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In this article we offer initial insights into the fairly new interdisciplinary and international domain of robotics in Christian religious practice. We are a group of scholars in media ethics, practical theology/religious education, and human computer interaction, who have been engaged in this discourse since 2017.
A natural starting point is our study of BlessU2, a “blessing robot,” a device which received considerable recognition from the global public at the Wittenberg 500th reformation anniversary in 2017. We thus begin with the results of this study. Secondly, we will briefly address the relevant theses from Gabriele Trovato et al., as presented in their 2019 article on so-called theomorphic robots – followed by our interdisciplinary discussion of their approach. Finally, we draw conclusions for further work on the field of “religious robots.”
Somewhat more carefully: Section 1 offers starting points within the perspectives of Christian religious practice: here, the blessing robot is both cause and occasion for doing religion and theologizing in the context of existential questions (1.1). We continue with perceptions in the field of religion regarding “Discursive Design Theory” (1.2). The interaction of humans with computers as posing questions for theological standardization of religious practice is focused upon in 1.3. Section 2 reconstructs the HRI/HCI-initiative to develop theomorphic robots in a twofold manner, i.e., the idea of developing theomorphic robots (2.1) and the concept of theomorphic robots: Questions and objections (2.2). In this part of the article we raise discussion points concerning the relationship between technology and religion and the need for sharpening the understanding of religion within the research field. Section 3 closes with propositions and alternatives.
Neurodegeneration plays an essential role in Parkinson’s disease (PD). Several crucial neuronal pro-and antidegeneration markers were described to be altered in disease models accompanied by neurodegeneration. In the AAV1/2-A53T-aSyn PD rat model progressive time-dependent motor impairment and neurodegeneration in the nigrostriatal tract starting from 2 weeks after PD model induction could be found. Downregulation of Nrf2 in SN and nigrostriatal axon localization, a trend of Tau downregulation in SN and upregulation in axon localization in the AAV1/2-A53T-aSyn PD rat model were observed, indicating potential therapeutic value of these two molecular targets in PD. No alterations of SARM1 and NMNAT2 could be detected, indicating little relevance of these two molecules with our AAV1/2-A53T-aSyn rat model.
Plexus injury often occurs after motor vehicle accidents and results in lifelong disability with severe neuropathic pain. Surgical treatment can partially restore motor functions, but sensory loss and neuropathic pain persist. Regenerative medicine concepts, such as cell replacement therapies for restoring dorsal root ganglia (DRG) function, set high expectations. However, up to now, it is unclear which DRG cell types are affected by nerve injury and can be targeted in regenerative medicine approaches.
This study followed the hypothesis that satellite glial cells (SGCs) might be a suitable endogenous cell source for regenerative medicine concepts in the DRG. SGCs originate from the same neural crest-derived cell lineage as sensory neurons, making them attractive for neural repair strategies in the peripheral nervous system. Our hypothesis was investigated on three levels of experimentation. First, we asked whether adult SGCs have the potential of sensory neuron precursors and can be reprogrammed into sensory neurons in vitro. We found that adult mouse DRG harbor SGC-like cells that can still dedifferentiate into progenitor-like cells. Surprisingly, expression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, induced neurons developed a nociceptor-like phenotype expressing functional nociceptor markers, such as the ion channels TrpA1, TrpV1 and NaV1.9. In a second set of experiments, we used a rat model for peripheral nerve injury to look for changes in the DRG cell composition. Using an unbiased deep learning-based approach for cell analysis, we found that cellular plasticity responses after nerve injury activate SGCs in the whole DRG. However, neither injury-induced neuronal death nor gliosis was observed. Finally, we asked whether a severe nerve injury changed the cell composition in the human DRG. For this, a cohort of 13 patients with brachial plexus injury was investigated. Surprisingly, in about half of all patients, the injury-affected DRG showed no characteristic DRG tissue. The complete entity of neurons, satellite cells, and axons was lost and fully replaced by mesodermal/connective tissue. In the other half of the patients, the basic cellular entity of the DRG was well preserved. Objective deep learning-based analysis of large-scale bioimages of the “intact” DRG showed no loss of neurons and no signs of gliosis.
