sufficient offspring nurtured from ILC3- and IL-22-deficient mice was reduced.
View lessOpioid receptor ligands (opioids) represent the gold standard for severe pain treatment. However, approved opioids possess many serious side effects even if they are used according to medical instructions. As a result, opioid use leads to thousands of hospitalizations and deaths each year. Four different opioid receptor subtypes exist but approved opioids mainly activate the μ-opioid receptor (MOR). Activation of the κ-opioid receptor (KOR) emerged as a promising strategy to sufficiently alleviate pain while improving the side effect profile of opioids. However, KOR-related side effects and the high structural similarity of the opioid receptor subtypes renders rational drug design challenging. The opioid receptor system is not only involved in pain modulation, but also in the maintenance of mood homeostasis. Antagonism of the KOR recently emerged as a promising strategy for the development of novel antidepressants with fast-onset antidepressant effects that are also effective against suicidal intent. However, the chemical space of KOR selective antagonists is limited. No short-acting and selective KOR antagonists have been approved so far. This dissertation encompasses retrospective and prospective studies that focus on in silico techniques to address current questions in opioid research. The first three articles, conducted retrospective in silico analyses of HS-731, an opioid agonist, Compound A, a KOR/MOR dual antagonist, and Salvinorin A (SalA), a non-basic, KOR-selective agonist. These analyses aimed to improve our limited understanding of the factors that determine opioid receptor activity and selectivity. The investigated compounds are of high pharmacological interest. HS-731 represents an analgesic with a potentially improved safety profile due to its exclusion from the central nervous system (CNS). Compound A has a unique scaffold that may overcome the unfavorable kinetic profile of current antidepressant opioids. SalA represents a non-basic agonist with exceptional KOR selectivity that potentially binds dissimilar to basic opioids at the KOR. Protein-ligand binding modes of the compounds at the opioid receptor subtypes were predicted and selectivity determinants rationalized. Important protein-ligand interactions responsible for the opioid receptor activity of these ligands were highlighted. A new binding site for SalA above the typical morphinan binding site that is highly non-conserved was discovered. The SalA binding mode is consistent with structure-activity relationship (SAR) data and rationalizes the exceptional receptor subtype selectivity of SalA at the KOR. In addition, a partial agonism-inducing mechanism for the KOR was hypothesized involving an interaction between the extracellular oriented parts of the transmembrane helices 5 and 6. Typical opioids possess a basic amine moiety interacting with a conserved aspartate residue inside the binding site. Since the discover of non-basic and highly KOR-selective SalA, the design of non-basic opioids became a new and promising strategy for the selective activation of the KOR, circumventing MOR-associated severe side effects. However, the number of known non-basic opioids is strongly limited and rational drug design of new non-basic opioids is the hindered by the lack of understanding about the binding mode of SalA. In the fourth article included in this dissertation, a prospective 3D pharmacophore-based virtual screening campaign was conducted. The campaign was based on the SalA binding mode postulated in the third article. The aim was to determine new non-basic and selective KOR ligands. Pharmacological in vitro experiments confirmed two of the suggested compounds to activate the KOR with nanomolar potency and good subtype selectivity. One hit compound possesses a full agonistic and one compound shows a partial agonistic profile at the KOR. Both hit compounds share a novel spiro-moiety-containing scaffold. Overall, the findings of this dissertation provide new mechanistic insights into opioid receptor activation in the context of current progress in structural biology, and lay the basis for improved, rational drug design of safer analgesics and novel antidepressants with KOR activity.
View lessZiel dieser Studie war es, die viruzide Wirksamkeit von alternativen Desinfektionsverfahren für Oberflächen, ohne den Einsatz von Feucht- und Wischdesinfektion, zu ermitteln. Dabei wurde die Anwendung der Trockenvernebelung aerosolisierter Peroxyessigsäure (aPES), UV C-LED-Strahlung bei 233 und 270 nm sowie einer UV C-Quecksilberlampe bei 254 nm und Kaltplasmastrahlung untersucht. Für diesen Zweck wurden unbelebte Oberflächen wie Edelstahl, Glas und PVC gegenüber einem breiten Erregerspektrum aus behüllten und unbehüllten Viren getestet. Die Auswahl geeigneter Viren erfolgte einerseits anhand der DVV-RKI-Leitlinien, der DVG-Leitlinien und der DIN EN 17272:2020. Andererseits wurden gezielt noch weitere Viren (MS 2 Bakteriophage, Bovines Coronavirus) ausgewählt und hinsichtlich ihrer Eignung als Surrogate untersucht. Zur jeweiligen Anwendung der einzelnen Methoden wurden entsprechende Keimträger aus den unterschiedlichen Materialien eingesetzt, auf denen die virushaltigen Suspensionen angetrocknet wurden. Die anschließende Quantifizierung des Desinfektionserfolges erfolgte durch Abspülen der Keimträger und nachfolgender Titration auf empfänglichen Zellkulturen. Da sowohl der UV C-LED-Strahler mit einer Wellenlänge von 233 nm als auch der Kaltplasmastrahler primär für die Desinfektion von belebten Oberflächen vorgesehen sind, wurde deren zytotoxisches Potenzial sowie die antivirale Wirksamkeit auch auf 2D-Monolayerkulturen und einem etablierten 3D-Schleimhautmodell getestet.
View lessDie Gebärparese ist eine der wichtigsten Stoffwechselerkrankungen im peripartalen Zeitraum bei Holstein Friesian Kühen. Ursächlich dafür ist eine zu niedrige Kalziumkonzentration im Blut. Bei dem Auftreten von Milchfieber scheint die Serumphosphatkonzentration ebenfalls eine wichtige Rolle zu spielen.
In der vorliegenden Untersuchung wurden von 291 Kühen nach der Kalbung Blutproben über einen Zeitraum von 24 Stunden entnommen und die Werte von Kalzium und anorganischem Phosphat im Serum erfasst. Mit Hilfe der dokumentierten Daten soll der Verlauf von Phosphat post partum im Blutserum im Tagesprofil dargestellt werden und herausgestellt werden, welchen Einfluss kuhassoziierte und managementassoziierte Faktoren auf die Phosphatkonzentration im Serum haben.
