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Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
Slack (sequence like a Ca2+ -activated K + channel; also termed Slo2.2, Kcnt1, or KNa 1.1) is a Na+ -activated K + channel that is highly expressed in the peripheral and central nervous system. Previous studies have shown that Slack is enriched in the isolectin B4binding, non-peptidergic subpopulation of C-fiber sensory neurons and that Slack controls the sensory input in neuropathic pain. Recent single-cell RNA-sequencing studies suggested that Slack is highly co-expressed with transient receptor potential (TRP) ankyrin 1 (TRPA1) in sensory neurons. By using in situ hybridization and immunostaining we confirmed that Slack is highly co-localized with TRPA1 in sensory neurons, but only to a minor extent with TRP vanilloid 1. Mice lacking Slack globally or conditionally in sensory neurons (SNS-Slack─/─ ), but not mice lacking Slack conditionally in neurons of the spinal dorsal horn (Lbx1-Slack─/─ ), displayed increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. Patch-clamp recordings with cultured primary neurons and in a HEK-293 cell line transfected with TRPA1 and Slack revealed that Slack-dependent K + currents are modulated in a TRPA1-dependent manner. Taken together, these findings highlight Slack as a modulator of TRPA1-mediated activation of sensory neurons.
Furthermore, we investigated the contribution of Slack in the spinal dorsal horn to pain processing. Lbx1-Slack ─/─ mice demonstrated normal basal pain sensitivity and Complete Freund’s Adjuvant-induced inflammatory pain. Interestingly, we observed a significantly increased spared nerve injury (SNI)-induced neuropathic pain hypersensitivity in Lbx1-Slack ─/─ mutants compared to control littermates. Moreover, we tested the effects of pharmacological Slack activation in the SNI model. Systemic and intrathecal, but not intraplantar administration of the Slack opener loxapine significantly alleviated SNI-induced hypersensitivity in control mice, but only slightly in Lbx1Slack ─/─ mice, further supporting the inhibitory function of Slack in spinal dorsal horn neurons in neuropathic pain processing.
Altogether, our data suggest that Slack in sensory neurons controls TRPA1-induced pain, whereas Slack in spinal dorsal horn neurons inhibits peripheral nerve injury induced neuropathic pain. These data provide further insights into the molecular mechanisms of pain sensation.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
Ob Klimawandel oder Luftverschmutzung: Die chemischen und physikalischen Prozesse in der Atmosphäre haben wichtige Auswirkungen auf die menschliche Gesundheit und Ökosysteme. Dabei ist die Atmosphäre mehr als ein Gemisch aus Stickstoff, Sauerstoff, Wasserdampf, Helium und Kohlenstoffdioxid. Es gibt zahlreiche Spurengase, deren Gesamtanteil am Volumen weniger als 1 % ausmacht. In dieser Arbeit werden Stickstoffoxide, Schwefeldioxid, Kohlenstoffmonoxid und Schwefelsäure näher betrachtet, die im Rahmen der flugzeugbasierten Messkampagne Chemistry of the Atmosphere: field experiment in Europe (CAFE-EU)/BLUESKY gemessen wurden.
Die Stickstoffoxide NO und NO2, als NOx zusammengefasst, besitzen hauptsächlich anthropogene Quellen, allen voran fossile Verbrennung und industrielle Prozesse. Zwischen NO und NO2 besteht ein photochemisches Gleichgewicht, sodass in der Atmosphäre vor allem NO2 in relevanten Konzentrationen vorkommt; dies wirkt aufgrund der Bildung von Salpetersäure, HNO3, in wässriger Lösung beim Einatmen ätzend und ist entsprechend gesundheitsschädlich. Troposphärisches Ozon, O3, wesentlicher Bestandteil von Sommersmog, wird hauptsächlich durch die Reaktion von NO mit Peroxiden (HO2 und RO2) gebildet. In der Stratosphäre entstehen NOx hauptsächlich durch die Photodissoziation von Lachgas, N2O, das aufgrund seiner langen Lebenszeit von der Tropo- in die Stratosphäre transportiert werden kann und dort die wichtigste Stickstoffquelle darstellt. In der Stratosphäre tragen NOx zum katalytischen Abbaumechanismus des Ozons bei (Bliefert, 2002; Seinfeld and Pandis, 2016).