This study suggests that concepts for regenerative medicine for restoring DRG function need at least two translational research directions: reafferentation of existing DRG units or full replacement of the entire multicellular DRG structure. For DRG replacement, SGCs of the adult DRG are an attractive endogenous cell source, as the multicellular DRG units could possibly be rebuilt by transdifferentiating neural crest-derived sensory progenitor cells into peripheral sensory neurons and glial cells using Neurog1 and Neurog2.
In dieser Arbeit wurde die Krankheitsprogression im Parkinson-Mausmodell hm2α-SYN-39 mit zunehmendem Alter charakterisiert. Die Mäuse wurden in 4 Altersgruppen (2-3, 7-8, 11-12, 16-17 Monate) mit motorischen Verhaltenstests auf einen Parkinson-Phänotyp untersucht. Zudem erfolgten Untersuchungen des dopaminergen Systems zur Detektion von neurochemischen Veränderungen und einer Neurodegeneration im nigrostriatalen Trakt. Weiterhin wurden neuroinflammatorische Prozesse des adaptiven und angeborenen IS in der SN und im Striatum mittels immunhistochemischer Färbungen beurteilt.
Ein Parkinson-Phänotyp in diesem Mausmodell zeigte sich nur leicht ausgeprägt, sodass der Rotarod- und Zylinder-Test lediglich den Hinweis auf eine nicht-signifikante Einschränkung der Motorik erbrachte. Dennoch ergab die stereologische Quantifizierung TH- und Nissl-positiver Zellen in der SNpc der hm2α-SYN-39 Mäuse eine altersabhängige, signifikant-progrediente Reduktion der dopaminergen Neurone mit zunehmendem Alter. Eine signifikant niedrigere TH-positive Zellzahl dieser tg Mäuse zeigte sich ab einem Alter von 16-17 Monaten verglichen zu gleichaltrigen wt Tieren. Dagegen war die Neurodegeneration im Striatum etwas weniger ausgeprägt. Die tg Mäuse präsentierten im Alter von 16-17 Monaten eine nicht-signifikante Erniedrigung der dopaminergen Terminalen verglichen zu gleichaltrigen wt Tieren. Ein DA-Mangel im Striatum der tg Mäuse konnte mittels HPLC bestätigt werden. Bis zum Alter von 16-17 Monaten wurde eine signifikante Reduktion der DA-Level von 23,2 % verglichen zu gleichaltrigen wt Mäusen gezeigt. Außerdem erniedrigt waren die striatalen Level von NA und 5-HAT bei tg Mäusen, passend zu den bisherigen Ergebnissen bei Parkinson-Patienten.
Immunhistochemische Untersuchungen einer Neuroinflammation im nigrostriatalen Trakt ergaben eine tendenziell erhöhte Infiltration von CD4- und CD8-positiven T-Zellen bei hm2α-SYN-39 Mäusen mit zunehmendem Alter, wobei die Infiltration CD8-positiver Zellen ausgeprägter war als bei CD4-positiven Zellen. Eine noch deutlichere neuroinflammatorische Reaktion zeigte das angeborene IS. Hierbei ergab die immunhistologische Quantifizierung CD11b-positiver mikroglialer Zellen einen hochsignifikanten Anstieg im nigrostriatalen Trakt bei hm2α-SYN-39 Mäusen schon im jungen Alter.
Zusammenfassend präsentierte dieses Parkinson-Mausmodell eine langsam-progrediente Parkinson-Pathologie mit begleitender Neuroinflammation im nigrostriatalen Trakt während des Alterns, wobei die Immunantwort der mikroglialen Zellen zu einem früheren Zeitpunkt einsetzte als die T-Zellinfiltration und Neurodegeneration. Dieses Mausmodell bietet zahlreiche Möglichkeiten zur zukünftigen Erforschung der Pathophysiologie beim MP. Generell weist diese Arbeit auf eine bedeutende Rolle neuroinflammatorischer Prozesse in der Krankheitsprogression der Parkinsonerkrankung hin und soll dazu ermutigen Neuroinflammation durchaus intensiver in tg Tiermodellen zu untersuchen.
Kanurennsport ist in Deutschland eine der erfolgreichsten olympischen
Sommersportarten und hat mit 12 potenziellen Goldmedaillenchancen eine hohe
Bedeutung für den deutschen Spitzensport. In der nationalen als auch
internationalen wissenschaftlichen Forschung ist Kanurennsport jedoch bis dato
unzureichend untersucht. Dabei stellt Kanurennsport als eine der wenigen vorrangig
durch die Oberkörpermuskulatur angetriebenen Sportarten eine Besonderheit dar.
