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Insights into the function of DNA repair factors MRN and ATM
Get PDFDNA double strand breaks (DSB) are a particularly deleterious threat to genomic integrity throughout all domains of life. DSBs can cause chromosomal aberrations, tumorigenesis and cell death if left unre-paired and are caused by either endogenous or exogenous sources. Cells rely on efficient detection, repair and response upon occurrence of DSBs. In eukaryotes, DSBs are mostly repaired by either end joining pathways or homologous recombination (HR). HR, in contrast to the end joining pathways, en-ables error-free DSB repair in presence of a template sister chromatid. The Mre11-Rad50-Nbs1 (MRN) complex recognizes and tethers DNA ends, even if they are obstructed by proteins to initiate HR. In order to respond to DSBs, the MRN complex recruits and activates the signaling kinase Ataxia-telangiectasia mutated (ATM), that belongs to the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family. Activated ATM in turn initiates the cellular DNA damage response (DDR). Mre11 and Rad50 are highly conserved and form a topology-specific, ATP-dependent nuclease complex that pro-cesses DNA ends but leaves genomic DNA intact. The eukaryote specific Nbs1 subunit finetunes MRN’s endonuclease activity by providing interaction with proteins (e.g. CtIP). Apart from its nucleo-lytic activity, MRN has a scaffolding function that promotes DNA end tethering, repair foci formation and possibly signal amplification.
Although the complex has been studied for more than two decades, a model that integrates both MRN’s enzymatic and scaffolding functions has not yet been established. In the first part of the thesis, such a model was elaborated by combining both structural and biochemical data from this and previ-ous studies. A cryo-electron microscopy (cryo-EM) structure of the Chaetomium thermophilum (Ct)MRN catalytic head domain in its ATPγS-bound state not only clarifies its atomic architecture but also reveals how a core part of Nbs1 stabilizes and possibly locks the Mre11 dimer. In this structure significant parts of the Rad50 coiled-coils were resolved in a rod configuration, stabilized by several interaction points. A previously uncharacterized C-terminal Mre11 domain, denoted bridge could fur-ther stabilize the rod configuration. The rod configuration and the bridge domain restrict access to the Rad50 DNA binding site. Biochemical analysis revealed the Rad50 DNA binding site is extremely specific for DNA ends. However, an additional, eukaryote-specific DNA binding site at the C-terminus of Mre11 enables binding to internal DNA. The Rad50 coiled-coil domains are linked at the apex via a zinc hook dimerization motif to form a large proteinaceous ring/rod. Cryo-EM data and crystal structures ex-plained how two MRN complexes can tether DNA ends via dimerization of these apical domains. In vivo assays indicate that mutation of the apex tethering element negatively impacts DSB repair.
Mutations in DDR pathways allow cancer cells to cope with increased replication and genotoxic stress. For this reason, proteins involved in DDR were described to be promising targets in cancer therapy. Due to its central role in DSB induced DDR, ATM is an auspicious target for drug development. Howev-er, lack of ATM high-resolution structures, as well as atomic details of small molecule inhibitor binding modalities hampered the application of structure-based drug design. In the second part of the thesis, the binding modalities of two ATP-competitive ATM-inhibitors were described. This project was a col-laborative work with Merck KGaA, that provided a novel inhibitor (M4076) with improved pharmacoki-netics. Comparison of the inhibitor-bound kinase active sites with the likewise resolved ATPγS-bound active site explains the high affinities that were determined in biochemical assays. Superposition and sequence alignment of the ATM kinase active site with other PIKK active sites enables to rationalize the molecular reasons for selectivity. In biochemical assays, IC50 values of the inhibitors for ATM, PIKKs and CHK2 showed high selectivity towards ATM. The binding of the inhibitors stabilized the N-terminal solenoid domain of ATM, this enabled the generation of a high-resolution structure of the entire ATM protein. The quality of the map allowed the identification of two zinc binding sites that possibly stabi-lize loops and the generation of a near-complete ATM structure. Taken together, the structural data provides the framework for structure-based ATM inhibitor design and allows mapping of cancer muta-tion as well as functionally important protein interaction sites
Langzeitergebnisse der Phakoemulsifikation mit Aspiration bei Pferden mit Uveitis-bedingter Katarakt
Get PDFDas Prisenrecht und seine Umsetzung in der Seekriegsführung Frankreichs, Englands und der Generalstaaten der Niederlande in den Kriegen 1672–1713
Get PDFIn dieser Dissertation werden die komplexen Dynamiken des Prisenrechts und seiner Anwendung durch die Seemächte Frankreich, England und die Niederlande zwischen 1672 und 1713 aufgezeigt. Geprägt von drei bedeutenden Konflikten – dem Holländischen Krieg, dem Neunjährigen Krieg und dem Spanischen Erbfolgekrieg –, illustriert die Arbeit die Verflechtung von Seekriegsführung und maritimer Rechtspraxis.