Die Untersuchung wurde auf einem Milchviehbetrieb in Mecklenburg-Vorpommern mit 2260 Kühen im Zeitraum von Juli 2014 bis März 2015 durchgeführt. Dazu wurden von den Tieren ab der 1. Laktation Blutproben im zeitlichen Abstand von 2 Stunden entnommen. Die Tiere wurden je nach Fragestellung in unterschiedliche Gruppen randomisiert eingeteilt und haben innerhalb der Gruppen in einem ähnlichen Zeitraum gekalbt.
Es wird festgestellt, dass die Laktationszahl einen wesentlichen Einfluss auf die Serumphosphatkonzentration hat. Tiere ab der 3. Laktation zeigen signifikant niedrigere Werte. In der vorliegenden Untersuchung haben einige Tiere ab der 2. Laktation einen Kalziumbolus erhalten. Diese Behandlungsmethode scheint jedoch keinen Einfluss auf die Konzentration von Phosphat zu haben. Auch die Gabe eines Phosphor-Bolus im Vergleich zu einem Kalzium-Bolus zeigt keine signifikanten Unterschiede auf die Phosphatkonzentration. Hingegen zeigen Tiere, die eine Kalzium-Infusion i.v. unmittelbar nach der Kalbung erhalten haben, einen deutlich schnelleren Anstieg der Serumphosphatwerte und somit höhere Werte über einen Verlauf von 24 Stunden. Ebenso haben eine reduzierte Melkhäufigkeit und eine reduzierte Menge des ermolkenen Kolostrums einen signifikanten Einfluss auf die Serumphosphatwerte. Weiteren Tieren wurde p.p. ein Kalbetrunk verabreicht. Diese Behandlungsmethode weist keinen signifikanten Einfluss auf die Phosphatkonzentration auf.
Ein weiterer signifikanter Einflussfaktor auf die Serumphosphatkonzentration ist die Wahl des Blutgefäßes zur Probenentnahme. In der vorliegenden Arbeit sind die Konzentrationen von Phosphat bei Blutproben aus der V. jugularis deutlich geringer, als bei Proben aus der V./A. coccygea oder der Eutervene. Dies sollte bei der Blutprobenentnahme berücksichtigt werden.
In der Untersuchung wurde deutlich, dass Tiere mit einer Hypophosphatämie zu über 50 % ebenfalls eine Hypokalzämie aufweisen. Somit lässt sich ein Zusammenhang zwischen dem Auftreten einer Gebärparese und der Phosphatkonzentration nachweisen. Aufgrund des signifikanten Einflusses der Kalzium-Infusion auf die Serumphosphatkonzentration lässt dies die Schlussfolgerung zu, dass diese Maßnahme zur Behandlung einer Hypokalzämie ebenso sinnvoll für die Behandlung einer Hypophosphatämie erscheint. Die orale Gabe von Kalzium und Phosphor, beispielsweise in Form eines Bolus, kann hierbei ergänzend erfolgen.
Die untersuchten Tiere zeigen unabhängig von der Behandlung einen ähnlichen Verlauf der Phosphatwerte über einen Zeitraum von 24 Stunden nach der Kalbung. Es stellt sich hier die Frage, ob sich diese Tagesdynamik der Serumphosphatkonzentration unabhängig vom Verlauf der Kalziumkonzentration zeigt. Um dies abschließend zu klären, müsste man die Blutprobenentnahme über einen längeren Zeitraum fortsetzen.
View lessIn der Humanmedizin sowie im Distanzpferdesport wurde bereits festgestellt, dass nach Ausdauerbelastungen Anzeichen eines „Exercise induced cardiac fatigue“ auftreten können. Diese äußern sich in einer reduzierten diastolischen und zum Teil auch systolischen Myokardverformung und auch Myokardgeschwindigkeit. Im Vielseitigkeitssport wurden bisher noch keine Studien mit der Gewebedopplerechokardiographie sowie dem Speckle-Tracking durchgeführt. Ziel dieser Studie war es, letztendlich von 19 Vielseitigkeitspferden die Veränderungen der Funktion des Myokards sonographisch nach einem Geländeritt, nach 24 Stunden sowie im Laufe der Saison zu beurteilen. Die Pferde wurden dafür am Morgen vor dem Geländeritt, 30 Minuten danach sowie 24 Stunden später echokardiographisch untersucht. Alle Pferde wurden zuvor in der Winterpause vor der Saison klinisch und echokardiographisch untersucht und als herzgesund beurteilt. Die Untersuchungen für diese Studie erfolgten mehrmals in der kompletten Turniersaison. Es wurden Pferde in unterschiedlichen Turnierschwereklassen untersucht. Die Belastungseinheiten betrugen zwischen ca. 3.000 – 5.000 m. Das Durchschnittstempo betrug zwischen 400 – 570 m/min. Die Gewebedopplerechokardiographie gilt als ein Verfahren, um die Spitzengeschwindigkeiten des Myokards in der Systole und Diastole zu bestimmen. Die Ergebnisse des PW-Dopplers zeigten eine reduzierte diastolische Funktion des Myokards nach der Belastung. Da diese Messungen durch Vor- und Nachlast beeinflusst werden können, lässt es sich diskutieren, ob es sich hierbei um eine kardiale Ermüdung handelt oder es durch Veränderungen der Vor- und Nachlast nach der Belastung zu einer veränderten Funktion des Myokards gekommen ist. Eine ausreichende Erholung mit Normalisierung der Werte nach 24 Stunden war größtenteils gegeben. Es konnte zudem keine reduzierte systolische Funktionfestgestellt werden. Im Laufe der Untersuchungsmonate sind die Myokardgeschwindigkeiten zum Teil angestiegen. Da diese Veränderungen bereits bei trainierten Athleten nachgewiesen wurden, lässt dies auf einen Trainingseffekt im Laufe der Saison schließen. Mithilfe des 2D-Speckle-Trackings werden die lokalen Myokardfunktionen bzw. die Myokardverformungen beurteilt. Es konnte eine vermehrte kontraktile Funktion und Relaxation des Myokards nach der Belastung festgestellt werden. Die ermittelten Werte für Strain und Strain rate zeigen eine Erhöhung nach der Belastung. Der Vorteil dieses Verfahrens besteht in der Unabhängigkeit bei Volumenveränderungen des Herzens. Im Laufe der Saison hat sich die Strain und Strain rate jedoch reduziert. Diese Reduzierung kann auf den vermehrten Parasympathikus und reduzierten Sympathikus zurückgeführt werden. Dies wiederum kann durch den verbesserten Trainingsstatus der Pferde im Laufe der Saison verursacht sein. Zusammenfassend kann gesagt werden, dass die Ergebnisse dieser Studie für eine verminderte Myokardgeschwindigkeit in der frühen Diastole direkt nach der Belastung sprechen. Eine reduzierte diastolische Funktion nach der Belastung, die mithilfe des PW-Dopplers untersucht wurde, liegt vor, müsste jedoch unter Berücksichtigung von Volumenveränderungen bestätigt werden, bevor von einem eindeutigen „cardiac fatigue“ gesprochen werden kann. Die Erholung am nächsten Morgen war größtenteils gegeben. Im Laufe der Saison lassen die Ergebnisse auf einen positiven Trainingseffekt schließen. Die beiden Ultraschallverfahren, PW-Doppler und Speckle-Tracking, stellen sich zum Thema Leistungsdiagnostik als zwei geeignete Verfahren in der Pferdekardiologie dar.
