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Analysis of eukaryotic translation by integrating cryo-EM and ribosome profiling
Get PDFTranslational control plays a critical role in maintaining proteome homeostasis, and in influencing cellular differentiation, proliferation, growth and developmental pathways. Protein synthesis is closely linked to cellular metabolism and any aberrations in its regulation lead to diseased states. In this thesis presented here, translational regulation has been investigated in three different biological contexts by integrating two different techniques namely, cryo-electron microscopy and ribosomal profiling.
Translational regulation has been investigated in maturing dendritic cells using ribosome profiling and RNAseq respectively. Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In the immature state (immature DC), they have the ability to monitor the environment and upon encountering antigens they mature to launch immune responses. Here, a defined cytokine mixture combined with TLR agonist (R848) has been used for in vitro DC maturation. Upon induction of maturation, pathways such as the ‘TNF signaling pathway’, the ‘cytokine-cytokine receptor interaction’ and the ‘IL-17 signaling pathway’ were up regulated both at the level of transcriptome and translatome respectively. Transcripts encoding for proteins involved in oxidative phosphorylation pathway were strongly repressed at the later stages of DC maturation (24 h). As observed in previous studies transcripts encoding for ribosomal proteins, antigen processing and presentation were also translationally up-regulated at 4 h while being translationally repressed at the 24 h time point. Transcripts of the glycolytic pathway are also translationally repressed at the 24 h time-point. Further, during the course of DC maturation, globally there was increased ribosome occupancy in the 5’ UTR. During the later stages of DC maturation, down regulation of ABCE1 led to accumulation of post-termination ribosomes in the 3’ UTR. Moreover, ribosome occupancy in the 3’ UTR showed strong correlation to its GC content.
Ski proteins function as accessory factors and are essential for exosome function, which mediates the 3’ to 5’ mRNA decay pathway. Non-stop transcripts are primarily decayed via the 3’ to 5’ pathway. It has been shown here that the Ski complex, interacts with the ribosome independent of Ski7. Ribosomal profiling of 80S-Ski-complexes revealed a fraction of longer footprints, and contained more poly-A containing footprints. Further, RNAseq analysis of the purified 80S-Ski-complexes revealed strong asymmetric distribution of reads, where more reads mapped towards the 5’ end of the transcripts. Also, transcripts with shorter half-life (< 5 min) and with more non-optimal codon content showed enrichment for Ski-80S footprints. This hinted at the possibility that Ski complex might interact with ribosomes for turnover of canonical transcripts via the 3’-5’ decay pathway.
The endoplasmic reticulum (ER) is responsible for properly modifying and folding most of the secretory and membrane proteins. Its functioning capacity is challenged during stressful circumstances such as in hypoxia, calcium imbalance and viral infection. Unfolded protein response (UPR) is the cellular mechanism that is activated to alleviate the ER stress. UPR acts via three main pathways in mammals, and of this IRE1α-XBP1u branch is the most evolutionarily conserved. XBP1u contains a C-terminal ribosomal pausing site and plays a critical role in mediating UPR. Using cryo-EM, XBP1u has been visualized in the ribosomal exit tunnel. Structural characterization revealed that XBP1u forms a turn in the vicinity of the peptidyl transferase center and causes a subtle distortion of the base C4398 to inhibit ribosomal activity. This explains the temporary nature of the ribosomal arrest mediated by XBP1u. During ribosomal pausing, HR2 of XBP1u is being recognized by SRP, but it fails to successfully engage with the Sec61 translocon. XBP1u has evolved with an intermediate ribosomal pausing strength, but this allows it to be efficiently targeted by SRP onto the Sec61 translocon, albeit without gating it
Precision spectroscopy of the 2S-6P transition in atomic deuterium
Get PDFDie Quantenelektrodynamik (QED) bildet die Grundlage aller anderen Quantenfeldtheorien, auf denen das Standardmodell der Teilchenphysik aufgebaut ist. Derzeit ist klar, dass unser fundamentales Naturverständnis unvollständig ist, sodass erwartet wird, dass das Standardmodell um neue Teilchen oder Wechselwirkungen verändert oder erweitert werden muss. Eine Möglichkeit, diese Grenzen der Grundlagenphysik zu erforschen, ist die Durchführung von Präzisionsmessungen. Diese Arbeit untersucht die Präzisionslaserspektroskopie von Deuterium, wo die Übergangsenergien zwischen verschiedenen Energiezuständen des an den Kern gebundenen Elektrons mit Techniken wie ultrastabilen Lasern und dem Frequenzkamm genau gemessen werden können. Aufgrund der Einfachheit der wasserstoffähnlichen Atome können ihre Energieniveaus anhand der QED-Theorie für gebundene Zustände genau berechnet werden, und mit dem Experiment mit der relativen Genauigkeit in der Größenordnung von verglichen werden. Ein solcher Vergleich zwischen Theorie und Experiment ist mit der Bestimmung von Naturkonstanten verbunden, die als Parameter in die Theorie eingehen. Erst wenn mehr unabhängige Messungen als Parameter vorliegen, kann die Theorie überprüft werden.