Eine detaillierte quantitative und qualitative Analyse von 361 thematisch relevanten Dokumenten enthüllt grundlegende externe und interne Auswirkungen auf die Praxis des Prisenrechts, insbesondere des Konterbanderechts. Dieses Rechtsinstitut spielte eine prägende Rolle auch über die folgenden 150 Jahre, bevor darüber in der Pariser Seerechtsdeklaration von 1856 eine Harmonisierung erzielt wurde. Die Untersuchung beleuchtet zudem das Spannungsfeld zwischen dem jeweiligen nationalen Streben nach Rechtssicherheit in der Anwendung des Prisenrechts und dem vorrangigen Interesse privater Kaperer an der Gewinnerzielung.
Dieses Buch richtet sich an Historiker, Juristen und alle, die ein umfassendes Verständnis für die komplizierten Aspekte der maritimen Handelskriegsführung und deren rechtlichen Rahmen suchen.
Bernd Lehmann promovierte nach seinen beruflichen Tätigkeiten in der deutschen Marine einschließlich eines Studiums an der United States Naval Postgraduate School Monterey/CA mit dem M.Sc. in ‘Operations Research‘ sowie bei der Studieneinrichtung der NATO in Den Haag/NL mit der vorliegenden Dissertation im Fach Frühneuzeitliche Geschichte an der Ludwig-Maximilians-Universität München. Sein Forschungsschwerpunkt liegt auf der Einbeziehung des Prisenrechts in der nationalen Sicherheits- und Verteidigungspolitik der Staaten seit dem Vertrag von Utrecht 1713 bis in die heutige Zeit
Modulation of human dendritic cell phenotype and function in response to yellow fever vaccination and SARS-CoV-2 infection
Get PDFAcute viral infections caused by RNA viruses such as flaviviruses (Yellow Fever, Dengue, West Nile) or SARS coronaviruses (SARS-CoV, SARS-CoV-2) represent a major global health threat. The recent worldwide pandemic of SARS-CoV-2 has shown the importance of researching viral infections to identify immune defense mechanisms against these pathogens and understand immune-mediated pathology.
In this study, live-attenuated yellow fever 17D (YF17D) vaccination was used as a model of an acute self-limited RNA virus infection in humans, and the role of dendritic cells (DCs) and monocyte subsets in the innate immune response was elucidated using multi-parametric flow cytometry, RNA sequencing, and in vitro experiments. Blood was sampled from vaccinees at time points before (day 0) and after (days 3, 7, 14, 28) YF17D vaccination to analyze the kinetics of the innate immune response.
After vaccination with YF17D, DCs and monocytes in the peripheral blood showed concerted activation indicated by the upregulation of CD86 and PD-L1 on the cell surface on day 7 in all DC and monocyte populations (cDC1, cDC2, DC3, tDC, pDC, and classical, intermediate, and non-classical monocytes). At the same time, a robust interferon-induced response was detected in these cells marked by upregulation of Siglec 1 mRNA and surface expression in most cell types - except tDCs and pDCs - and increased expression of a multitude of interferon-induced genes (ISG). A common set of ISGs, consisting of OAS1, OASL, OAS3, RSAD2, IFIT3, IFIT1, and EIF2AK2, was concertedly upregulated in all antigen-presenting subsets analyzed, with peak expression on day 7 after vaccination. Besides this common gene signature induced by vaccination, cell-type-specific effects were also seen, indicating that each DC subset plays a unique role in the innate immune response to YF17D vaccination. Interestingly, DC3, a population with high similarities to both classical monocytes and cDC2, and marked by a CD1c+ CD5- CD14+/- phenotype, showed higher similarity to cDC2 in their transcriptomic response to YF17D vaccination than to classical monocytes, with common gene sets of ISGs and genes relevant for MHC I presentation significantly upregulated on day 7 after vaccination in both cell populations.
