Refine
Has Fulltext
- yes (481)
Is part of the Bibliography
- yes (481)
Year of publication
- 2023 (481) (remove)
Document Type
- Doctoral Thesis (481) (remove)
Keywords
- Tissue Engineering (8)
- Herzinsuffizienz (7)
- Thrombozyt (7)
- Schlaganfall (6)
- Topologischer Isolator (6)
- Angststörung (5)
- Biene (5)
- Brustkrebs (5)
- Chemische Synthese (5)
- Maschinelles Lernen (5)
Institute
- Graduate School of Life Sciences (130)
- Theodor-Boveri-Institut für Biowissenschaften (26)
- Medizinische Klinik und Poliklinik I (22)
- Institut für Anorganische Chemie (17)
- Lehrstuhl für Orthopädie (17)
- Institut für Pharmazie und Lebensmittelchemie (16)
- Physikalisches Institut (16)
- Institut für Organische Chemie (15)
- Medizinische Klinik und Poliklinik II (15)
- Institut für Psychologie (13)
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (12)
- Neurologische Klinik und Poliklinik (12)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (11)
- Neurochirurgische Klinik und Poliklinik (10)
- Institut für Geographie und Geologie (9)
- Julius-von-Sachs-Institut für Biowissenschaften (9)
- Klinik und Poliklinik für Anästhesiologie (ab 2004) (9)
- Missionsärztliche Klinik (9)
- Rudolf-Virchow-Zentrum (9)
- Graduate School of Science and Technology (8)
- Institut für Experimentelle Biomedizin (8)
- Institut für Theoretische Physik und Astrophysik (8)
- Klinik und Poliklinik für Mund-, Kiefer- und Plastische Gesichtschirurgie (8)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (8)
- Institut für Informatik (7)
- Institut für Pharmakologie und Toxikologie (7)
- Institut für Physikalische und Theoretische Chemie (7)
- Klinik und Poliklinik für Strahlentherapie (7)
- Deutsches Zentrum für Herzinsuffizienz (DZHI) (6)
- Fakultät für Biologie (6)
- Institut für Virologie und Immunbiologie (6)
- Institut für diagnostische und interventionelle Radiologie (Institut für Röntgendiagnostik) (6)
- Kinderklinik und Poliklinik (6)
- Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I) (6)
- Klinik und Poliklinik für Unfall-, Hand-, Plastische und Wiederherstellungschirurgie (Chirurgische Klinik II) (6)
- Medizinische Fakultät (6)
- Institut für Geschichte der Medizin (5)
- Klinik und Poliklinik für Hals-, Nasen- und Ohrenkrankheiten, plastische und ästhetische Operationen (5)
- Klinik und Poliklinik für Kinder- und Jugendpsychiatrie, Psychosomatik und Psychotherapie (5)
- Pathologisches Institut (5)
- Betriebswirtschaftliches Institut (4)
- Frauenklinik und Poliklinik (4)
- Institut für Anatomie und Zellbiologie (4)
- Institut für Hygiene und Mikrobiologie (4)
- Institut für Klinische Neurobiologie (4)
- Institut für Klinische Transfusionsmedizin und Hämotherapie (4)
- Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie (4)
- Klinik und Poliklinik für Nuklearmedizin (4)
- Klinik und Polikliniken für Zahn-, Mund- und Kieferkrankheiten (4)
- Fakultät für Chemie und Pharmazie (3)
- Graduate School of the Humanities (3)
- Institut Mensch - Computer - Medien (3)
- Institut für Allgemeinmedizin (3)
- Institut für Geschichte (3)
- Institut für Klinische Epidemiologie und Biometrie (3)
- Institut für Kulturwissenschaften Ost- und Südasiens (3)
- Institut für Molekulare Infektionsbiologie (3)
- Institut für Pädagogik (3)
- Institut für Rechtsmedizin (3)
- Poliklinik für Zahnerhaltung und Parodontologie (3)
- Abteilung für Forensische Psychiatrie (2)
- Augenklinik und Poliklinik (2)
- Graduate School of Law, Economics, and Society (2)
- Institut für Humangenetik (2)
- Institut für Mathematik (2)
- Institut für Sonderpädagogik (2)
- Juristische Fakultät (2)
- Klinik und Poliklinik für Thorax-, Herz- u. Thorakale Gefäßchirurgie (2)
- Poliklinik für Kieferorthopädie (2)
- Poliklinik für Zahnärztliche Prothetik (2)
- Urologische Klinik und Poliklinik (2)
- Volkswirtschaftliches Institut (2)
- Abteilung für Parodontologie (in der Poliklinik für Zahnerhaltung und Parodontologie) (1)
- Fakultät für Humanwissenschaften (Philos., Psycho., Erziehungs- u. Gesell.-Wissensch.) (1)
- Graduate Schools (1)
- Institut für Altertumswissenschaften (1)
- Institut für Funktionsmaterialien und Biofabrikation (1)
- Institut für Internationales Recht, Europarecht und Europäisches Privatrecht (1)
- Institut für Politikwissenschaft und Soziologie (1)
- Institut für Sportwissenschaft (1)
- Institut für Strafrecht und Kriminologie (1)
- Institut für Systemimmunologie (1)
- Institut für deutsche Philologie (1)
- Klinik für Anaesthesiologie (bis 2003) (1)
- Lehrstuhl für Biochemie (1)
- Physiologisches Institut (1)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (2)
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Anthropology Department University of Tennessee, Knoxville (1)
- Bungando Medical Centre, Mwanza, Tanzania (1)
- CHC Würzburg (Comprehensive Hearing Center) (1)
- CUHAS Catholic university of health and allied science, Mwanza, Tanzania (1)
- Caritas-Krankenhaus Bad Mergentheim (1)
- Carl-Ludwig-Institut für Physiologie, Universität Leipzig (1)
- Center for Computational and Theoretical Biology (CCTB), Universität Würzburg (1)
- Chair of Experimental Biomedicine I (1)
Die Pyridoxal-5‘-Phosphat Phosphatase (PDXP), auch bekannt als Chronophin (CIN), ist eine HAD-Phosphatase, die beim Menschen ubiquitär exprimiert wird und eine entscheidende Rolle im zellulären Vitamin-B6-Metabolismus einnimmt. PDXP ist in der Lage Pyridoxal-5‘-Phosphat (PLP), die co-enzymatisch aktive Form von Vitamin B6, zu dephosphorylieren. In-vivo Studien mit Mäusen zeigten, dass die Abwesenheit von PDXP mit verbesserten kognitiven Leistungen und einem verringerten Wachstum von Hirntumoren assoziiert ist. Dies begründet die gezielte Suche nach einem pharmakologischen Inhibitor für PDXP. Ein Hochdurchsatz-Screen legte nahe, dass 7,8-Dihydroxyflavon (7,8-DHF) hierfür ein potenzieller Kandidat ist. Zahlreiche Studien beschreiben bereits vielfältige positive neurologische Effekte nach in-vivo Administration von 7,8-DHF, allerdings bleibt der genaue Wirkmechanismus umstritten und wird bis dato nicht mit PDXP in Zusammenhang gebracht. Ziel dieser Arbeit ist es, die Inhibition von PDXP durch 7,8-DHF näher zu charakterisieren und damit einen Beitrag zur Beantwortung der Frage zu leisten, ob PDXP an den 7,8-DHF-induzierten Effekten beteiligt ist.