Der Vergleich zwischen Theorie und Laser-Spektroskopie im Deuterium betrifft die Ryd-berg-Konstante und den Deuteronen-Ladungsradius . Dies erfordert mindestens zwei Messungen der verschiedenen Übergangsfrequenzen, um diese Konstanten zu bestimmen, und mehr Messungen, um die Theorie zu testen. Im Gegensatz zum Wasserstoff gibt es bei Deuterium nur wenige ausreichend genaue Messungen der Übergänge. In dieser Arbeit wird die erste Untersuchung des 2S-6P-Übergangs in Deuterium vorgestellt, die mit der bestehenden Frequenzmessung des 1S-2S-Übergangs kombiniert werden kann, um und zu erhalten. Zusammen mit der Messung des 2S-2P-Übergangs von myonischem Deuterium stellt diese Bestimmung einen Theorietest dar. Ein solcher Vergleich ist wichtig, um die anhaltende Diskrepanz zwischen dem Ergebnis aus myonischem Deuterium und dem Durchschnitt früherer Daten aus elektronischem Deuterium, sowie die Spannungen zwischen den jüngsten Ergebnissen aus der Wasserstoffspektroskopie, zu beleuchten.
Im Gegensatz zu Wasserstoff wird die Präzisionsspektroskopie des 2S-6P-Übergangs in Deuterium durch die gleichzeitige Anregung unaufgelöster Hyperfeinstruktur-Komponenten erschwert, was zur unaufgelösten Quanteninterferenz führen kann. Diese Arbeit untersucht die möglichen systematischen Effekte, die mit dieser Komplikation verbunden sind. Zusammen mit analytischen störungstheoretischen Modellen werden Supercomputersimulationen durchgeführt, um diese Effekte zu analysieren. Es wird gezeigt, dass die Quanteninterferenz für alle 2S-P-Übergänge in Deuterium stark unterdrückt wird, wodurch Präzisionsmessungen dieser Übergänge möglich werden. Darüber hinaus wird ein weiterer Effekt in Deuterium im Vergleich zu Wasserstoff untersucht, der sich aus der Lichtkraft ergibt, die auf die Atome in der stehenden Welle des Spektroskopielichts wirkt. Trotz zusätzlicher Zustandsvielfalt durch die gleichzeitige Anregung unaufgelöster Hyperfeinkomponenten wird gezeigt, dass diese sogenannte ``Lichtkraftverschiebung'' mit dem gut verstandenen Effekt im Wasserstoff vergleichbar ist.
Die größte Herausforderung bei der Messung des 2S-6P-Ein-Photonen-Übergangs in Deuterium ist die Doppler-Verschiebung erster Ordnung. Ein großer Teil dieser Arbeit befasst sich daher mit dem verbesserten aktiven faserbasierten Retroreflektor (AFR), der eine Technik zur Unterdrückung dieser Verschiebung darstellt. Der zentrale Teil des AFR ist der Faserkollimator, der für die Erzeugung hochwertiger gegenläufiger Laserstrahlen erforderlich ist. Die Entwicklung und Charakterisierung eines solchen Kollimators für die nahe ultraviolette Wellenlänge des 2S-6P-Übergangs ist eine der wichtigsten Errungenschaften des verbesserten AFR. Die Ergebnisse dieser Arbeit können für andere Anwendungen von Interesse sein, bei denen eine hohe Strahlqualität oder wellenfront-zurückverfolgende Strahlen wichtig sind. Darüber hinaus werden die Einschränkungen der AFR untersucht, die sich aus polarisationserhaltenden Singlemode-Fasern ergeben. Neben anderen Verbesserungen wurde eine Polarisationsüberwachung der Spektroskopielaserstrahlen implementiert. Es werden verschiedene Charakterisierungsmessungen vorgestellt, um die Leistungsfähigkeit des verbesserten AFR zu demonstrieren.