In vitro experiments using a reporter YF17D virus and flow cytometric detection of YF17D-RNA showed that all DCs and monocytes from the peripheral blood can be infected by YF17D. Infected cells exhibited upregulation of activation markers and secretion of cytokines and chemokines such as type I IFN and CXCL10. Since the viral infection rate was not very high, this direct infection of antigen-presenting cells (APCs) could be used as a mechanism to induce cell activation and allow for antigen presentation. After blocking the IFN-α/β receptor in vitro, high-er YF17D infection rates were found in DCs and monocytes, indicating that the type I interfer-on response is essential in controlling viral replication of YF17D in APCs. Therefore, the strong ISG response found in the transcriptome of peripheral DC and monocyte subsets indicates the induction of an antiviral state in the peripheral blood and suggests highly efficient viral control after vaccination.
This posed the question, how viral antigens can be delivered to and efficiently presented by DCs if infection of DCs by YF17D is highly restricted by type I IFNs. It was therefore investigated whether the infection of DCs is more efficient when they are in contact with other infected cells. Indeed, a higher infection rate of DCs and monocytes was achieved after coculture with YF17D-infected cell lines, and this effect depended on cell-to-cell contact. Thus, contact of DCs with YF17D-susceptible cells at the injection site could promote infection and activation of DCs, leading to the presentation of viral antigens to T cells in the draining lymph node.
To put the efficient innate immune response to YF17D vaccination into context with another acute RNA virus infection, the innate immune responses to YF17D and SARS-CoV-2 infection were compared. The SARS-CoV-2 viral infection leads to the disease called COVID-19 with different degrees of disease severities and, in some patients, even death. In the early phase after infection, a slow and inefficient control of the virus by the innate immune system may lead to a delayed response which is characterized by hyperinflammation and causes severe immune pathology systemically and in the lung.
Compared to the YF17D vaccination, patients with more severe COVID-19 showed low expression of costimulatory molecule CD86 in the cDC2, DC3, and monocytes in the peripheral blood. In contrast, non-hospitalized patients with a mild COVID-19 disease progression showed an upregulation of CD86 similar to what was observed in YF17D vaccinees. The downregulation of CD86 in severe COVID-19 was accompanied by upregulation of PD-L1, which is known to interact with PD-1 on T cells, thereby regulating their inhibiting activation. This altered phenotype of peripheral APCs coincided with a reduced capability of DC3 and monocytes isolated from the blood of COVID-19 patients to stimulate autologous T cell activa-tion and proliferation in vitro, thereby revealing functional impairment of circulating DCs and monocytes in this disease. An increase of Ki67+ cells in both YF17D vaccinees and COVID-19 patients, together with temporary reductions in cDC1 and cDC2 frequencies in the peripheral blood, indicated an increased turnover of the blood DC compartment in acute viral infection. While in YF17D vaccinees only a temporary relative reduction of cDC1 and cDC2 was ob-served, absolute DC numbers and also pDCs were reduced in the peripheral blood of COVID-19 patients. The depletion was transient in patients with a mild disease progression and long-lasting in patients with severe COVID-19.