Schwefeldioxid, SO2, ist ein toxisches Gas, dessen atmosphärische Quellen hauptsächlich anthropogen sind, nämlich fossile Verbrennung und industrielle Prozesse; Senken sind trockene und feuchte Deposition, wobei letztere zu saurem Regen führen kann. Seit den 1980ern sinken die globalen SO2-Emissionen. SO2 kann in der Atmosphäre zu Sulfat und Schwefelsäure oxidiert werden, was Hauptbestandteil des Wintersmogs ist. Der wichtigste Mechanismus ist die Oxidation mit dem Hydroxylradikal, OH˙, unter Beteiligung von Wasserdampf. In der Stratosphäre ist Carbonylsulfid, OCS, die wichtigste Schwefelquelle, da es analog zum N2O dank seiner langen Lebenszeit von der Tropo- in die Stratosphäre transportiert werden kann (Bliefert, 2002; Seinfeld und Pandis, 2016). Typische Konzentrationen von Schwefelsäure sind 105 cm–3 nachts und 107 cm–3 tagsüber in der Troposphäre sowie 105 cm–3 tagsüber in der Stratosphäre (Clarke et al., 1999; Weber et al., 1999; Fiedler et al., 2005; Arnold, 2008; Kürten et al., 2016; Berresheim et al., 2000).
Kohlenstoffmonoxid, CO, ist ein toxisches Gas, das zu gleichen Teilen durch direkte Emissionen (v.a. Biomasseverbrennung und fossile Verbrennung) und In-situ-Oxidation (v.a. von Methan, Isopren und industriellen Kohlenwasserstoffen) in die Atmosphäre gelangt. Die Hauptsenke ist die Reaktion mit OH˙ in der Troposphäre. Seit 2000 sinkt die globale CO-Konzentration (Bliefert, 2002).
Doch neben Gasen sind auch Aerosolpartikel fester Bestandteil des Gemisches Luft, welche luftgetragene feste oder flüssige Teilchen sind. Primäre Aerosolpartikel werden direkt als solche in die Atmosphäre emittiert, während sekundäre Aerosolpartikel in der Atmosphäre gebildet werden, indem gasförmige Vorläufersubstanzen mit geringer Flüchtigkeit auf primären Partikeln kondensieren oder durch Zusammenclustern und Anwachsen komplett neue Partikel bilden. Aerosolpartikel ermöglichen als Wolkenkondensationskeime erst die Bildung von Wolken und wirken somit – neben ihrem direkten reflektierenden Effekt – durch Änderung der Wolkenbedeckung und -eigenschaften insgesamt kühlend aufs Klima und beeinflussen die lokalen und globalen Wasserkreisläufe. Doch sie haben auch negative Auswirkungen auf die menschliche Gesundheit und sind für eine Verkürzung der durchschnittlichen Lebensdauer in Regionen mit hohen Feinstaubbelastungen verantwortlich (Seinfeld und Pandis, 2016; Bellouin et al., 2020; World Health Organization, 2016).
Neben den bisher betrachteten neutralen, also ungeladenen Gasen und Partikeln sind Ionen in der Gasphase sowie geladene Partikel ebenfalls Bestandteil der Atmosphäre. Sie spielen bei vielen atmosphärischen Prozessen eine wichtige Rolle, wie etwa bei Gewittern, Radiowellenübertragung und ionen-induzierter Nukleation von Aerosolpartikeln. Die Hauptquellen für Ionisation in der Tropo- und Stratosphäre ist die galaktische kosmische Strahlung, die entgegen ihrem Namen hauptsächlich aus Protonen und α-Partikeln (primäre Partikel genannt) besteht und in der Erdatmosphäre durch Kollision mit Luftmolekülen Teilchenschauer von sekundären Partikeln (u.a. Myonen, Pionen und Neutrinos) hervorruft. Die primären und sekundären Partikel können die Luftmoleküle ionisieren unter Entstehung von N+, N2+, O+, O2+ und Elektronen. Sauerstoff reagiert rasch mit letzteren zu O– und O2–. Diese Kationen und Anionen reagieren weiter, bis Ionenclustern der Summenformeln (HNO3)n(H2O)mNO3– und H+(H2O)n(B)m gebildet werden, wobei B Basen wie Methanol, Aceton, Ammoniak oder Pyridin sind. Weitere Ionisationsquellen sind der Zerfall des Radioisotops 222Rn in Bodennähe und ionisierende Solarstrahlung oberhalb der Stratosphäre. Atmosphärische Ionen haben zwei wichtige Senken: die Wiedervereinigung, auch Rekombination genannt, bei der sich ein Kation und ein Anion gegenseitig neutralisieren sowie das Anhaften an Aerosolpartikeln. Letztere Senke ist vor allem in der Troposphäre aufgrund der relativ hohen Konzentration an Aerosolpartikeln relevant (Arnold, 2008; Viggiano und Arnold, 1995; Bazilevskaya et al., 2008; Hirsikko et al., 2011).