Schließlich wird in dieser Arbeit eine vorläufige Messung des 2S-6P-Übergangs in Deuterium vorgestellt. Für diese Messung wurde ein neuer Kryostat in die Apparatur eingebaut, der die Stabilität des Spektroskopiesignals durch reduzierte Temperaturschwankungen verbessert. Die Erzeugung des kryogenen Deuterium-Atomstrahls wurde in Abhängigkeit von der Düsentemperatur analysiert, was eine wichtige Studie für künftige Spektroskopiemessungen darstellt. Darüber hinaus wurden für die Präzisionsmessung verschiedene systematische Effekte untersucht, darunter die Fehlausrichtung des Atomstrahls und die elektrischen Streufelder. Es wird gezeigt, dass eine Präzisionsmessung des 2S-6P-Übergangs in Deuterium mit einer ähnlichen Unsicherheit wie in Wasserstoff machbar ist. Nach der vorläufigen Unsicherheitsabschätzung kann die 2S-6P-Übergangsfrequenz in Deuterium auf \SI{1.7}{kHz} bestimmt werden, was einer relativen Genauigkeit von entspricht. Zusammen mit der 1S-2S-Messung kann dieses Ergebnis bereits die genauesten Bestimmungen des Deuteronenradius und der Rydberg-Konstante aus dem elektronischen Deuterium ermöglichen, sodass die Unsicherheiten für die Rydberg-Konstante und den Deuteronenradius bzw.~\delta r_d \simeq \SI{0.002}{fm} betragen. Dieses Ergebnis bildet die Grundlage für eine zukünftige Präzisionsmessung, bei der die 2S-6P-Übergangsfrequenz mit ähnlicher Genauigkeit wie bei Wasserstoff bestimmt werden soll, was und \delta r_d \simeq \SI{0.0007}{fm} entsprechen würde. Der Vergleich mit dem Ergebnis von myonischem Deuterium würde es dann erlauben, die QED-Theorie für gebundene Zustände auf dem Niveau von zu testen.Quantum electrodynamics (QED) forms the basis for all other quantum field theories, upon which the Standard Model of particle physics is constructed. Currently, it is clear that our fundamental understanding of nature is incomplete, such that the Standard Model is expected to be modified or extended by new particles or interactions. One way to explore these frontiers of fundamental physics is to perform precision measurements. This thesis studies the precision laser spectroscopy of deuterium, where the transition energies between different energy states of the electron bound to the nucleus can be accurately measured with techniques such as ultra-stable lasers and the frequency comb. Due to the simplicity of hydrogen-like atoms, their energy levels can be precisely calculated from bound-state QED and confronted with the experiment with the relative accuracy on the order of . Such a comparison between theory and experiment is linked to the determination of fundamental constants, which enter the theory as parameters. Only if more indepedendent measurements are available than there are parameters, the theory can be tested.
The comparison between theory and laser spectroscopy in deuterium concerns the Rydberg constant and the deuteron charge radius . This requires at least two different transition frequency measurements to determine those constants, and more measurements to test the theory. Contrary to hydrogen, only few accurate enough transition frequency measurements are available in deuterium. This thesis presents the first study of the 2S-6P transition in deuterium, which can be combined with the existing 1S-2S transition frequency measurement to obtain and . Together with the 2S-2P transition measurement from muonic deuterium, this determination provides a theory test. Such a comparison is important to shine light on the persisting discrepancy between the result from muonic deuterium and the average of previous data from electronic deuterium, as well as tensions between the recent results from hydrogen spectroscopy.