Hierzu wurde der Effekt von 7,8-DHF auf die enzymatische Aktivität von rekombinant hergestelltem, gereinigtem PDXP in in-vitro Phosphatase-Assays charakterisiert. Um die Selektivität von 7,8-DHF gegenüber PDXP zu untersuchen, wurden fünf weitere HAD-Phosphatasen getestet. Unter den analysierten Phosphatasen zeigte einzig die dem PDXP nah verwandte Phosphoglykolat Phosphatase (PGP) eine geringer ausgeprägte Sensitivität gegen 7,8-DHF. Ein Vergleich von 7,8-DHF mit sechs strukturell verwandten, hydroxylierten Flavonen zeigte, dass 7,8-DHF unter den getesteten Substanzen die höchste Potenz und Effektivität aufwies. Außerdem wurde eine Co-Kristallisation von PDXP mit 7,8-DHF durchgeführt, deren Struktur bis zu einer Auflösung von 2,0 Å verfeinert werden konnte. Die in der Kristallstruktur identifizierte Bindungsstelle von 7,8-DHF an PDXP wurde mittels verschiedener, neu generierter PDXP-Mutanten enzymkinetisch bestätigt. Zusammenfassend zeigen die hier beschriebenen Ergebnisse, dass 7,8-DHF ein direkter, selektiver und vorwiegend kompetitiver Inhibitor der PDXP-Aktivität ist, mit einer IC50 im submikromolaren Bereich.
Die Ergebnisse dieser in-vitro Untersuchungen motivieren zu weiterer Forschung bezüglich der 7,8-DHF-vermittelten Inhibition der PDXP-Aktivität in Zellen, um die Frage beantworten zu können, ob PDXP auch in-vivo ein relevantes Target für 7,8-DHF darstellt.
The discovery, heterologous expression, and characterization of channelrhodopsin-2 (ChR2) – a light-sensitive cation channel found in the green alga Chlamydomonas reinhardtii – led to the success of optogenetics as a powerful technology, first in neuroscience. ChR2 was employed to induce action potentials by blue light in genetically modified nerve cells. In optogenetics, exogenous photoreceptors are expressed in cells to manipulate cellular activity. These photoreceptors were in the beginning mainly microbial opsins. During nearly two decades, many microbial opsins and their mutants were explored for their application in neuroscience. Until now, however, the application of optogenetics to plant studies is limited to very few reports. Several optogenetic strategies for plant research were demonstrated, in which most attempts are based on non-opsin optogenetic tools. Opsins need retinal (vitamin A) as a cofactor to generate the functional protein, the rhodopsin. As most animals have eyes that contain animal rhodopsins, they also have the enzyme - a 15, 15'-Dioxygenase - for retinal production from food-supplied provitamin A (beta-carotene). However, higher plants lack a similar enzyme, making it difficult to express functional rhodopsins successfully in plants. But plant chloroplasts contain plenty of beta-carotene. I introduced a gene, coding for a 15, 15'-Dioxygenase with a chloroplast target peptide, to tobacco plants. This enzyme converts a molecule of β-carotene into two of all-trans-retinal. After expressing this enzyme in plants, the concentration of all-trans-retinal was increased greatly. The increased retinal concentration led to increased expression of several microbial opsins, tested in model higher plants. Unfortunately, most opsins were observed intracellularly and not in the plasma membrane. To improve their localization in the plasma membrane, some reported signal peptides were fused to the N- or C-terminal end of opsins. Finally, I helped to identify three microbial opsins -- GtACR1 (a light-gated anion channel), ChR2 (a light-gated cation channel), PPR (a light-gated proton pump) which express and work well in the plasma membrane of plants. The transgene plants were grown under red light to prevent activation of the expressed opsins. Upon illumination with blue or green light, the activation of these opsins then induced the expected change of the membrane potential, dramatically changing the phenotype of plants with activated rhodopsins.
This study is the first which shows the potential of microbial opsins for optogenetic research in higher plants, using the ubq10 promoter for ubiquitous expression. I expect this to be just the beginning, as many different opsins and tissue-specific promoters for selective expression now can be tested for their usefulness. It is further to be expected that the here established method will help investigators to exploit more optogenetic tools and explore the secrets, kept in the plant kingdom.