View lessMigräne tritt bei Frauen zwei- bis dreimal häufiger auf als bei Männern. Zudem sind Migräneattacken bei Frauen länger und schwerer, was insgesamt zu einer stärkeren Beeinträchtigung im Vergleich zu Männern führt. Die Ursachen für solche geschlechtsabhängigen Unterschiede sind vielschichtig und betreffen sowohl psychosoziale als auch biologische Aspekte. Ein tiefergehendes Verständnis geschlechtsabhängiger Mechanismen ist notwendig, um die Versorgung von Migränepatient:innen zu verbessern, spezifische therapeutische Ansätze zu entwickeln und die Lebensqualität der Betroffenen zu steigern. Die in dieser Habilitationsschrift vorgestellten Studien tragen dazu bei, wichtige geschlechtsspezifische Aspekte in der Pathophysiologie, Diagnostik, Behandlung und sozialer Wahrnehmung von Migräne zu charakterisieren. Ein zentrales Neuropeptid in der Migränepathophysiologie ist Calcitonin Gene-Related Peptid (CGRP). In einer prospektiven Kohortenstudie konnten wir erstmalig unterschiedliche CGRP-Konzentrationen bei Frauen mit Migräne und gesunden Kontrollprobandinnen, basierend auf ihren Sexualhormonprofilen, nachweisen. Höhere perimenstruelle CGRP-Konzentrationen bei Migränepatientinnen im Vergleich zu gesunden Frauen weisen auf eine verstärkte perimenstruelle Freisetzung von CGRP hin. Diese Ergebnisse bieten somit eine mögliche pathophysiologische Erklärung für die erhöhte Anfälligkeit für Migräneattacken in dieser Zyklusphase. CGRP spielt auch in der Pathophysiologie der Endometriose, einer häufigen Komorbidität bei Frauen mit Migräne, eine bedeutende Rolle. In einer weiteren Kohortenstudie konnten wir die Hypothese einer hormonbedingten Fehlregulation des CGRP-Signalweges bei komorbiden Patientinnen bestätigen. Obwohl keine signifikanten Unterschiede in den absoluten CGRP-Spiegeln festgestellt wurden, zeigte sich, dass Frauen mit beiden Erkrankungen während der Menstruation einen Anstieg der CGRP-Konzentrationen aufwiesen, was auf eine besondere pathophysiologische Relevanz von CGRP bei komorbiden Patientinnen hinweist. Ein weiterer geschlechtsspezifischer Aspekt in der Behandlung von Frauen mit Migräne betrifft die Verwendung von hormonhaltigen Kontrazeptiva, die den Migräneverlauf beeinflussen können. Eine deutschlandweite Umfrage unter Gynäkolog:innen ergab, dass diese nahezu immer das Vorhandensein von Migräne vor einer Verschreibung von Kontrazeptiva berücksichtigen. Die Zurückhaltung bei der Verschreibung von östrogenhaltigen Kontrazeptiva bei Migräne mit Aura entspricht den gültigen Leitlinien. Angesichts der Tatsache, dass Gynäkolog:innen ebenfalls therapeutische Entscheidungen für Patientinnen mit Migräne treffen, betont diese Studie die Notwendigkeit einer guten Zusammenarbeit zwischen beiden Disziplinen. Veränderungen des hormonellen Zustandes während der Schwangerschaft können zu Änderungen von Kopfschmerzeigenschaften führen. Unsere klinische Datenerhebung zeigte, dass Migräne die häufigste Ursache für kopfschmerzbedingte Vorstellungen in der Rettungsstelle der Charité während der Schwangerschaft ist. Dennoch wurde bei über 40% der Patientinnen eine sekundäre Kopfschmerzursache diagnostiziert. Diese Ergebnisse unterstreichen die Notwendigkeit einer gründlichen Anamnese, klinischen und neurologischen Untersuchung sowie gegebenenfalls weiterer Diagnostik, um potenziell lebensbedrohliche Kopfschmerzursachen nicht zu übersehen. Abschließend konzentrierte sich eine prospektive Befragung auf die Wahrnehmung von stereotypen Migränebildern in den Medien. Die Ergebnisse zeigen, dass solche Bilder von Patient:innen mit Migräne und Mitarbeitenden im Gesundheitswesen als nur mäßig realistisch wahrgenommen werden. Insbesondere Bilder mit jungen Frauen wurden als am wenigsten realistisch empfunden, was die dringende Notwendigkeit einer Überarbeitung und Verbesserung solcher Darstellungen verdeutlicht.