In contrast to hydrogen, precision spectroscopy of the 2S-6P transition in deuterium is complicated by the simultaneous excitation of unresolved hyperfine components, possibly leading to unresolved quantum interference. This thesis studies the possible systematic effects associated with this complication. Along with analytical perturbative models, supercomputer simulations are performed to analyze these effects. It is shown, that quantum interference is strongly suppressed for all 2S-P transitions in deuterium, making precision measurements of these transitions possible. Furthermore, another effect is studied in deuterium compared to hydrogen, which arises from the light force acting on the atoms in the standing wave of the spectroscopy light. Despite additional state manifolds from the simultaneous excitation of unresolved hyperfine components, it is shown that this so-called ``light force shift'' is comparable to the well understood effect in hydrogen.
The main challenge of measuring the one-photon 2S-6P transition in deuterium is the first-order Doppler shift. Therefore, a large part of this thesis contributes to the improved active fiber-based retroreflector (AFR), which is a technique to suppress this shift. The central part of the AFR is the fiber collimator, which is required to produce high-quality counter-propagating laser beams. Designing and characterizing such a collimator for the near ultra-violet wavelength of the 2S-6P transition is one of the main achievements of the improved AFR. The results of this work can be of interest to other applications where a high beam quality or wavefront-retracing beams are important. Furthermore, the limitations of the AFR arising from single-mode polarization-maintaining fibers are investigated. Along with other improvements, a polarization monitor of the spectroscopy laser beams has been implemented. Various characterization measurements are presented to demonstrate the performance of the improved AFR.
Finally, this thesis presents a preliminary measurement of the 2S-6P transition in deuterium. For this measurement, a new cryostat has been installed in the apparatus, which improves the stability of the spectroscopy signal due to reduced temperature fluctuations. The cryogenic deuterium atomic beam generation has been analyzed in dependence on the nozzle temperature, which is an important study for future spectroscopy measurements. Furthermore, for the precision measurement different systematic effects have been investigated, including the atomic beam misalignment and the stray electric fields. It is demonstrated that a precision measurement of the 2S-6P transition in deuterium with a similar uncertainty than in hydrogen is feasible. According to the preliminary uncertainty budget, the 2S-6P transition frequency in deuterium can be determined to \SI{1.7}{kHz}, which corresponds to relative accuracy. Together with the 1S-2S measurement, already this result can enable the most accurate determinations of the deuteron radius and the Rydberg constant from the electronic deuterium with the uncertainties on the Rydberg constant and the deuteron radius of and \delta r_d \simeq \SI{0.002}{fm}, respectively. This result sets the stage for a future precision measurement, where the 2S-6P transition frequency is expected to be determined with the similar accuracy as in hydrogen, which would correspond to and \delta r_d \simeq \SI{0.0007}{fm}. The comparison to the result from muonic deuterium would then allow to test bound-state QED at the level of
Retrospektive Analyse der bullösen Autoimmundermatosen Pemphigus vulgaris und Pemphigus foliaceus mit Hilfe von Desmoglein 1 und 3 zur Entwicklung neuer, nebenwirkungsarmer Therapiestrategien
Get PDFCausal explanations - how to generate, identify, and evaluate them
Get PDFThe main goal of this dissertation is to provide a solid foundation for a formalization of Inference to the Best Explanation (IBE). This foundation consists of three major components. First, an intuitively adequate and formally precise model of causal explanation. Secondly, an intuitively adequate and formally precise measure of (causal) explanatory power. And third, an intuitively adequate and formally precise criterion of proportionality that is able to identify the most appropriate level of specificity for a causal explanation. While the first component makes it possible to generate and identify causal explanations reliably, the second and third components make it possible to evaluate the strength or quality of causal explanations, which is crucial for identifying the best of a set of competing causal explanations
Characterization of recombinant Modified Vaccinia virus Ankara delivering African swine fever virus proteins
Get PDFGeschlechtsabhängige Expression von Glucocorticoid-Rezeptoren und Histonmodifikationen der Plazenta bei Intrauteriner Wachstumsrestriktion
Get PDFInteractome mapping with enzyme-mediated proximity labeling and establishment of DHFR* reporter dependent protein fragment complementation assay for plant research
Get PDFReal time in vivo investigation of nanoparticle dynamics and immune response during ventilator-assisted nanoparticle inhalation
Get PDFMany lung diseases have been associated with exposure to various factors of air pollution including (nano)particles. In the past, research investigating cell-particle interactions was mainly based on cultured cells or ex-vivo tissues. Such experimental methods cannot reproduce in full, the complex reactions of the immune system, which might be triggered by nanoparticles (NPs) in vivo. To visualize and measure in real-time the cellular pulmonary innate immune response elicited by different inhaled NPs, we apply state of the art intravital microscopy on the peripheral alveolar region of the murine lung, in combination with ventilator-assisted inhalation of nebulized Quantum-Dot and carbon black NP aerosols.