This dissertation explores the local gazetteers of West Lake that were compiled by literati of the Ming dynasty. In 1547, the first West Lake gazetteer was published by the local literatus of Hangzhou, Tian Rucheng 田汝成. In the late sixteenth and early seventeenth centuries, accompanying the huge enthusiasm for West Lake and the flourishing of its tourism, the production of West Lake gazetteers reached its peak. This trend, however, was reduced by the turmoils in the last years of the Ming and the dynastic transition, a period when West Lake had also experienced destruction. Nevertheless, the practice was resumed in the first decades of the Qing dynasty by some literati who had survived the disasters. One prominent work of this period was compiled by the Ming loyalist and “remnant subject” Zhang Dai 張岱, who wrote an author’s preface in 1671. This dissertation can be divided into two parts. The first part focuses on the editorial principles of compilers, e.g., which materials are included, how they are organized and presented. It explores various possible intentions of the compilers, such as scholarly and documentary, practical and oriented toward tour-guiding, didactic and educational, and personal and nostalgic ones. The second part focuses on some of the perceptions, attitudes, and values of literati focusing on West Lake. The discourses analyzed in this part include West Lake as a hybrid between metropolitan city and sheer wilderness, as a national symbol and object of nostalgia of the lost dynasty, and as a place of pleasure-seeking and indulgence. While a discourse often had a long tradition and historical development, the emphasis of the study is on the late sixteenth and early seventeenth centuries, i.e., the late Ming.
Ziel dieser Studie war es, zu untersuchen, ob dendritische Zellen eine Rolle beim ischämischen Schlaganfall spielen. Zur Beantwortung dieser Fragestellung wurde ein Mausmodell gewählt, in dem es nach Administration von Diphterietoxin zur selektiven Depletion CD11c positiver Zellen kommt (C.FVB-Tg(Itgax-DTR/EGFP)57Lan/J). Hierbei wird der Diphterietoxinrezeptor unter dem CD11c Promotor (ITGAX) exprimiert. Aufgrund der Wiederherstellung dendritischer Zellen nach ca. 24 Stunden waren wiederholte Applikationen von Diphterietoxin notwendig. Die Zusammensetzung anderer Immunzellen wurde dabei im Wesentlichen nicht geändert.
Für eine Schlaganfallinduktion wurde eine tMCAO (transient middle cerebral artery occlusion) durchgeführt. Hierbei wird durch Okklusion der A. cerebri media mittels Verschlussfilament für 30 oder 60 Minuten ein Schlaganfall im Mediastromgebiet induziert.
Es wurden unterschiedliche Verschlusszeiten, Zeitpunkte und Depletionsraten untersucht. In keinem der Versuchsansätze kam es zu einer signifikanten Veränderung des Schlaganfallvolumens nach Depletion CD11c positiver Zellen.
Mittels quantitativer real-time PCR wurde die Expression unterschiedlicher Zytokine nach tMCAO und CD11c-Depletion untersucht. An Tag 1 nach Schlaganfallinduktion und hoher Depletionsrate ergab sich eine Verminderung der Expression von IL-1β und IL-6, während an Tag 3 und niedriger Depletionsrate die Expression dieser Zytokine nach CD11c-Depletion zunahm. Grund hierfür könnte die Expression dieser Zytokine durch andere Zellen des Immunsystems, wie etwa neutrophile Granulozyten oder Mikroglia/Makrophagen sein, die möglicherweise einer regulatorischen Funktion durch die Interaktion von Dendritischen Zellen und regulatorischen T-Zellen unterliegen. Weitere experimentelle Ansätze sind notwendig, um diese Fragestellung beantworten zu können.
TGF-β zeigte durchgehend in allen Versuchsanordnungen eine verminderte Expression nach der Depletion dendritischer Zellen. Es ist naheliegend, dass dieses neuroprotektiv-regulatorische Zytokin direkt einer Produktion durch dendritische Zellen oder von nachfolgend aktivierten T-Zellen unterliegt.
In immunhistochemischen Studien konnte des Weiteren keine Änderung des Immigrationsverhaltens von CD11b+ Zellen ins Gehirn gesehen werden.
Diese Studie unterliegt jedoch einigen Limitationen. So stellte sich im Laufe der Experimente heraus, dass die wiederholte Applikation von Diphterietoxin zu einer erhöhten Mortalität der Versuchstiere führte. Nach Fertigstellung der Experimente erschien hierzu eine Publikation, welche die wiederholte Administration von DTX und die Entwicklung einer Myokarditis im gewählten Mausmodell in Zusammenhang brachte.
Auditorische Hirnstammimplantate (ABI stellen die einzige Option der Hörrehabilitation bei bilateraler retrocochleärer Ertaubung dar. Die Implantate sind insbesondere in ihrer größten Nutzergruppe - Neurofibromatose Typ 2 Patienten - für ihr sehr variables Hörergebnis bekannt.
Die Evozierbarkeit und die Qualität der intraoperativ abgeleiteten elektrisch evozierten auditorischen Hirnstammantworten wird als möglicher Einflussfaktor auf das Outcome diskutiert. Bisher gelten weder für die Frage des Einsatzes an sich, noch für die Methodik oder die Analyse und Bewertung der EABR in der ABI-Chirurgie einheitliche Konzepte. Ziel dieser Studie ist die detaillierte Analyse der intraoperativ registrierten EABR während ABI-Implantation bei NF2-Patienten.
Zudem stellt Beurteilung der Hörfunktion mit ABI bei NF2-Patienten stellt aufgrund oftmals begleitender Symptomatik der Grunderkrankung eine besondere Herausforderung dar. Sprachtests allein spiegeln die Hörfunktion in dieser Patientengruppe nicht immer umfassend wider. Die in dieser Studie angewendete Würzburger Skala für Implantat-Hören soll dieser Problematik gerecht werden, indem Ergebnisse eines etablierten Sprachtests mit der klinischen Kommunikationsfähigkeit kombiniert werden.
Zusammenfassung der Hauptergebnisse:
Nach intraoperativer Stimulation mittels ABI zeigten sich EABR-Antworten mit null bis 3 Vertex-positiven Peaks (P1, P2, P3), welche in dieser Kohorte im Mittel nach 0,42 ms (P1), 1,43 ms (P2) bzw. 2,40 ms (P3) auftraten. Eine 2-Peak Wellenform war in dieser Studie die am häufigsten beobachtete Morphologie (78,8%). Bei der Stimulation unterschiedlicher Elektrodenkontakte zeigten sich Unterschiede in der EABR-Wellenmorphologie. Alle Antworten konnten in eine der fünf Kategorien der Würzburger EABR-Klassifikation eingeordnet werden.
Für die Latenz von P2 konnte eine statistisch signifikante Korrelation mit der Tumorausdehnung nach Hannover Klassifikation gezeigt werden.