Die vorgestellten Arbeiten tragen neue Erkenntnisse zu zahlreichen geschlechtsabhängigen Unterschieden in der Pathophysiologie, Diagnostik und gesellschaftlichen Wahrnehmung von Migräne bei. Sexualhormone sind entscheidend an der Migränepathophysiologie beteiligt, und künftige Arbeiten werden sich darauf konzentrieren, hormonabhängige entzündliche, vaskuläre und neuronale Prozesse der Migränepathophysiologie genauer zu charakterisieren. Die Berücksichtigung geschlechtsspezifischer Aspekte im biopsychosozialen Modell der Migräne ist von herausragender Bedeutung, um dieses komplexe Krankheitsbild gründlicher zu verstehen und eine optimale Versorgung von Patient:innen aller Geschlechter in jeder Lebensphase zu gewährleisten.
View lessPrimärer Leberkrebs – hepatozelluläres (HCC) und cholangiozelluläres Karzinom (CCA) - ist die Tumorerkrankung, die nach dem Lungenkarzinom für den Verlust der meisten Lebensjahre weltweit verantwortlich gemacht wird. Unter einem Drittel der Patienten kann kurativen Therapien, wie der Leberresektion zugeführt werden, aber ihr Outcome ist durch eine hohe perioperative Morbidität und Mortalität sowie hohe Rezidivraten limitiert, sodass ein hoher Bedarf besteht, präoperativ PatientInnen besser zu stratifizieren. In dieser Habilitationsschrift wurden prognostische Parameter für primäre Lebertumoren nach einer Leberteilresektion in kurativer Intention untersucht. Hierbei lag ein Fokus auf dem selteneren, und gerade in Europäischen Kollektiven unterrepräsentiertem, Cholangiokarzinom. Bisherige prognostische Parameter haben einen starken Fokus auf Tumor-Parameter, wie Genmutationen und pathologische Kriterien gelegt. Einzelnukleotidpolymorphismen können sich sowohl auf Prozesse der Wirtszellen, als auch auf die Interaktion des Wirtes mit dem Tumor auswirken, zum Beispiel in Bereichen der Immunologie und Neoangiogenese. Hierbei fanden wir für IL-1b, IL-8, und den IL-8 Rezeptor, Polymorphismen, die hochgradig mit der onkologischen und allgemeinen Prognose assoziiert war. Somit kann die Analyse von Genopymorphismen die Abschätzung onkologischer Outcomes auch für das CCA unterstützen.
Im zweiten Teil dieser Arbeit wurde anhand präoperativer Computertomographie (CT)-Bilder die Körperzusammensetzung basierend auf dem Muskel-und Fettkompartiment. Wichtige Erkenntnisse hierbei waren, dass während isolierte konventionelle Parameter wie die Sarkopenie oder die viszerale oder subkutane Fettleibigkeit keinen isolierten prognostischen Stellenwert hatten, hatten Patient*innen mit Sarkopener Obesität ein dramatisch verkürztes Überleben. Somit konnten unsere Daten eine – gerade im Kontext der weltweiten Übergewichtsepidemie immer prävalenter werdende – Hochrisikogruppe aufzeigen, die bei konventioneller klinischer Betrachtung durch BMI nicht aufgefallen wäre.
Zusammenfassend konnte diese Arbeit angeborene als auch akquirierte Wirtfaktoren als prognostisch relevant für das chirurgisch therapierte Cholangiokarzinom herausstellen. Unsere Ergebnisse können dank ihrer präoperativen Erhebbarkeit (Einzelnukleotidpolymorphismen aus peripherem Blut, body composition aus präoperativen CT Bildern) prognostisch unterstützend wirken und in Zukunft helfen, vulnerable Patient*innengruppen aufzeigen.
View lessBiodegradable poly(lactide-co-glycolide) (PLGA) microparticles enable the controlled parenteral administration of drugs. Commonly used microencapsulation methods require the dissolution of PLGA in organic solvents. The removal of these solvents during the manufacturing process is essential to obtain solid microparticles. In addition, a low residual solvent content must be achieved in the final product to ensure storage stability and patient safety. In this dissertation the solvent removal during the preparation of microparticles with the O/W solvent extraction/evaporation method was investigated. In this method, an organic (O) phase containing drug, PLGA and organic solvent (dichloromethane or ethyl acetate) was emulsified in an aqueous polyvinyl alcohol solution (W) by a classical batch process or by microfluidic flow focusing. The effect of various formulation and process parameters was investigated in order to optimize the properties of PLGA microparticles with regard to minimal residual solvent levels at high drug loadings and encapsulation efficiencies and optimal drug release profiles. The initial removal of organic solvent had a great effect on the properties of the microparticles and is usually controlled by two steps, the solvent dissolving in the continuous aqueous phase and the solvent evaporation rate. One-step dilution and continuous diafiltration were investigated to control the initial solvent extraction. The latter replaced the normally required large-volume manufacturing equipment and the O/W emulsion transfer. Accelerated extraction of the PLGA solvent dichloromethane shortened the process time until the microparticles were solidified and thus collectable. Increasing the diafiltration rate increased the particle size, porosity and drug loading of the microparticles. The encapsulation efficiency of risperidone was significantly increased from 62 % to up to 80 % in PLGA 503H microparticles and from 27 % to up to 75 % in PLGA 753S microparticles. A slower and more uniform solidification of the end-capped and higher lactide content PLGA 753S was identified as the reason for the increased drug loss without diafiltration. The residual dichloromethane content was not affected by diafiltration, but decreased by increasing the temperature, because the final solvent removal was limited by the diffusivity within the PLGA phase. Accelerated solvent extraction by diafiltration did not affect the in-vitro release of risperidone from both PLGA 503H and PLGA 753S microparticles. The effect of diafiltration on the extent of dexamethasone burst release depended on the drug loading. Increased porosity enhanced percolation if a large amount of undissolved drug crystals was present in the PLGA matrix. By increasing the process temperature during diafiltration, the unwanted burst release could be reduced because of a reduced microparticle porosity. Due to its complete miscibility with water, methanol as a co-solvent was extracted significantly faster than dichloromethane from the dispersed phase. Increasing the methanol content above 2.5 % (w/w) resulted in an increased porosity, drug loss and burst release. The final dichloromethane extraction was faster, although methanol had already been completely extracted from the PLGA-phase. The residual dichloromethane content after 24 h was reduced from 2.4 % (w/w) (without methanol) to below 0.5 % (w/w) if ≥ 2.5 % (w/w) methanol were used as a co-solvent. The resulting porosity did not explain this, because, if caused by diafiltration, it had no effect on the residual solvent content. Therefore, the effect of