Fluorescent Quantum-Dot NPs (with carboxyl-, amine-PEG- or PEG-surface modifications) became visible within seconds after the onset of inhalation and accumulated as distinct fluorescent spots at the alveolar walls.
As early as 60 min after inhalation, a deposited dose of 16 cm2/g (NP surface area /mass lung) elicited an increase in neutrophil numbers only for cQDs, but neither for aPEG-QDs nor PEG-QDs. Neutrophils preferentially arrested in microvessels in close proximity to the site of cQD deposition, where they exhibited probing and crawling behavior, followed by rapid translocation into the alveolar space, where neutrophils ingested cQDs. This early immune response was not specific to cQDs as a comparable increase in neutrophil numbers was also observed upon inhalation of an equivalent dose of carbon black NPs (CNP), a typical component of urban air pollution.
The neutrophil response was accompanied by resident alveolar macrophages (AMs) increasingly internalizing cQDs up to the maximum observation time of 90 min, and again only little uptake was detected for aPEG-QDs. Similar results were observed in in vitro experiments using the AM-like M-HS cell line.
Analysis of neutrophil numbers in a 30 µm radius (which roughly corresponds to one alveolar diameter) around cQD-positive AMs clearly showed increased neutrophil amounts, whereas no local increase in neutrophil numbers was detected neither around cQD-negative AMs, nor close to cell-free cQD, thus indicating a central role of AMs in the initiation of a spatially restricted innate-immune response.
Intriguingly, cQDs as well as CNPs increased the migration velocity of AMs in the alveoli of exposed mice, whereas aPEG-QDs exposure decreased AM crawling velocity, as compared to sham controls.
In order to decipher the chain of effects leading to NP-induced neutrophil recruitment, we applied specific blocking antibodies and inhibitors in order (1) to weaken AM-epithelial bonds to inhibit AM migration and affect NP uptake (anti-ICAM1 and anti-LFA1 mAbs), (2) to inhibit NP uptake and AM stimulation via Complement 5a receptor 1 (anti-CD88 mAbs) and Fcγ receptor (anti-CD64 mAbs), (4) to block NP-induced ROS formation via N-acetyl cysteine (NAC) application, (5) to prevent NP induced cellular degranulation (cromolyn), (6) and to assess if fast acting inflammatory mediator release (TNF-α) contributes to the NP induced immune cell recruitment.
These mechanistic studies suggest that the induction of NP-elicited neutrophil recruitment depends on several key events:
1. NP-induced neutrophil recruitment is initiated by AMs and related to NP internalization/uptake by AMs.
2. The particle uptake efficacy depends on three factors: (1) NP surface modification; (2) the speed of macrophage patrolling movement in the alveoli; (3) AMs phagocytic/internalizing capability.
(1) PEGylation of QDs which are deposited in alveoli renders the NPs invisible to AMs via avoiding protein corona formation and impairs subsequent NP-induced neutrophil recruitment.
(2) Alveolar AM patrolling mainly involves ICAM-1 and LFA-1 interactions and can be blocked by respective antibodies applied to the airway side but not to the vascular side. Blocking ICAM-1 and LFA-1 also effectively impaired particle-triggered neutrophil recruitment.
(3) The macrophage receptors C5aR1 and FcγRI mediate particle internalization by AMs. Blocking C5aR1 or FcγRI also completely blocks Neutrophile recruitment.