Ein zentraler Forschungsschwerpunkt ist seit einigen Jahrzehnten die Erforschung
der optimalen Verteilung der Trainingsintensität (engl. training intensity distribution;
TID) für die Leistungsentwicklung von Ausdauerathlet:innen. Häufig wird die
Trainingsintensität hierzu in einem Drei-Zonen-Modell kategorisiert, bei dem Zone
(Z) 1 einer Intensität unterhalb der aeroben Schwelle, Z2 der Intensität zwischen
der aeroben und anaeroben Schwelle und Z3 Intensitäten oberhalb der anaeroben
Schwelle entspricht. Forschungsergebnisse weisen darauf hin, dass sich die TID
nicht nur in Abhängigkeit von Sportart, Belastungsform, Trainingsstatus und
Saisonphase unterscheidet, sondern auch in Abhängigkeit von der eingesetzten
Quantifizierungsmethode (z.B. Herzfrequenz, Geschwindigkeit, Wattleistung, etc.).
Für die Sportart Kanurennsport besteht bezüglich TID-Forschung großer
Nachholbedarf, da bisherige Untersuchungen ausschließlich in Ausdauerportarten
stattfanden, die hauptsächlich den Unterkörper (z.B. Radfahren, Laufen) oder Oberund
Unterkörper (Schwimmen, Rudern) in die Vortriebsgenerierung einbinden.
Bislang fehlen Informationen zu rein aus dem Oberkörper angetriebenen
Sportarten.
Als Grundlage für die Bestimmung der Trainingsintensitätszonen werden in
Trainingspraxis und Forschung Stufentests zur Bestimmung der maximalen
Sauerstoffaufnahme sowie der Leistung an der aeroben und anaeroben
ventilatorisch- und/oder laktatbasierten Schwelle angewandt. Die Stufentest werden
im Kanurennsport aktuell vorrangig mittels Labordiagnostik auf dem Kanu-
Ergometer durchgeführt, da diese weniger stark durch die diversen Umwelteinflüsse
(Wind, Wellen, Temperatur, Strömung, etc.) beeinträchtigt wird. Jedoch gibt es
Hinweise, dass die Belastung auf dem Ergometer biomechanisch und physiologisch
von der auf dem Wasser im Kanurennsport abweicht, sodass deren Mehrwert für
die Diagnostik und die Trainingsplanung in Frage zu stellen ist.
Ziel der vorliegenden kumulativen Dissertation war es
(1) zu untersuchen, inwiefern eine laborbasierte Leistungsdiagnostik einer
feldbasierten im Kanurennsport entspricht (Studie 1) und daraufhin die
Methoden der Leistungsdiagnostik für die Studien 2 und 3 zu wählen; und
(2) erste wissenschaftliche Erkenntnisse zur TID und deren
Quantifizierungsmethodik in der Sportart Kanurennsport zu gewinnen
(Studie 2 & 3).
Diese sollten dann mit dem Wissensstand aus Sportarten, die obere und untere
Extremitäten (z.B. Biathlon, Rudern) bzw. primär die unteren Extremitäten (z.B.
Radsport, Laufen) für den Vortrieb einsetzen, abgeglichen werden.
Zusammenfassend konnte zunächst in Studie 1 aufgrund von Unterschieden in der
VO2, der Muskeloxygenierung im Musculus biceps brachii sowie im subjektiven
Belastungsempfinden dargestellt werden, dass sich eine Belastung auf dem
Wasser von der auf dem Ergometer unterscheidet und somit eine wasserbasierte
Leistungsdiagnostik im Kanurennsport vorzuziehen ist.
Die Ergebnisse aus den Studien 2 und 3 zeigten, dass die TID im Saisonverlauf
variiert und im Mittel einen hohen Anteil (80–90%) niedrigintensiven Trainings (Z1)
aufwies, wobei in der Vorbereitungsphase eine pyramidale TID Struktur (Z1>Z2>Z3)
und in der Wettkampfvorbereitung die Tendenz zu einer vermehrt polarisierten
Struktur (Z1>Z3>Z2) gefunden wurde. Somit weisen die Ergebnisse trotz der
physiologischen sowie biomechanischen Unterschiede zu Sportarten, die Oberbzw.