Die Einstufung des Hörergebnisses mit ABI in NF2 nach Ergebnis im MTP-Test und nach Kommunikationsfähigkeit im Alltag unterschied sich in 7 von 22 Fällen (31,2%) um eine Kategorie. Bei der Einordnung in die Würzburger Skala für Implantat-Hören zeigte sich nach Diskussion der divergenten Fälle in 2 Fällen die Kategorisierung zugunsten des Ergebnisses im MTP-Test und in 5 Fällen zugunsten des Ergebnisses der Kommunikationsfähigkeit im Alltag.
Nützliches Hören mit ABI konnte in 95,5% der Patienten gezeigt werden, davon erzielten 68,2% Sprachverständnis.
Die Auslösbarkeit reproduzierbarer intraoperativer EABRs konnte in 95,5% Hörvermögen hervorsagen.
N-heterocyclic carbenes (NHC) are utilized for the stabilization of reactive compounds, for the activation of strong bonds, and as ligands in transition metal chemistry. In contrast to neutral NHCs, few examples of anionic or even dianionic NHCs are known. One approach for the synthesis of anionic carbenes is the deprotonation of neutral or anionic precursors, bearing Lewis acids instead of alkyl or aryl substituents. Following this strategy, novel anionic and dianionic NHCs, featuring weakly coordinating fluorinated borane and phosphorane substituents or coordinating tricyanoborane substituents were synthesized within the scope of this thesis. These carbenes possess unprecedented stabilities compared to related species. Furthermore, their electronic and steric properties can be directly adjusted by the type of Lewis acid attached. Their potential as ligands with highly shielding weakly coordinating substituents next to the carbene coordination center was demonstrated by the syntheses of the respective NHC selenium adducts and NHC gold(I) complexes. In contrast anionic NHCs with coordinating tricyanoborane moieties have an outstanding potential as ditopic ligands with coordination being possible at the carbene center and via the cyano groups. Their beneficial ligand properties were demonstrated by the syntheses of the respective NHC selenium adducts and NHC nickeltricarbonyl complexes. The combination of electronic properties, the large buried volume, the negative charge, the possibility to act as ditopic or ligands with weakly coordinating groups, and the ease of accessibility render borane- and phosphorane functionalized NHCs unique novel ligands. A further project of this PhD thesis deals with the steric properties of Lewis acids. Therefore, an easy-to-apply model was designed to quantify the steric demand of Lewis acids. Using the results of this evaluation, a second model was developed which judges the steric repulsion in Lewis acid/base adduct formation for arbitrary sets of acids and bases.
Aging is known to be a risk factor for structural abnormalities and functional decline in the nervous system. Characterizing age-related changes is important to identify putative pathways to overcome deleterious effects and improve life quality for the elderly. In this study, the peripheral nervous system of 24-month-old aged C57BL/6 mice has been investigated and compared to 12-month-old adult mice. Aged mice showed pathological alterations in their peripheral nerves similar to nerve biopsies from elderly human individuals, with nerve fibers showing demyelination and axonal damage. Such changes were lacking in nerves of adult 12-month-old mice and adult, non-aged humans. Moreover, neuromuscular junctions of 24-month-old mice showed increased denervation compared to adult mice. These alterations were accompanied by elevated numbers of macrophages in the peripheral nerves of aged mice. The neuroinflammatory conditions were associated with impaired myelin integrity and with a decline of nerve conduction properties and muscle strength in aged mice.
To determine the pathological impact of macrophages in the aging mice, macrophage depletion was performed in mice by oral administration of CSF-1R specific kinase (c-FMS) inhibitor PLX5622 (300 mg/kg body weight), which reduced the number of macrophages in the peripheral nerves by 70%. The treated mice showed attenuated demyelination, less muscle denervation and preserved muscle strength. This indicates that macrophage-driven inflammation in the peripheral nerves is partially responsible for the age-related neuropathy in mice.
Based on previous observations that systemic inflammation can accelerate disease progression in mouse models of neurodegenerative diseases, it was hypothesized that systemic inflammation can exacerbate the peripheral neuropathy found in aged mice. To investigate this hypothesis, aged C57BL/6 mice were intraperitoneally injected with a single dose of lipopolysaccharide (LPS; 500 μg/kg body weight) to induce systemic inflammation by mimicking bacterial infection, mostly via activation of Toll-like receptors (TLRs). Altered endoneurial macrophage activation, highlighted by Trem2 downregulation, was found in LPS injected aged mice one month after injection. This was accompanied by a so far rarely observed form of axonal perturbation, i.e., the occurrence of “dark axons” characterized by a damaged cytoskeleton and an increased overall electron density of the axoplasm. At the same time, however, LPS injection reduced demyelination and muscle denervation in aged mice. Interestingly, TREM2 deficiency in aged mice led to similar changes to LPS injection. This suggests that LPS injection likely mitigates aging-related demyelination and muscle denervation via Trem2 downregulation.
Taken together, this study reveals the role of macrophage-driven inflammation as a pathogenic mediator in age-related peripheral neuropathy, and that targeting macrophages might be an option to mitigate peripheral neuropathies in aging individuals. Furthermore, this study shows that systemic inflammation may be an ambivalent modifier of age-related nerve damage, leading to a distinct type of axonal perturbation, but in addition to functionally counteracting, dampened demyelination and muscle denervation. Translationally, it is plausible to assume that tipping the balance of macrophage polarization to one direction or the other may determine the functional outcome in the aging peripheral nervous system of the elderly.