3. NP-triggered neutrophil recruitment requires cellular degranulation and can be inhibited by cromolyn treatment.
4. Recruitment of neutrophils further requires TNFα as an anti-TNFα application into the airways to reduce the neutrophilic inflammatory response.
5. Scavenging ROS via NAC also decreased cQDs-induced neutrophil recruitment to some extent but was less effective than ICAM-1/LFA-1 blocking.
Overall, our data indicate a close relation between AM activity (phagocytosis, migration) and the rapid and site-specific recruitment of neutrophils during the early phase (1h and 24h) of particle inhalation, demonstrating a specific role of AMs in triggering the immune response by different NPs
Pulsed-interleaved MINFLUX super-resolution microscopy
Get PDFLight microscopy has become a powerful tool to investigate structures, dynamics, and interactions in natural science such as cell biology. Especially fluorescence microscopy has seen a rapid development during the last decades. The emergence of far-field fluorescence super-resolution microscopy even overcame the fundamental diffraction limit, enabling imaging with high contrast and specificity of structures below 200 nm. With less than a tenth of the photons needed compared to previous super-resolution methods, MINFLUX is the most recent development to push the resolution limit to truly molecular dimensions. This is achieved by combining the excitation information of a structured illumination featuring a minimum with the respective emission information. While MINFLUX enables the visualization of structures and dynamics with 1 nm precision, the size of a fluorophore, only individual fluorophores are localized, thus information about their environment is missing. The method of choice to report about the environment of a fluorophore is the fluorescence lifetime. To this end, a combination of MINFLUX coupled with the fluorescence lifetime would vastly increase the information wealth of MINFLUX localizations.
In this thesis, I built a pulsed-interleaved MINFLUX (pMINFLUX) that extends the nanometer precise localizations of MINFLUX with the fluorescence lifetime domain while additionally simplifying the technological complexity of pMINFLUX. I demonstrated the performance of this setup using DNA origami structures which act as nanoruler. The unprecedented combination of fluorescence lifetime and nanometer precise localizations was employed in four novel methodologies to make the investigation of structures, interactions, dynamics, and their interplay on the nanometer length scale more accessible.
In combination with graphene energy transfer (GET), I extended the nanometer precise lateral localizations of MINFLUX to the third dimension, by using the fluorescence lifetime encoded axial distance information for nanometer precise 3D super-resolution microscopy. I demonstrated the resolution of GET-pMINFLUX on DNA origami structures using DNA-PAINT with axial precisions below 0.4 nm. DNA-PAINT was used to generate stochastic blinking, which is necessary for the localization method MINFLUX to resolve distances smaller than the diffraction limit. To increase the imaging speed and overcome issues with high fluorescent background of DNA-PAINT, I established local-PAINT (L-PAINT). In contrast to DNA-PAINT, L-PAINT imager strands have two binding sequences with a designed binding hierarchy such that the L-PAINT DNA-strand binds longer on one side. This allows the fluorescent dye-modified second end of the strand to locally scan for binding sites at a rapid rate.
While MINFLUX is able to give insight into structural information, the interplay with the environment remains unknown as only individual fluorophores are localized. I addressed this problem by first combining Förster resonance energy transfer (FRET) with MINFLUX to simultaneously localize the donor dye and map the distance to an acceptor dye. With the multilateration of several donor dye positions I localized the acceptor dye with a full width half maximum of 0.17 nm. To overcome the limited working range of FRET of 2-10 nm, I developed a pMINFLUX lifetime multiplexing approach. pMINFLUX lifetime multiplexing uses the fluorescence lifetime to colocalize two spectrally similar dyes without photo-switching over a large field-of-view. Beyond the FRET range, pMINFLUX lifetime multiplexing enabled the co-localization of two dyes by separating their fluorescence intensities according to their fluorescence lifetimes. I demonstrated this in simulation and experiment using two independent L-PAINT pointer systems, whose dynamics were imaged with nanometer precision. Inside the FRET range, two dyes were co-localized using a newly developed combined phasor-microtime gating approach. As a result, the combination of both multiplexing approaches closed the resolution gap between single-molecule FRET and co-tracking