Additionally, a cell population lacking markers of lymphocytes, granulocytes, DCs, and mono-cytes but expressing HLA-DR and Ki67 was also found to be increased in the peripheral blood of COVID-19 patients, but not YF17D vaccinees, as a sign of dysregulated myelopoiesis. Fur-thermore, in COVID-19 patients – but not in YF17D vaccinees – a subset of CD14+ DC3 expanded within the DC3 population and correlated with inflammatory markers and the accumulation of activated Tfh and B cells. Therefore, the innate immune response in COVID-19 patients seems to be a critical factor influencing inflammatory and adaptive immune responses. Dysregulation of innate immune cells as seen by the altered phenotype, impaired function and long-lasting reduction found in DCs and monocytes could lead to an increased susceptibility to sec-ondary infections as a consequence of severe COVID-19. While YF17D vaccination induces a transient coordinated response of blood APC subsets with a peak on day 7 after vaccination, the responses in COVID-19 are long-lasting and show unusual phenotypes of monocytes and DCs accompanied by functional impairment
Impact of human alpha-synuclein overexpression on the nigrostriatal dopaminergic neurotransmission
Get PDFAlpha-synuclein is a small 140 amino acid presynaptic protein associated with everal neurodegenerative disorders such as Parkinson’s disease (PD). In idiopathic PD, abnormally misfolded wild-type proteins aggregate in the cytosol of certain neurons, forming what is the main component of Lewy bodies (LBs) - a major PD hallmark. Besides its role in regulating synaptic vesicle functions, alpha-synuclein appears to modulate dopamine (DA), a neurotransmitter particularly important in PD symptoms manifestation. DA can be found sparsely distributed in the brain and is predominantly expressed in midbrain neurons. From the midbrain, two main dopaminergic (DAergic) pathways densely innervate the dorsal portion of the striatum. Degeneration of such DAergic terminals is known to dysregulate DA homeostasis in the striatum. Despite several decades of PD research, the physiological influence of alpha-synuclein on DAergic neurotransmission in the two main areas of the dorsal striatum is still poorly understood. These areas are the dorsomedial striatum (DMS), mainly receiving innervation from the ventral tegmental area, and the dorsolateral striatum (DLS), which receives input from the substantia nigra pars compacta and is more susceptible to neurodegeneration. To clarify how alpha-synuclein may interfere with DAergic neurotransmission in either area, fast-scan cyclic voltammetry experiments were carried out in the DMS and DLS of transgenic mice overexpressing human alpha-synuclein (Tg) at twelve, six and three months of age. Additionally, pharmacological assays, behavioural tasks, and ex vivo immunofluorescence staining were performed to support the electrophysiological ex vivo results.
In this thesis, it is hypothesized that due to its intrinsic characteristics, alpha-synuclein preferentially interferes with DA neurotransmission dynamics in the dorsolateral region of the striatum. Data analysis confirmed that overexpression of human alpha-synuclein differentially interfered with normal DA release in the DLS in an age‐dependent manner. At older ages (twelve-month-old Tg mice), decreased evoked DA release and slower DA uptake kinetics were observed. In addition, alterations in dopamine transporter (DAT) distribution, which appeared as increasing amounts of DAT-positive clumps, were found only in the DLS. Moreover, at pre-symptomatic stages (six-month-old) DA neurotransmission appeared to be stabilised before DA disruption. Surprisingly, at younger ages (three-month-old) increased electrically evoked DA release was also recorded in the DLS. Such changes were in line with the motor learning enhancement observed in their behavioural phenotype. In addition, DA uptake appeared to be impaired as evidenced by reduced extracellular DA withdrawal. Further pharmacological experiments demonstrated that such alterations were mediated by DAT.
In summary, the present findings indicate that abnormal DAergic neurotransmission and function of DLS can be identified before the onset of structural pathologies in a model of transgenic expression of human alpha-synuclein. Depending on the progression of the pathology, human alpha-synuclein has different impacts on neurotransmission, initially enhancing it but impairing it at later stages. It is here proposed that assessment of DLS function by non-invasive brain imaging and neuropsychological techniques might be relevant in early PD diagnosis and help design appropriate therapeutic intervention
DNA nanostructures in advanced plasmonic applications
Get PDFChiral structures can be found anywhere from particle physics over electromagnetism to chemistry and biology. Although circular dichroism (CD) has long been used to trace changes of protein configurations or chiral molecular states, chiral plasmonic nanostructures have only shown their potential in recent years. The ability to design nanostructures with tailored geometries and specific functions on the nanoscale is one of the possibilities offered by DNA nanotechnology.
In this thesis, we use DNA origami to assemble model meta-molecules from multiple plasmonic particles, accurately positioned in space. This approach allows us to build up varying molecular geometries piece by piece and study their impact on their surrounding optical near fields, confirming numerical simulations elucidating the intricate chiral optical fields in complex architectures. With this, we studied the emergence of CD signatures by step-wise constructing of a gold nanohelix, composed of single spherical nanoparticles. In this, we were able to show the effects of varying geometries by implementing sign flipping signals through addition or removal of single particles.
We furthermore studied the effects of a spherical transmitter particle situated within a pair of nanorods in a chiral geometry. The transmitter particle not only enhances the CD response and causes a redshift of the plasmonic resonance frequency of the nanorods, it also triggers the emergence of an additional chiral signal at the resonance frequency of the nanosphere.
In conclusion, we present an approach of utilizing specific plasmonic heating effects to construct a novel type of DNA nanowalker: a nanowheel. Unlike previous DNA walkers, which are based on the process of strand displacement, this nanowheel is fueled by light and could therefore potentially reach significantly higher speeds than any previous attempts