Ober- und Unterkörper bei der Vortriebsgenerierung einsetzen, eine
vergleichbare TID Struktur im Kanurennsport auf. Es ist zu vermuten, dass der
geringe Impact auf das Skelettmuskelsystem und die damit einhergehende
Möglichkeit, sehr hohe Trainingsvolumen mit der vergleichsweise kleinen
Oberkörpermuskulatur zu verwirklichen, diese TID-Struktur bedingen. Zudem
konnte dargestellt werden, dass die Wahl der Quantifizierungsmethode (extern vs.
intern; basierend auf physiologischen Parametern vs. Wettkampftempo) die
Darstellung der TID beeinflusst. Für eine adäquate Vergleichbarkeit und den
gezielten Einsatz muss insofern in der Forschung wie auch in den Sportarten ein
Konsens über die Wahl der Quantifizierungsmethode erarbeitet werden. Es scheint
zudem empfehlenswert die TID-Quantifizierungsmethode anhand der
Trainingsphase auszuwählen, wobei sich in der allgemeinen und spezifischen
Vorbereitungsperiode vorzugsweise eine TID Quantifizierung nach physiologischen
Kenngrößen empfiehlt. Hierbei erscheint ein Mix aus HF-basierter Analyse für Z1
sowie für längere Belastungen in Z2 und geschwindigkeitsbasierter Analyse für Z3
sowie kürzere Belastungen der Z2 zweckmäßig. In der Wettkampfvorbereitung stellt
sich dann zusätzlich eine Zoneneinteilung basierend auf dem Wettkampftempo als
sinnvoll dar.
Aufgrund der starken intra- und interindividuellen Variation der TID ist der
individuelle Mehrwert der auf dem Gruppenmittelwert basierenden Ergebnisse
jedoch zu hinterfragen und weist auf den Bedarf nach einer individuelleren
Betrachtung der TID und ihrer Effekte hin. Genauso stellt sich ein starker Einfluss
der allgemeinen physischen Aktivität sowie psychischer Belastungen auf die TID
und ihre Effekte dar, der wiederrum die Notwendigkeit eines holistischen
Betrachtungsansatzes für zukünftige Forschung aufzeigt. Außerdem gibt es im
Allgemeinen eine große Wissenslücke in Bezug auf Athletinnen in der TIDForschung,
weshalb die bisherigen Erkenntnisse für die Trainingsgestaltung
weiblicher Athleten mit Vorsicht behandelt werden müssen.
Various types of cancer involve aberrant cell cycle regulation. Among the pathways responsible for tumor growth, the YAP oncogene, a key downstream effector of the Hippo pathway, is responsible for oncogenic processes including cell proliferation, and metastasis by controlling the expression of cell cycle genes. In turn, the MMB multiprotein complex (which is formed when B-MYB binds to the MuvB core) is a master regulator of mitotic gene expression, which has also been associated with cancer. Previously, our laboratory identified a novel crosstalk between the MMB-complex and YAP. By binding to enhancers of MMB target genes and promoting B-MYB binding to promoters, YAP and MMB co-regulate a set of mitotic and cytokinetic target genes which promote cell proliferation. This doctoral thesis addresses the mechanisms of YAP and MMB mediated transcription, and it characterizes the role of YAP regulated enhancers in transcription of cell cycle genes.
The results reported in this thesis indicate that expression of constitutively active, oncogenic YAP5SA leads to widespread changes in chromatin accessibility in untransformed human MCF10A cells. ATAC-seq identified that newly accessible and active regions include YAP-bound enhancers, while the MMB-bound promoters were found to be already accessible and remain open during YAP induction. By means of CRISPR-interference (CRISPRi) and chromatin immuniprecipitation (ChIP), we identified a role of YAP-bound enhancers in recruitment of CDK7 to MMB-regulated promoters and in RNA Pol II driven transcriptional initiation and elongation of G2/M genes. Moreover, by interfering with the YAP-B-MYB protein interaction, we can show that binding of YAP to B-MYB is also critical for the initiation of transcription at MMB-regulated genes. Unexpectedly, overexpression of YAP5SA also leads to less accessible chromatin regions or chromatin closing. Motif analysis revealed that the newly closed regions contain binding motifs for the p53 family of transcription factors. Interestingly, chromatin closing by YAP is linked to the reduced expression and loss of chromatin-binding of the p53 family member Np63. Furthermore, I demonstrate that downregulation of Np63 following expression of YAP is a key step in driving cellular migration.