The light-gated cation channel Channelrhodopsin-2 was discovered and characterized in 2003. Already in 2005/2006 five independent groups demonstrated that heterologous expression of Channelrhodopsin-2 is a highly useful and simply applicable method for depolarizing and thereby activating nerve cells. The application of Channelrhodopsin-2 revolutionized neuroscience research and the method was then called optogenetics. In recent years more and more light-sensitive proteins were successfully introduced as “optogenetic tools”, not only in neuroscience. Optogenetic tools for neuronal excitation are well developed with many different cation-conducting wildtype and mutated channelrhodopsins, whereas for inhibition of neurons in the beginning (2007) only hyperpolarizing ion pumps were available. The later discovered light-activated anion channels (anion channelrhodopsins) can be useful hyperpolarizers, but only at low cytoplasmic anion concentration. For this thesis, I optimized CsR, a proton-pumping rhodopsin from Coccomyxa subellipsoidea, which naturally shows a robust expression in Xenopus laevis oocytes and plant leaves. I improved the expression and therefore the photocurrent of CsR about two-fold by N-terminal modification to the improved version CsR2.0, without altering the proton pump function and the action spectrum. A light pulse hyperpolarised the mesophyll cells of CsR2.0-expressing transgenic tobacco plants (N. tabacum) by up to 20 mV from the resting membrane potential of -150 to -200 mV. The robust heterologous expression makes CsR2.0 a promising optogenetic tool for hyperpolarization in other organisms as well. A single R83H point-mutation converted CsR2.0 into a light-activated (passive) proton channel with a reversal potential close to the Nernst potential for intra-/extra-cellular H+ concentration. This light-gated proton channel is expected to become a further useful optogenetic tool, e.g. for analysis of pH-regulation in cells or the intercellular space. Ion pumps as optogenetic tools require high expression levels and high light intensity for efficient pump currents, whereas long-term illumination may cause unwanted heating effects. Although anion channelrhodopsins are effective hyperpolarizing tools in some cases, their effect on neuronal activity is dependent on the cytoplasmic chloride concentration which can vary among neurons. In nerve cells, increased conductance for potassium terminates the action potential and K+ conductance underlies the resting membrane potential in excitable cells. Therefore, several groups attempted to synthesize artificial light-gated potassium channels but 2 all of these published innovations showed serious drawbacks, ranging from poor expression over lacking reversibility to poor temporal precision. A highly potassium selective light-sensitive silencer of action potentials is needed. To achieve this, I engineered a light-activated potassium channel by the genetic fusion of a photoactivated adenylyl cyclase, bPAC, and a cAMP-gated potassium channel, SthK. Illumination activates bPAC to produce cAMP and the elevated cAMP level opens SthK. The slow diffusion and degradation of cAMP makes this construct a very light-sensitive, long-lasting inhibitor. I have successfully developed four variants with EC50 to cAMP ranging from 7 over 10, 21, to 29 μM. Together with the original fusion construct (EC50 to cAMP is 3 μm), there are five different light- (or cAMP-) sensitive potassium channels for researchersto choose, depending on their cell type and light intensity needs.
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.
RNA is one of the most abundant macromolecules and plays essential roles in numerous biological processes. This doctoral thesis consists of two projects focusing on RNA structure and RNA-RNA interactions in viral genome packaging. In the first project I developed a method called Functional Analysis of RNA Structure (FARS-seq) to investigate structural features regulating genome dimerization within the HIV-1 5’UTR. Genome dimerization is a conserved feature of retroviral replication and is thought to be a prerequisite for binding to the viral structural protein Pr55Gag during genome packaging. It also plays a role in genome integrity and evolution through recombination, and is linked to a structural switch that may regulate genome packaging and translation within cells. Despite its importance for HIV-1 replication, the RNA signals regulating genome dimerization, and the molecular mechanism leading to the selection of the genome dimer over the monomer for packaging are incompletely understood. The FARS-seq method combines RNA structural information obtained by chemical probing with single nucleotide resolution profiles of RNA function obtained by mutational interference. In this way, we found nucleotides that were critical for dimerization, especially within the well-characterized dimerization motif within stem-loop 1 (SL1). We also found stretches of nucleotides that enhanced genome dimerization upon mutation, suggesting their role in negatively regulating dimerization. A structural analysis identified distinct structural signatures within monomeric and dimeric RNA. The dimeric conformation displayed the canonical transactivation response (TAR), PolyA, primer binding site (PBS), and SL1-SL3 stem-loops, and contained a long range U5-AUG interaction. Unexpectedly, in monomeric RNA, SL1 was reconfigured into long- and short-range base-pairings with PolyA and PBS, respectively. Intriguingly, these base pairings concealed the palindromic sequence needed for dimerization and disrupted the internal loop in SL1 previously shown to contain the major packaging motif for Pr55Gag. We therefore rationally introduced mutations into PolyA and PBS, and showed how these regions regulate genome dimerization, and the binding of Pr55Gag in vitro, as well as genome packaging into virions. These findings give insights into late stages of the HIV-1 life cycle and a mechanistic explanation for the link between RNA dimerization and packaging.
In the second project, I developed a proximity ligation and high-throughput sequencing-based method, RNA-RNA seq, which can measure direct (RNA-RNA) and indirect (protein-mediated) interactions. In contrast to existing methods, RNA-RNA seq is not limited by specific protein or RNA baits, nor to a particular crosslinking reagent. The genome of influenza A virus contains eight segments, which assemble into a “7+1” supramolecular complex. However, the molecular details of genome assembly are poorly understood. Our goal is to use RNA-RNA seq to identify the sites of interaction between the eight genomic RNAs of influenza, and to use this information to define the quaternary RNA architecture of the genome. We showed that RNA-RNA seq worked on model substrates, like the HIV-1 Dimerization Initiation Site (DIS) RNA and purified ribosome, as well as influenza A virus infected cells.
The universal two-child policy was introduced by the central government of China in 2016 to respond to the country’s deteriorating population problems, but it was soon replaced by a three-child policy in 2021 given that it failed to continuously boost fertility in Chinese society. This dissertation empirically investigates the implementation of universal two-child policy in three Chinese major cities. Based on the data collected through semi-structured interviews with leaders of local family planning agencies, it finds that local officials are primarily devoted to coping with the discontent of the bereaved single-child parents (shidu families), which is an unexpected consequence of the historical one-child policy, rather than working on the tasks regarding birth encouragement. The dissertation suggests understanding the implementation of China’s population policy within the framework of both historical and rational choice institutionalism. The target responsibility system as an effective tool of the central authority drives local agents to fix their attention at tasks that have larger impact on their career. The shifted focus in the implementation of the universal two-child policy is a result of local officials’ emphasis on the task of maintaining social stability. Shidu families are deemed as a salient threat to social order because their discontent with the state support has incurred continuous petitions at both the national and local level, which would severely undermine local officials’ career advancement. However, in the meantime, stability maintenance is found to have become alienated as reflected by the rising costs and that it replaced birth support to be the focus of local family planning agents in the universal two-child policy era. Since the conflict between the shidu group and the state is unlikely to be resolved, the future population policy design and enforcement will continue to be constrained by the shidu problem.