alcohols in the continuous phase on the solvent extraction was investigated further. The final removal of the residual solvent from the microparticles was performed by wet extraction and secondary drying methods. With decreasing residual solvent content, the solvent removal became significantly slower as the diffusivity in the PLGA matrix decreased due to a decreasing plasticizing effect and free volume. A higher molecular weight and end-capping of PLGA increased the residual solvent content due to an increasing affinity for dichloromethane, viscosity and droplet/particle size. Increasing the lipophilicity of PLGA with a higher lactide content did not increase the residual dichloromethane content because a slower droplet solidification facilitated the final solvent extraction. The removal of residual dichloromethane was more efficient with alcoholic wet extraction, followed by aqueous wet extraction at elevated temperature and vacuum drying of the microparticles. Aqueous wet extraction reduced the residual dichloromethane content of risperidone-loaded PLGA 503H microparticles to 2.43 % (w/w) (20 °C) and 0.03 % (w/w) (35 °C) in 24 h. The elevated temperature promoted the risperidone-caused degradation of PLGA resulting in visible microparticle erosion and a decrease of risperidone encapsulation efficiency from 88 % to 75 %. Early filtration and subsequent vacuum drying of the solidified microparticles prevented this unwanted erosion of microparticles and drug loss. The residual dichloromethane content in filtered PLGA 503H microparticles was only reduced from about 5 % (w/w) to 4.34 % (w/w) (20 °C) and 3.20 % (w/w) (35 °C) after 18 h vacuum drying because of the missing plasticizing effect of water. Redispersing filtered wet microparticles in alcoholic media improved the residual dichloromethane extraction. The potential of different extractants was explained by the Gordon-Taylor equation and Hansen solubility parameters. Short-chain monohydric alcohols decreased the glass transition temperature of PLGA more than polyhydric alcohols, water or dichloromethane. Ethanol had the greatest plasticizing effect of all investigated solvents. A higher chain length of monohydric alcohols increased the affinity to PLGA and thus the solvent absorption, but also the tendency of agglomeration of the microparticles. Extraction in methanol: or ethanol:water mixtures efficiently reduced the residual dichloromethane content from 4 - 7 % (w/w) to 0.5 - 2.3 % (w/w) within 1 h and 0.08 - 0.18 % (w/w) within 6 h. Increasing the alcohol content and temperature promoted microparticle aggregation and drug loss. An alcohol vapor-assisted fluidized bed drying process for microparticles was developed to avoid the loss of encapsulated drug and thus utilize the potential of alcoholic extraction of the solvent residues from the microparticles. By regulating the alcohol concentration and the temperature of the purge gas, the alcohol absorption and the residual solvent and alcohol removal were controlled. Methanol proved to be particularly efficient in the developed setup due to its high volatility, molecular mobility and PLGA-affinity. The absorbed methanol was easily removed by alcohol-free fluidized bed or vacuum drying. While alcohol-free fluidized bed drying decreased the residual dichloromethane content only from about 7 % (w/w) to 6.4 % (w/w) (18 °C) or 0.7 % (w/w) (45 °C) within 24 h, 140 mg/L methanol vapor in the purge gas decreased the residual dichloromethane content to 0.11 % (w/w) in 2 h and removed it completely within 6 h. Methanol vapor also removed efficiently residual ethyl acetate from the microparticles (0.11 % (w/w) after 6 h), which is an alternative PLGA solvent to dichloromethane. Encapsulated risperidone increased the methanol absorption and thus contributed to microparticle plasticization. A high initial residual water content, which favored microparticle aggregation, was completely removed in less than 1 h by alcohol-free fluidized bed drying, which enabled the subsequent alcohol vapor-assisted removal of the residual organic solvent. Alcohol vapor-assisted fluidized bed drying was introduced as a promising alternative to established residual solvent removal methods, accelerating microparticle preparation without negatively affecting drug loading or release profile. A continuous process for the preparation of monodisperse PLGA microparticles by microfluidic flow-focusing was developed as an alternative to the discontinuous preparation by the classical batch process. For methodological reasons, smaller O/W phase ratios (1:2 - 1:8) were used compared to the batch process (≥ 1:20). The resulting limited initial solvent extraction resulted in a low risperidone encapsulation efficiency of only 19 - 21 % at a theoretical drug loading of 30 %. The buffering of the continuous phase to alkaline pH increased encapsulation efficiency up to 93 % but led to a change in the microfluidic droplet formation and thus microparticle size. This could be counteracted by reducing the total flow rate and the amount of stabilizer in the continuous phase. The in-vitro release of risperidone was delayed from microparticles prepared by microfluidic compared to similar-sized polydisperse microparticles prepared by the batch process probably because of a more homogeneous drug distribution within the PLGA-matrix. A continuous dilution-based solvent extraction process was developed, and the effect on droplet shrinkage examined with flow microscopy. A phase ratio of at least 1:100 after dilution and a sufficient extraction time (75 s at 1:100 and 15 s at 1:500) were necessary to shrink the droplets to the particle size of the final microparticles. Increasing the temperature or adding methanol to the continuous phase improved the dichloromethane extraction but impaired the process stability due to the formation of gas bubbles or sticking of droplets/particles. A tangential flow filtration (TFF) process was developed to separate the dichloromethane-enriched continuous phase from the microparticles. In conclusion, the effect of various formulation and process parameters on the removal of solvents in extraction/evaporation methods and on critical properties of the final microparticles such as drug loading and drug release was investigated. Methods were developed which accelerate both the initial and final removal of the organic solvents, thus shortening the total process time. In addition, high encapsulation efficiencies and desirable drug release profiles (e.g., low burst release) were achieved. This research contributes to know-how in the manufacturing of biodegradable PLGA microparticles by the solvent extraction/evaporation method through a detailed investigation of various single and combined solvent removal processes and their effects on the resulting properties of PLGA microparticles.