Together, the findings of this thesis provide insights into the role of YAP in the chromatin changes that contribute to the oncogenic activities of YAP. The overexpression of YAP5SA not only leads to the opening of chromatin at YAP-bound enhancers which together with the MMB complex stimulate the expression of G2/M genes, but also promotes the closing of chromatin at ∆Np63 -bound regions in order to lead to cell migration.
Companies are expected to act as international players and to use their capabilities to provide customized products and services quickly and efficiently. Today, consumers expect their requirements to be met within a short time and at a favorable price. Order-to-delivery lead time has steadily gained in importance for consumers. Furthermore, governments can use various emissions policies to force companies and customers to reduce their greenhouse gas emissions. This thesis investigates the influence of order-to-delivery lead time and different emission policies on the design of a supply chain. Within this work different supply chain design models are developed to examine these different influences. The first model incorporates lead times and total costs, and various emission policies are implemented to illustrate the trade-off between the different measures. The second model reflects the influence of order-to-delivery lead time sensitive consumers, and different emission policies are implemented to study their impacts. The analysis shows that the share of order-to-delivery lead time sensitive consumers has a significant impact on the design of a supply chain. Demand uncertainty and uncertainty in the design of different emission policies are investigated by developing an appropriate robust mathematical optimization model. Results show that especially uncertainties on the design of an emission policy can significantly impact the total cost of a supply chain. The effects of differently designed emission policies in various countries are investigated in the fourth model. The analyses highlight that both lead times and emission policies can strongly influence companies' offshoring and nearshoring strategies.
Fear and anxiety disorders – interaction of AVP and OXT brain systems with the serotonergic system
(2023)
Anxiety disorders pose a great burden onto society and economy and can have devastating consequences for affected individuals. Treatment options are still limited to psychopharmacotherapy originally developed for the treatment of depression and behavioral therapy. A combination of genetic traits together with aversive events is most likely the cause of these diseases. Gene x environment studies are trying to find a link between genetic traits and specific negative circumstances. In a first study, we focused on social anxiety disorder (SAD), which is the second most-common anxiety disorder after specific phobias. We used a social fear conditioning (SFC) paradigm, which is able to mimic the disease in a mouse model. We wanted to investigate protein levels, as well as mRNA expression of immediate early genes (IEGs), to determine brain areas affected by the paradigm. We also included genes of the vasopressin (AVP)-, oxytocin (OXT)-, neuropeptide Y (NPY)-, and the serotonin system, to investigate the effects of SFC on neurotransmitter gene expression levels in brain regions related to social as well as fear-related behavior. AVP and OXT regulate a lot of different social and anxiety-related behaviors, both positive and negative. Finding a link between different neurotransmitter systems in the development of anxiety disorders could help to identify potential targets for new treatment approaches, which are desperately needed, because the rate of patients not responding to available treatment is very high.
We were able to show altered gene expression of the IEGs cFos and Fosl2, as well as a change in number and density of cFOS-positive cells in the dorsal hippocampus, indicating an influence of SFC on neuronal activity. Our results reveal a possible involvement of anterior dentate gyrus (DG), as well as cornu ammonis area 1 (CA1) and CA3 in the dorsal hippocampus during the expression of social fear. Contrary to our hypothesis, we were not able to see changes in neuronal activity through expression changes of IEGs in the amygdala. Significant higher IEG immunoreactivity and gene expression in the dorsal hippocampus of animals without fear conditioning (SFC-), compared to animals with fear conditioning (SFC+), indicate an involvement of different hippocampal regions in two possible scenarios. Either as elevated gene expression in SFC- animals compared to SFC+ animals or as reduction in SFC+ animals compared to SFC- animals. However, this question cannot be answered without an additional control of basal IEG-activity without social interaction. The NPY system in general and the neuropeptide y receptor type 2 in particular seem to be involved in regulating the response to social fear, mostly through the septum region. In addition to that, a possible role for the induction of social fear response could be identified in the serotonergic system and especially the serotonin receptor 2a of the PVN.