Motivated by the perceived great potential of chiral polymers, the presented work aimed at the investigation of synthesis, solubility and optical activity of chiral poly(2,4-disubstituted-2-oxazoline)s. A novel polymeric carrier based on ABA-type triblock copolymers poly(2-oxazoline)s with chiral and racemic hydrophobic blocks was developed for the formulation of chiral and achiral drugs (Fig. 5.1). Poly(2-methyl-2-oxazoline) (pMeOx) was used as hydrophilic A block, and poly(2-ethyl-4-ethyl-2-oxazoline) (pEtEtOx) and poly(2-propyl-4-methyl-2-oxazoline) (pPrMeOx) were used as hydrophobic B blocks. Curcumin (CUR), paclitaxel (PTX) and chiral/racemic ibuprofen (R/S/RS-IBU) were applied as model drugs. Nanoformulations were prepared consisting of these triblock copolymers and model drugs. ...
SUMOylation, as a post-translational modification, plays a crucial role in several biological processes. Small ubiquitin-like modifier (SUMO) proteins can be reversibly linked to the lysine residues located within specific motifs on numerous target proteins, leading to the change of stability, localization, activity of target proteins, mostly by promoting or interfering with the interaction with other molecules. Consequently, it can regulate gene transcription, migration, cell cycle progression, cellular responses to stress, and tumorigenesis.
NFATc1 belongs to the Nuclear Factor of Activated T-cells (NFAT) transcription factor family, which is dephosphorylated and translocates to the nucleus upon cell stimulation, which provokes Ca2+ signalling. NFAT plays a crucial role in the development and function of the immune system. NFATc1 has three SUMOylation sites at the position of aa 349, 702, and 914. In our previous study, we demonstrated that point mutations performed on the SUMOylation sites on all three or only at the lysine residues K702 and K914 lead to enhanced expression of IL-2 in vitro. To evaluate the function of SUMOylation of NFATc1 on T cell-mediated immunity in vivo, we not only generated a transgenic mouse strain (NFATc1/ΔS+ mouse) by point mutations from Lysine to Arginine on the two SUMOylation sites within exon 10 of Nfatc1 to prevent their SUMOylation, but in combination created another mouse strain (NFATc1/ΔBC+ mouse) that is completely Nfatc1 exon 10-ablated by using the LoxP/Cre system. In NFATc1/ΔS+ T cells, we observed enhanced IL-2 production and less IL-17A and IFN-γ expression. In line with exon 10 bearing the relevant SUMO sites, NFATc1/ΔBC+ CD4+ T cells behaved similarly as NFATc1/ΔS+ ones. The mechanism is that elevated IL-2 secretion can counteract the expression of IL-17A and IFN-γ via STAT5 and Blimp-1 induction. Afterwards, Blimp-1 suppressed IL-2 itself as well as Bcl2A1. Next, we performed two disease models with our NFATc1/ΔS+ mice. In a major mismatch model for acute graft-versus-host disease, we found that the mice transplanted with NFATc1/ΔS+ CD3+ T cells developed less severe disease, and T cells proliferated less due to increased Tregs. Moreover, when transferring 2D2.NFATc1/ΔS+ Th1 plus Th17 cells to Rag1-/- mice to induce experimental autoimmune encephalitis, we also observed ameliorated disease compared to animals with transferred WT T cells as well as increased Tregs.
Taking all data together, the deficiency in SUMOylation of NFATc1 leads to an elevated IL-2 secretion in T cells and subsequent activation of STAT5, which competes with STAT3 to inhibit IL-17A production and promotes Treg expansion, as well as to an enforcement of Blimp-1 expression, which suppresses IFN-γ and IL-2 expression. Consequently and despite a short phase of enhanced IL-2 secretion, the deficiency of SUMOylation on NFATc1 can protect from autoreactive and alloreactive diseases.
Moreover, to further understand the function of SUMOylation of NFATc1 in humans, we started by establishing an in vitro 3D culture system for tonsil organoids, which was successful in the presence of feeder cells, along with IL-4 and IL-7 cytokines. To confirm that our 3D tonsil organoids can respond to real antigens, we used CMV peptides and peptides of spike proteins from Covid-19 as real antigens, and co-cultured with tonsil organoids, which indeed can generate memory cells and plasmablasts. In the end, we also compared 3D to 2D cultures. Although the total numbers of all B cell subsets were much less in 3D culture than that in 2D culture, still, it indicates that this in-vitro culture system has its limitation, while being usable to produce the similar results as 2D did. Therefore, this 3D culture system can be used as a platform to investigate NFATc1/ΔS+ or NFATc1/ΔBC+ TFH and TFR cells in the dynamic of human GC responses.
Die Technik der strukturierten Beleuchtungsmikroskopie (structured illumination microscopy, SIM) ist eine etablierte ultrastrukturelle Aufnahmemethode, die der hochauflösenden Visualisierung intrazellulärer Strukturen dient. In der Ophthalmologie findet diese Art der Bildgebung bisher wenig Anwendung.
SIM ermöglicht die histologische Darstellung retinaler Strukturen, wie der Zellen des humanen retinalen Pigmentepithels (RPE). In den Zellen des RPE reichern sich Granula an, die für die Autofluoreszenz-Bildgebung von Bedeutung sind. Anhand der Morphologie und autofluoreszierenden Merkmale lassen sich grundsätzlich drei Granulatypen im RPE unterscheiden: Melanosomen (M), Melanolipofuszin (ML)- und Lipofuszin (L)-Granula. Die Anwendung der SIM ermöglicht die präzise Darstellung und Differenzierung dieser autofluoreszierenden Strukturen, sowie die Bestimmung ihrer Anzahl und Lokalisation.