View lessZwischen Mitte der 1840er-Jahre und dem Deutsch-Französischen Krieg von 1870/71 avancierte der Kurort Baden-Baden zum führenden Modebad Europas und zu einem bedeutenden interkulturellen Zentrum. Der ausgeprägte französische Einfluss brachte der Stadt auf beiden Seiten des Rheins den Ruf einer "französischen Kolonie" und einer "Filiale von Paris" ein, wobei dies unterschiedlich interpretiert wurde. Die vorliegende Dissertationsschrift geht der Entwicklung dieses einzigartigen Ortes der deutsch-französischen Kulturbeziehungen im Spannungsfeld zwischen Kosmopolismus und erstarkendem Nationalismus nach. Sie analysiert die vielfältigen Faktoren, die zur Entstehung der "Sommerhauptstadt Europas" beitrugen, und identifiziert Phänomene und Grenzen des Austauschs und Kulturtransfers, welche die gesamte Bäderkultur prägten.
View lessThe satellite gravimetry missions GRACE and its successor GRACE-FO measure large-scale mass redistributions in the Earth system, including changes in terrestrial water storage, ice shelves, the oceans and atmosphere as well as the solid Earth. As the measurements are typically accumulated over one month before the computation of a gravity solution, high-frequency mass variations need to be accounted for using a-priori background models. The Atmosphere and Ocean Dealiasing Level 1B (AOD1B) data product routinely provides the necessary simulated non-tidal mass variations in the atmosphere and oceans. While progress towards better background models has been made over the past years, residual errors in the simulated atmospheric and oceanic mass variations are still among the largest limiting factors to the overall GRACE accuracy. As a result, further improvements of these data-products are directly linked to improvements in the final gravity solutions and thus subsequent geophysical analyses. This thesis summarizes the most recent improvements to the AOD1B product, including an updated ocean model configuration considering the effects of self-attraction and loading, an updated atmospheric component as well as an improved post-processing and tidal analysis. All of these changes are shown to result in a better representation of the simulated mass variations and are thus combined into a new release RL07 of AOD1B. Additionally, the new release is examined in terms of its stability and consistency over long and short time-scales. Spurious trends, low-frequency signals or bias jumps in the background model data can, if unaccounted for, introduce biases into the global gravity solutions which are prone to be interpreted erroneously in subsequent geophysical analyses. Consequently, the analysis focuses on signals that might be induced into the final gravity field solutions such as trends but also possible discontinuities caused by changes is the employed atmospheric datasets. It is shown that AOD1B RL07 is free from such artifacts and can thus safely be used as a background model in satellite gravimetry. Finally, an analysis of the residual errors in AOD1B RL07 is presented. The estimation is based on ensemble statistics derived from different atmospheric reanalyses. For the oceans, the impact of both the forced and intrinsic variability is investigated through differences in simulation experiments. The atmospheric and oceanic information is then combined to produce a new time-series of true errors, called AOe07, which is applicable in combination with AOD1B RL07 and can be used both as stochastic information in the gravity field retrieval process, as well as in simulation studies in preparation for future satellite gravimetry missions.
View lessDie Erzeugung und Aufrechterhaltung der synaptischen Übertragung zählen zu den energieaufwändigsten Phänomenen in der Biologie. Das Gehirn ist deswegen auf ein ununterbrochenes Angebot an Glukose und Sauerstoff angewiesen. Störungen in der Nutrientenversorgung von Neuronen führen zu chronischer Neurodegeneration und/oder zum akuten neuronalen Tod. Die in dieser Habilitation zusammengefassten Studien untersuchten den Energieverbrauch der synaptischen Übertragung studiert und den Einfluss von Anästhetika auf den Neurometabolismus. Ein besseres Verständnis über den energetischen Preis einzelner synaptischen Prozesse könnte uns helfen, neue neuroprotektive Therapien zu entwickeln. Anästhetika rufen stärkste reversible Effekte in der synaptischen Übertragung hervor und senken dadurch den neuronalen Energiebedarf. Die neurometabolischen Effekte der Anästhetika sind vielfaltig, nicht eindeutig verstanden und sowohl neuroprotektiver als auch neurotoxischer Natur. In mehreren experimentellen Studien untersuchten wir die Effekte von drei gängigen Narkosemitteln (Propofol, Isofluran und Sevofluran) ex vivo und in vivo im Gehirn von Wistar-Ratten. Zu diesem Zweck wurden elektrophysiologische Messungen, Sauerstoffmessungen, Autofluoroszenzmessungen, molekularbiologische Techniken und Computermodellierung kombiniert. 40 Im Ergebnis konnten wir feststellen, dass nur hohe Konzentrationen der Anästhetika bzw. eine sehr tiefe Narkose die oxydative Phosphorylierung im neuronalen Gewebe senken. Für Propofol waren diese Effekte teilweise mit einer direkten Hemmung von mitochondrialen Enzymen verbunden. Bei Isofluran und Sevofluran wurde die Senkung des Energieverbrauchs vor allem durch eine Hemmung der synaptischen Übertragung bzw. der Netzwerkaktivität hervorgerufen. Isofluran hatte von allen getesteten Narkotika die stärkste Wirkung auf den Neurometabolismus. Wir haben des Weiteren zum ersten Mal eine spezifische Hemmung der Natrium-Pumpe im Hirngewebe durch Isofluran beschrieben. Dies könnte nicht nur der Grund für den starken Effekt von Isofluran auf den neuronalen Energieverbrauch sein, sondern auch eine Störung der Ionhomöostase im Gehirn während einer tiefen Narkose erklären. Ein neuroprotektiver oder neurotoxischer Effekt von Anästhetika lässt sich in unseren Untersuchungen nicht eindeutig feststellen. Da schwerwiegende Effekte im Metabolismus und Gewebehomöostase nur unter hohen Konzentrationen bzw. bei der tiefen Anästhesie beobachtet werden sollte diese durch Neuromonitoring vermieden werden. Eine Senkung des Metabolismus im Gehirn zwecks Neuroprotektion ist möglich aber die notwendige Narkosetiefe birgt Risiken durch weitere unspezifische Effekte auf die Neuronen.