In a second study we focused on changes in the serotonergic system. A polymorphism in the human serotonin transporter (5-HTT) gene is associated with higher risks for the development of anxiety disorders. This makes the 5-HTT a widely used target to study possible causes and the development of anxiety disorders. In mice, a genetically induced knockout of the 5-Htt gene is associated with increased anxiety-like behavior. High amounts of stress during pregnancy, also known as prenatal stress, significantly increase the risk to develop psychiatric disorders for the unborn child. We utilized a prenatal stress paradigm in mice heterozygous for the 5-Htt gene. Some of the animals which had been subjected to prenatal stress showed noticeably “unsocial” interaction behavior towards conspecifics. Again, we were searching for links between the serotonergic system and AVP- and OXT systems. Through quantitative gene expression analysis, we were able to show that both AVP and OXT neuromodulator systems are affected through prenatal stress in female mice, but not in male mice. The 5-Htt genotype seems to be only slightly influential to AVP, OXT or any other neurotransmitter system investigated. Gene expression of AVP and OXT brain systems is highly influenced through the estrous cycle stages of female mice. Additionally, we analyzed the AVP and OXT neuropeptide levels of mice with different 5-Htt genotypes and in both sexes, in order to see whether the production of AVP and OXT is influenced by 5-Htt genotype. On neuropeptide level, we were able to identify a sex difference for vasopressin-immunoreactive (ir) cells in the PVN, with male mice harboring significantly more positive cells than female mice.
All presented studies aimed on the improvement of the quality analysis of already monographed drugs. Thereby different LC methods were applied and coupled to i.e., the UV/VIS detector, the CAD or a hyphenation of these detectors, respectively. The choice of the chromatographic system including the detector was largely dependent on the physicochemical properties of the respective analytes.
With the risk-assessment report on the API cetirizine we presented an exemplary tool, that can help to minimize the risk of the occurrence of unexpected impurities. An in- deep analysis of each step within synthesis pathway by means of reaction matrices of all compounds was performed. It is essential to understand the complete impurity profile of all reactants, solvents, and catalysts and to include them in the matrix. Finally, the API of this synthesis was checked if all impurities are identified by this tool. Of note, a shortcoming of such a targeted approach is that impurities can still occur, but they are not captured. This disadvantage can be partially compensated by non-targeted approaches if they are performed in parallel with the other studies that represent most of the impurities. However, this work also shows that even in a supposedly simple synthesis, potentially hundreds of by-products can be formed. For each of them, it must be decided individually whether their formation is probable or how their quantity can be minimized in order to obtain APIs, that are as pure as possible.
In the dapsone project it was aimed to replace the existing old Ph. Eur. TLC method with a modern RP-HPLC method. This was successful and since Ph. Eur. 10.6, the method developed in this work, became a valid monograph. Within the revision process of the monograph, the individual limits for impurities were tightened. However, this new method needs HPLC instrumentation, suitable to perform gradients. As this is not always available in all control laboratories, we also developed an alternative, more simple method using two different isocratic runs for the impurity analysis. The obtained batch results of both, the new pharmacopoeial method and the more simple one, were in a comparable order of magnitude. Furthermore, within the method development stage of the Ph. Eur. method, we could identify one unknown impurity of the impurity reference by high-resolution MS/MS analysis.
Also, in the baclofen project it was aimed to replace the existing Ph. Eur. method with the introduction of an additional impurity to be quantified. A corresponding method was developed and validated. However, due to the harmonization process of the pharmacopoeias, it is currently not used. In addition, we tried to find further, non- 116
SUMMARY
chromophoric impurities by means of the CAD. However, except for one counterion of an impurity, no further impurities were found. Also, the aforementioned new impurity could not be detected above the reporting threshold in the batches analyzed. As the only individually specified impurity A is also present at a low level, it can be concluded that the examined batches of baclofen are very pure.