Ziel der Arbeit ist die Darstellung der im humanen RPE lokalisierten Granula mithilfe der SIM. Anhand der unterschiedlichen Autofluoreszenz (AF) der Granula können diese innerhalb des RPE-Zellkörpers klassifiziert, sowie deren Anzahl und Dichte analysiert werden. Diese Analyse wird in Altersgruppen und Retinalokalisationen differenziert. Zudem sind direkte Vergleiche zwischen der Histologie (SIM, ex vivo) und klinischen Aufnahmen (Fundusautofluoreszenz, in vivo) kaum existent. Durch Ermittlung der Gesamt-AF pro Zelle in Korrelation zu der intrazellulären Granuladichte und -verteilung soll eine neue Interpretationsebene ermöglicht werden.
Diese Arbeit soll helfen anhand der gewonnenen Daten die Stoffwechselmechanismen der Retina und deren Einfluss auf die Fundusautofluoreszenz (FAF) besser verstehen zu können. Sie soll insbesondere dazu beitragen bestehende und neue klinische FAF-Bildgebungsverfahren zu validieren, die Diagnostik pathologischer Prozesse der Retina zu optimieren und sowohl eine möglichst frühe Erkennung als auch präzise Prognostik zu ermöglichen.
Zudem sollen die Daten eine belastbare Basis darstellen, um die mit einem hohen Zeitaufwand verbundene manuelle Zellanalyse einer geschulten künstlichen Intelligenz zu überlassen. Damit könnte der Analyseprozess von Gewebeproben immens beschleunigt werden und in seiner Effizienz maximiert werden.
Diese Arbeit beschäftigt sich mit der Synthese und Charakterisierung von mehrfach perfluoralkylierten Verbindungen.
Das erste Kapitel behandelt die Kupfer-vermittelte mehrfache Pentafluorethylierung von aromatischen Systemen, sowie die Untersuchung deren Folgechemie. Dabei wurden iodierte Aryl-Derivate mit dem Kupferorganyl CuC2F5 umgesetzt. Mit dieser Methode konnten symmetrisch und unsymmetrisch pentafluorethylierte Anilin- und Phenol-Derivate dargestellt werden. Die Anilin-Derivate wurden zu den entsprechenden Diazoniumsalzen umgesetzt, welche zunächst vollständig charakterisiert und anschließend deren Folgechemie untersucht wurde.
Das zweite Kapitel behandelt die Darstellung von Bis(trifluormethyl)sulfon durch elektrochemische Fluorierung mittels des Simons-Prozesses. Dabei wurde der Umsatz der Reaktion durch Verwendung der teilfluorierten Verbindung (Trifluormethyl)methylsulfon als Startmaterial optimiert.
This work presents excited state investigations on several systems with respect to experimental
spectroscopic work. The majority of projects covers the temporal evolution of
excitations in thin films of organic semiconductor materials. In the first chapters, thinfilm
and interface systems are build from diindeno[1,2,3-cd:1’,2’,3’-lm]perylene (DIP)
and N,N’-bis-(2-ethylhexyl)-dicyanoperylene-3,4:9,10-bis(dicarboximide) (PDIR-CN2)
layers, in the third chapter bulk systems consist of 4,4’,4”-tris[(3-methylphenyl)phenylamino]
triphenylamine (m-MTDATA), 4,7-diphenyl-1,10-phenanthroline (BPhen) and
tris-(2,4,6-trimethyl-3-(pyridin-3-yl)phenyl)borane (3TPYMB). These were investigated
by aggregate-based calculations. Careful selection of methods and incorporation
of geometrical relaxation and environmental effects allows for a precise energetical assignment
of excitations. The biggest issue was a proper description of charge-transfer
excitations, which was resolved by the application of ionization potential tuning on
aggregates. Subsequent characterization of excitations and their interplay condenses
the picture. Therefore, we could assign important features of the experimental spectroscopic
data and explain differences between systems.
The last chapter in this work covers the analysis of single molecule spectroscopy on
methylbismut. This poses different challenges for computations, such as multi-reference
character of low-lying excitations and an intrinsic need for a relativistic description.
We resolved this by combining complete active space self-consistent field based methods
with scalarrelativistic density-functional theory. Thus we were able to confidently
assign the spectroscopic features and explain underlying processes.
Emotional shifts are often a fundamental part of the narrative experience and engrained into the schematic structures of stories. Recent theoretical work suggests that these shifts are key for narrative influence and are interconnected with transportation, a known mechanism of narrative effects. Empirical research examining this proposition is still scarce, inconclusive, and lacking measures that assess the experience of emotional shifts throughout a narrative to explain effects. This thesis aims to contribute to this research lacuna and investigates the link between emotional shifts, transportation, and story-consistent outcomes using different methods to measure emotional shifts in the moment they occur (Manuscript #1 and #2), and using various narrative stimuli (audiovisual, written, auditive).
Manuscript #1 uses real-time-response (RTR) measurement to examine the relationship of valence shifts experienced during film viewing with transportation and post-exposure self-reported emotional flow. Manuscript #2 reports a pilot study and two experiments in which a self-probed emotional retrospection task is used to measure the number and intensity of emotional shifts during reading. I investigate the effect of reviews on transportation, the link between transportation and emotional shifts, and their respective associations with story-consistent attitudes, social sharing intentions, and donation behavior. In Manuscript #3, narrative structures are manipulated. Two experiments examine the effects of audio stories with shifting (positive-negative-positive) vs. positive-only emotional trajectories on the experience of happiness- and sadness-shifts, transportation, and post-exposure emotional flow.
Transportation was positively linked to valence shifts (M#1), and the number and intensity of emotional shifts (M#2), and emotional flow (M#1, M#3). In M#3, transportation was predicted by shifts in happiness, but not sadness. Emotional flow was linked to shifts in happiness, sadness, and RTR valence (M#1, M#3). Emotional shifts and transportation were associated with social sharing intentions, but only transportation was linked to some story-consistent attitudes (affective attitudes in particular).