View lessNeurotransmission, essential for sensory perception, motor control, cognition, and behavior, occurs at synapses, where neurotransmitters are released from the presynaptic neuron into the synaptic cleft, triggering responses at the postsynaptic cell. At chemical synapses, neurotransmitter release involves the fusion of synaptic vesicles (SVs) with the presynaptic membrane, necessitating compensatory membrane retrieval, termed synaptic vesicle endocytosis, to reform and refill SVs for subsequent fusion cycles. Despite the recognized role of the Actin cytoskeleton in synaptic vesicle endocytosis, the precise mechanisms governing Actin polymerization and its function within presynaptic nerve terminals remain poorly understood. Here, we delineate the pivotal role of Actin regulatory diaphanous-related formins mDia1/3 and small Rho GTPases, RhoA/B and Rac1, in orchestrating synaptic vesicle recycling at rodent central synapses. Employing optical recordings of presynaptic membrane dynamics, ultrastructural and proteomic analyses, in combination with genetic/pharmacological manipulations, we demonstrate that mDia1/3 localize to the presynaptic membrane, proximal to the endocytic machinery, and govern the formation of presynaptic filamentous Actin structures (F-Actin). Loss of F-Actin due to perturbation of mDia1/3 results in significant alterations in presynaptic architecture, impacting plasma membrane homeostasis. Furthermore, our findings highlight that in the absence of mDia1/3, downregulation of RhoA and activation of Rac1 drive a compensatory response to mitigate the disruption of formin-mediated Actin dynamics. Besides modulating Rho GTPase signaling, we find that mDia1/3 negatively regulate the complex signaling network mediated by the mechanistic target of rapamycin complex 2 (mTORC2). We identify mTORC2 activation to be inversely coupled to the kinetics of SV recycling, likely through the modulation of cytoskeletal dynamics. In conclusion, our study elucidates that the endocytic cytoskeleton is governed by interdependent signaling pathways involving the small Rho GTPases RhoA/B and Rac1, as well as the action of mDia1/3 formins, operating within feed forward loops. The dynamics of the Actin cytoskeleton integrate mechanical regulation of synaptic membrane morphology with biochemical signaling mediated by mTORC2 and Rho GTPases to orchestrate the kinetics of synaptic vesicle endocytosis.
View lessThe discovery of useful molecules and new molecular phenomena is one of the cornerstones of human progress. Until the last two centuries, this process was largely driven by empirical evidence and serendipitous discovery. The understanding of physical phenomena at the macro level, driven by Newtonian mechanics, electromagnetism, and thermodynamics, and at the micro level, driven by quantum mechanics, has allowed for a more targeted approach to the discovery of new functional molecules for various applications. Despite these advances, the pace at which such molecules are discovered lags behind the rate of demand for green catalysts, sustainable materials, and effective medicines. A significant factor influencing this is the vastness of the chemical space of molecules. It has been approximated that within this chemical space, there exist approximately 1060 organic molecules (with a molecular weight less than 500, containing atoms H, C, N, and S). This count will be several magnitudes higher if larger molecules and extended structures are taken into account. Cataloging the properties of these molecules is not currently possible with our computational existing capabilities, but it is essential to find better materials and more effective drugs. As a result, the search for methods that can help speed up the assessment of the properties of molecules and accelerate the discovery of new molecules is an issue of paramount importance in modern chemistry. Machine Learning (ML) algorithms for predicting chemical properties represent an important step in this direction. Not only are ML algorithms capable of learning accurate structure-property relationships, but they are also faster than experiments or quantum chemical simulations. Furthermore, some ML methods leverage the structure-property relationships learned from data to generate novel molecules with desired properties, providing a cost-efficient way to identify useful molecules for laboratory synthesis. The spectrum of a molecule is one such important molecular property that helps scientists identify different molecules without destroying them. Amongst the various techniques of spectroscopy, X-ray Absorption Spectroscopy (XAS) is a well established technique that provides information about the structure and composition of various materials. The identification of materials using XAS, however, is not straightforward and requires using a combination of experimental data and quantum-chemical calculations performed on large computing clusters. These computational evaluations are resource-intensive and one often needs several such calculations to achieve successful molecular identification. Access to methods that can accelerate the prediction of spectra through structure-property relationships in spectroscopy can greatly enhance the ability to identify compounds synthesized in laboratories. Therefore, a major part of this dissertation is dedicated to employing and understanding ML methods that speed up the prediction of spectra by learning structure-property relationships from data. This work lays a foundation for future applications, where ML models can be used in experimental setups to identify molecules from spectra without human intervention, thereby helping accelerate the synthesis and identification of novel compounds. One downside of ML applications is the lack of model interpretability, which decreases the trust of the end-users. Investigations in this dissertation focus on devising a technique that helps humans understand why ML models make certain predictions, thereby helping build trust between the ML model and its end user. The creation of chemical data for ML applications itself usually requires quantum chemical calculations that involve solving the Schrödinger equation. The time dependent Schrödinger equation (TDSE) helps understand the behavior of quantum systems and allows for the calculation of time-dependent properties of molecules. The area of research that concerns itself with techniques for solving the TDSE is termed quantum dynamics. Using computer simulations of numerical methods for solving this equation, researchers have modeled several quantum dynamical systems, which have improved our understanding of photo-catalysis (reactions driven by light), surface phenomena such as chemisorption, and chemical reaction pathways. The second part of this dissertation focuses on using ML methods to solve the TDSE. The TDSE, which is a partial differential equation (PDE) in space and time, is one of the many fundamental equations that help model the behavior of physical systems. Some other notable PDEs that play an important role in physics and engineering are the Navier-Stokes equation for modeling fluids, the Heat equation in thermodynamics, and the wave equation in acoustics. Numerical techniques for solving PDEs are based on the discretization of the coordinate space into finite elements. As the size and dimensions of the grids increase, these methods become computationally expensive. As a result, solving PDEs such as the TDSE for large molecular systems is computationally demanding or even impossible. Advances in ML for solving PDEs aim at accelerating the solution of PDEs through a data-driven approach. In the second part of this thesis, ML models were trained on simulation data from quantum dynamical systems. Once trained, these models are capable of providing accurate descriptions of the behavior of systems that were not seen during training. A key advantage of such methods is their ability to generate novel simulations accurately and at high speed. As a proof of concept, the work in this dissertation shows how this speed can be exploited for downstream applications in quantum dynamics.