The use of universal detectors, such as the CAD can be particularly interesting for compounds with no chromophore or those with only a weak chromophore. Therefore, we decided to take a closer look at the impurity profile of acarbose. Currently, acarbose and its impurities are being studied by low wavelength UV detection at 210 nm. Therefore, the question arose whether there are no other impurities in the API that do not show absorption at this wavelength. CAD, which offers consistent detection properties for all non-volatile compounds, is ideally suited for this purpose. However, it was not so easy to use the CAD together with the UV detector, for example, as a hyphenated detection technique, because the Ph. Eur. method uses phosphate buffers. However, this is non-volatile and therefore inappropriate for the CAD. Therefore, an attempt was made to replace the buffer with a volatile one. However, since this did not lead to satisfactory results and rather the self-degradation process of the stationary phase used could be observed by means of the CAD, it was decided to switch to alternative stationary phases. A column screening also revealed further difficulties with acarbose and its impurities: they show an epimerization reaction at the end of the sugar chain. However, since one wanted to have uniform peaks in the corresponding chromatograms, one had to accelerate this reaction significantly to obtain only one peak for each component. This was best achieved by using two stationary phases: PGC and Amide-HILIC. Impurity-profiling methods could be developed on each of the two phases. In addition, as expected, new impurities could be detected, albeit at a low level. Two of them could even be identified by spiking experiments as the sugar fragments maltose and maltotriose.
Taken together, it can be concluded, that this work has contributed significantly to the improvement of the quality analysis of monographed drugs. In addition to the presented general tool for the identification of potential impurities, one of the methods developed, had already been implemented to the Ph. Eur. In an effort to improve the CAD's universal detection capabilities, additional methods have also been developed. Further, new improved methods for the impurity profiling are ready to use.
In Nervensystemen bedürfen Informationsweitergabe und Gedächtnisformation eines präzisen Zusammenspiels von Synapsen in Zeit und Raum. Synaptische Transmission basiert strukturell auf mesoskopischen cytosolischen Kompartimenten an der präsynaptischen Membran, sogenannten Aktiven Zonen (AZ). Ihre Cytomatrix, bestehend aus zentralen Gerüstproteinen wie Rab3 interacting molecule (RIM), ermöglicht eine schnelle Freisetzung synaptischer Vesikel. Die Defizienz der lokal häufigsten Isoform RIM1α resultiert an einer komplexen zentralen Säugersynapse, die des hippocampalen Moosfaserboutons (MFB) zu im Cornu ammonis (CA)3 befindlichen Pyramidalzellen, in einer dezimierten Langzeitplastizität. Auf Verhaltensebene zeigen diese Mäuse eine reduzierte Lernfähigkeit.
Die vorliegende Dissertation widmet sich grundlegend der bisher unbekannten dreidimensionalen (3D) AZ-Ultrastruktur des MFB in akuten Hippocampusschnitten der adulten Wildtyp- und RIM1α-Knock-Out-Maus (RIM1α\(^{-/-}\)). In einer methodischen Entwicklungsphase wurde ein neuartiges, anspruchsvolles Protokoll der nahezu artefaktfreien (near to native) Synapsenpräparation am MFB mittels Hochdruckgefrierung und Gefriersubstitution sowie der 3D-Modellierung mittels Elektronentomographie etabliert. In einer zweiten Experimentier- und Analysephase ermöglichte die hochwertige synaptische Gewebeerhaltung in beiden Genotypen eine standardisierte, auf Programmierskripten basierte Quantifizierung der AZ-Ultrastruktur bis auf die Ebene eines individuell gedockten synaptischen Vesikels.
Dieser Dissertation gelingt der Nachweis, dass eine Defizienz von RIM1α zu einer multidimensionalen ultrastrukturellen Veränderung der AZ und ihres Vesikelpools am MFB führt. Neben einer Reduktion, Dezentralisierung und strukturellen Veränderung (eng) gedockter Vesikel – der ultrastrukturellen Messgrößen von unmittelbar freisetzungsfähigen Vesikeln – verdichtet sich der distaler lokalisierte Vesikelpool auf zugleich größeren, heterogenen AZ-Flächen mit erweitertem synaptischem Spalt. Vorliegende Untersuchungen tragen zum Verständnisgewinn über eine zentrale Rolle von RIM1α für das Docking und die Organisation von Vesikeln der AZ im MFB bei. Darüber hinaus stellen die präzisen ultrastrukturellen Analysen eine morphologische Grundlage für weiterführende Studien mit Hilfe modernster Techniken dar, beispielsweise über die Auswirkungen der geänderten RIM1α\(^{-/-}\) AZ-Ultrastruktur auf die präsynaptische Plastizität sowie in Korrelation zum Gedächtnis und Lernen der Tiere.