CRISPR-Cas systems are highly diverse and canonically function as prokaryotic adaptive immune systems. The canonical resistance mechanism relies on spacers that are complementary to the invaders' nucleic acids. By accidental incorporation or other mechanisms, prokaryotes can also acquire self-targeting spacers that are complementary to their own genome. As self-targeting commonly leads to lethal autoimmunity, the existence of self-targeting spacers poses a paradox. In Chapter 1, we provide an overview of the prevalence of self-targeting spacers, summarize how they can be incorporated, and which means can be employed by the host to evade lethal self-targeting. In addition, we outline alternative functions of CRISPR-Cas systems that are associated with self-targeting spacers. Whether CRISPR-Cas systems can efficiently target their own genome depends heavily on the presence of protospacer adjacent motifs (PAMs) next to the target region. In Chapter 2, we developed a method to determine PAM requirements. Thereby, we specifically focused on type I systems that engage multi-protein complexes, which are challenging to assess. Using the cell-free transcription-translation (TXTL) system, we developed an enrichment-based binding assay and validated its reliability by examining the well-known PAM requirements of the E. coli type I-E system. In Chapter 3, we applied the TXTL-based PAM assay to assess 16 additional CRISPR-Cas systems. These 16 systems included three CRISPR-Cas associated transposons (CASTs). CASTs are recently discovered transposons that employ CRISPR-Cas systems in a non-canonical function for the directed integration of the transposon. To further characterize CASTs in TXTL outside their PAM requirements, we reconstituted the transposition of CASTs in TXTL. In Chapter 4, we turned to non-canonical self-targeting CRISPR-Cas systems, which were already discussed in Chapter 1. While investigating how the plant pathogen Xanthomonas albilineans survives self-targeting by its two endogenous CRISPR-Cas systems, we identified multiple putative anti-CRISPR proteins (Acrs) in the genome of X. albilineans. Two of the Acrs, named AcrIC11 and AcrIF12Xal, inhibited degradation by their respective CRISPR-Cas systems but still retained Cascade-binding ability, and appear responsible for the lack of autoimmunity in X. albilineans. In summary, we developed new technologies that eased the investigation of non-canonical multi-component systems and, if applied to additional systems, might reveal unique properties that could be implemented in new CRISPR-Cas based tools.
Altersbedingte Makuladegeneration (AMD) ist weltweit die häufigste Ursache von irreversibler Erblindung des alternden Menschen. Mit der anti-VEGF-Behandlung steht für die deutlich seltenere feuchte AMD eine zugelassene Therapie bereit, die deutlich häufigere trockene AMD entzieht sich aktuell jedoch jeglicher Therapie. Ein zentraler Pathomechanismus der AMD ist der progrediente Untergang des retinalen Pigmentepithels (RPE). Die Rarifizierung und letztendlich Atrophie des RPEs führt zum Untergang der funktionellen Einheit aus RPE, Photorezeptoren und Bruch’scher Membran und somit zum irreversiblen Funktionsverlust. Ein möglicher therapeutischer Ansatz, der progredienten Atrophie des RPEs entgegenzuwirken, ist, das prinzipiell post- mitotischen RPE zur Proliferation anzuregen.
Grundlage unserer in vitro Untersuchungen ist das ARPE-19 Zellmodell. Um die Proliferation anzuregen wurden die RPE-Zellen mit E2F2, einem Zellzyklus- regulierendem Transkriptionsfaktor, transfiziert.
Zunächst wurde ein nicht-proliferatives RPE-Zellmodell mit spontanem Wachstumsarrest etabliert. Innerhalb von zwei Wochen konnte die Ausbildung von Zonulae occludentes als Zeichen der Integrität des adhärenten Zellmonolayers beobachtet werden. Die chemische Transfektion von E2F2 unter einem CMV-Promoter führte zur Überexpression von E2F2-Protein. Der proliferationssteigernde Effekt von E2F2 konnte durch die Proliferationsmarker Cyclin D1 sowie Ki67, dem Anstieg der BrdU-Aufnahme und der nach Transfektion mit E2F2 zunehmenden Gesamtzellzahl nachgewiesen werden.
Der Zellzahlerhöhung standen jedoch potentiell qualitative und funktionelle Einbußen entgegen. So zeigten sich nach Behandlung mit E2F2 die Zellviabilität reduziert und die Apoptoserate sowie die Permeabilität des Epithels erhöht. Diese Einschränkungen waren jedoch nur passager bis 7 Tage nach Transfektion sichtbar und reversibel. Unsere Ergebnisse weisen darauf hin, dass diese Defizite nicht durch E2F2 selbst, sondern durch das Transfektionsreagenz PEI bedingt waren. Weitere funktionelle Defizite könnten durch epithelial-mesenchymale Transition (EMT) verursacht werden. Hier zeigte sich durch E2F2 keine De-Differenzierung im Sinne einer typischen EMT-Marker- Expression.
Die vorliegende Arbeit zeigt in einem in vitro Zellmodell die Grundlagen eines vielversprechenden Ansatzes zur Therapie der trockenen AMD: Durch Überexpression eines den Zellzyklus regulierenden Gens (hier E2F2) wurde die RPE-Regeneration angeregt. Analog zur schon zugelassenen Gentherapie des RPEs bei RPE65-assoziierten Netzhautdystrophien durch den Transfer von funktionstüchtigem RPE65-Gen mittels Adeno-assoziierten Viren könnte mittels E2F2, übertragen mit einem lentiviralen Verktor, eine Stimulation des RPEs zur Proliferation möglich sein. Entscheidend ist der möglichst gute Struktur- und Funktionserhalt des Photorezeptor-Bruch-Membran-RPE Komplexes. Eine Therapie sollte daher in frühen Krankheitsstadien erfolgen, um die Progression zu fortgeschrittenen Erkrankungsstadien mit irreversiblem Funktionsverlust zu verzögern oder zu verhindern.
Diese Arbeit untersuchte die Wirkung von bakteriellen Substanzen auf essenzielle
thrombozytäre Funktionen. Bei den Substanzen handelte es sich um Toxine von
Bakterien, die mutmaßlich zur Pathogenese der Parodontitis beitragen.
Während LTX von Bakterium A. actinomycetemcomitans und C14 von F. nucleatum
zur Hemmung der Aggregation und zur Stimulation inhibitorischer Systeme beiträgt,
induziert LPS von P. gingivalis eine leichte Aktivierung der Thrombozyten,
gekennzeichnet durch eine gering verstärkte P-Selektin-Expression.