View lessDie Untersuchungen heben die Bedeutung des Tierschutzes in der Fleischindustrie, insbesondere in Schlachtbetrieben hervor, und spiegeln die öffentliche Debatte über den Schutz der Tiere während des Transports und der Schlachtung wider. Bereits vor dem Transport haben die Haltung und der Gesundheitszustand der Rinder einen erheblichen Einfluss auf das Wohlergehen der Tiere. Im Schlachtbetrieb kann das Verhalten der Mitarbeiter:innen oder die baulichen Bedingungen und die damit verbundenen Auswirkungen Stress bei den Tieren verursachen. Studien haben gezeigt, dass Stress sowohl durch Verhaltensbeobachtungen als auch durch die Messung physiologischer Parameter bewertet werden können. Eine schnelle und effektive Betäubung ist ein wichtiger Aspekt des Tierschutzes. Leicht überprüfbare Indikatoren für die Bewusstlosigkeit, wie der unmittelbare Zusammenbruch nachdem Bolzenschuss, Verlust des rhythmischen Atmens und des Kornealreflexes, werden routinemäßig am Schlachtbetrieb überwacht. Andere Aspekte, wie die Messung von Stresshormonen im Blut oder die Verwendung eines Elektrokardiogramms während der Betäubung, bieten wissenschaftliche Informationen für die Bewertung von Stress, sind aber in der Praxis nicht routinemäßig umsetzbar. Schulungen für Tiertransporteur:innen und Schlachthofmitarbeiter:innen tragen erheblich zur Stressreduzierung während des Umgangs mit Rindern bei, was wiederum das Wohl der Tiere während des Transports und der Schlachtung sowie die Fleischqualität verbessert. In einer Studie wurden zwei mehrsprachige Online-Schulungsmodule zur Schulung von Mitarbeiter:innen in deutschen Rinderschlachthöfen auf den Gebieten Tierverhalten und Umgang mit Rindern entwickelt und getestet. Die Teilnehmer:innen bewerteten das Programm positiv und die Ergebnisse deuten darauf hin, dass E-Learning zur Verbesserung des Wissens beiträgt, insbesondere wenn es auf Video-basiertem Lernen basiert. Die Untersuchung zeigte, dass es Verbesserungen in den Testergebnissen vom Prä- zum Post-Test in beiden teilnehmenden Gruppen gab. In einer weiteren Untersuchung wurde die gleiche Online-Schulung und deren Implementierung von Tierschutzbeauftragten und amtlichen Tierärzt:innen getestet und evaluiert. Die 25 an der Umfrage teilnehmenden Personen gaben den Schulungsmodulen hinsichtlich des Designs, der Lerninhalte und der Benutzerfreundlichkeit eine positive Bewertung. Insgesamt zeigen die Untersuchungen, dass gezielte Online-Schulungsprogramme das Potenzial haben, das Tierwohl zu fördern, Stress für Mitarbeiter:innen und Tiere zu reduzieren und die Arbeitsbedingungen insgesamt zu verbessern. Hier besteht die Notwendigkeit weiterer Forschung und Entwicklung von Online-Schulungsmodulen, um die Standards im Tierschutz während des Transports und der Schlachtung weiter zu verbessern.
View lessVivianite, a hydrated Fe(II) phosphate (Fe3(PO4)2·8H2O), is a mineral found globally in anoxic lacustrine or marine sediments, soils, associated with organic matter and in groundwater streams. It is a key phase controlling the cycling and immobilization of Fe(II) and phosphorus in these systems. Vivianite formation can aid in phosphorus recovery from wastewater; therefore, it is an important mineral for circular economy applications. This dissertation presents novel insights into the intricate processes governing the nucleation, growth, and phase transformations of vivianite. The nucleation of vivianite occurs via a transient intermediate phase – amorphous ferrous phosphate (AFEP), via a nonclassical pathway. AFEP is a metastable precursor phase which has a distinctly different local structure and composition (Fe3(PO4)2·4.7H2O), compared to vivianite. Through laboratory experiments and ex situ techniques, I demonstrated that AFEP precipitates from supersaturated solutions, undergoes subsequent hydration and structural transformations to transform into thermodynamically stable vivianite. The crucial role of water in this transformation is also highlighted. The stability of AFEP is inversely proportional to systemic (vivianite) supersaturation and temperature, suggesting AFEP occurrence to be a widely prevalent phenomenon in near-equilibrium (environmental) systems. Synchrotron-based small and wide-angle X-ray scattering experiments provide novel insights into the temporal evolution of vivianite crystallization via AFEP nanoparticles, with observations indicating concomitant processes of AFEP transformation and vivianite growth in solution – revealing a direct link between these two processes. Furthermore, the investigation of vivianite growth kinetics shows temperature-dependent dynamics, and a direct correlation between temperature and transformation rate. The rate of vivianite growth could be modeled via the JMAK model to derive the apparent activation energy (~ 51 kJ/mol) of this transformation. Temperature-dependent variations in crystal splitting also reveal new insights into varying vivianite crystal morphologies. Additionally, the thermally induced phase transformations of vivianite (amorphization and recrystallization into a distinctly new mineral phase), particularly under high-temperature conditions, including the critical influence of atmosphere on the resultant transformed phase compositions, is also reported here for the first time. This research provides new and significant insights for vivianite/mineral nucleation and growth mechanisms in aqueous solutions, materials synthesis and thermal stability of iron phosphates.
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