This Ph. D. thesis with the title "Characterisation of laser-driven radiation beams: Gamma-ray dosimetry and Monte Carlo simulations of optimised target geometry for record-breaking efficiency of MeV gamma-sources" is dedicated to the study of the acceleration of electrons by intense sub-picosecond laser pulses propagating in a sub-millimeter plasma with near-critical electron density (NCD) and resulting generation of the gamma bremsstrahlung and positrons in the targets of different materials and thickness.
Laser-driven particle acceleration is an area of increasing scientific interest since the recent development of short pulse, high-intensity laser systems. The interaction of intense high-energy, short-pulse lasers with solid targets leads to the production of high-energy electrons in the relativistic laser intensity regime of more than 1018 W /cm2. These electrons play the leading role in the first stage of the interaction of laser with matter, which leads to the creation of laser sources of particles and radiation. Therefore, the optimisation of the electron beam parameters in the direction of increasing the effective temperature and beam charge, together with a slight divergence, plays a decisive role, especially for further detection and characterisation of laser-driven photon and positron beams.
In the context of this work, experiments were carried out at the PHELIX laser system (Petawatt High-Energy Laser for Heavy Ion eXperiments) at GSI Helmholtz Center for Heavy-Ion Research GmbH in Darmstadt, Germany. This thesis presents a thermoluminescence dosimetry (TLD) based method for the measurement of bremsstrahlung spectra in the energy range from 30 keV to 100 MeV. The results of the TLD measurements reinforced the observed tendency towards the strong increase of the mean electron energy and number of super-ponderomotive electrons. In the case of laser interaction with long-scale NCD-plasmas, the dose caused by the gamma-radiation measured in the direction of the laser pulse propagation showed a 1000-fold increase compared to the high contrast shots onto plane foils and doses measured perpendicular to the laser propagation direction for all used combinations of targets and laser parameters.
In this thesis I present novel characterisation method using a combination of TLD measurements and Monte Carlo FLUKA simulations applicable to laser-driven beams. The thermoluminescence detector-based spectrometry method for simultaneous detection of electrons and photons from relativistic laser-induced plasmas initially developed by Behrens et al. (Behrens et al., 2003) and further applied in experiments at PHELIX laser (Horst et al., 2015) delivered good spectral information from keV energies up to some MeV, but as it was presented in (Horst et al., 2015) this method was not really suitable to resolve the content of photon spectra above 10 MeV because of the dominant presence of electrons. Therefore, I created new evaluation method of the incident electron spectra from the readings of TLDs. For this purpose, by means of MatLab programming language an unfolding algorithm was written. It was based on a sequential enumeration of matching data series of the dose values measured by the dosimeters and calculated with of FLUKA-simulations. The significant advantage of this method is the ability to obtain the spectrum of incident electrons in the low energy range from 1 keV, which is very difficult to measure reliably using traditional electron spectrometers.
The results of the evaluation of the effective temperature of super-ponderomotive electrons retrieved from the measured TLD-doses by means of the Monte-Carlo simulations demonstrated, that application of low density polymer foam layers irradiated by the relativistic sub-ps laser pulse provided a strong increase of the electron effective temperature from 1.5 - 2 MeV in the case of the relativistic laser interaction with a metallic foil up to 13 MeV for the laser shots onto the pre-ionized foam and more than 10 times higher charge carried by relativistic electrons.
The progressive simulation method of whole electron spectra described with two -temperatures Maxwellian distribution function has been developed and the results of dose simulations were compared with the acquired experimental data. The advanced feature of this method, which distinguishes it from the results of the simulation of the photon spectrum using the interaction with the target of mono-energetic electron beams (Nilgün Demir, 2013; Nilgün Demir, 2019) or the initial electron spectrum expressed as a function of one electron temperature (Fiorini, 2012), is the ability to simulate the initial electron spectrum described by the Maxwellian distribution function with two temperatures.
The important objective of this thesis was dedicated to the study and characterisation of laser-driven photon beams. In addition to this, the positron beams were evaluated. The investigation of bremsstrahlung photons and positrons spectra from high Z targets by varying the target thickness from 10 µm to 4 mm in simulated models of the interactions of electron spectra with Maxwellian distribution functions allowed to define an optimal thickness when the fluences of photons and positrons are maximal. Furthermore based on the results of FLUKA simulations the gold material was found to be the most suitable for the future experiments as e − γ target because of its highest bremsstrahlung yield.
Additionally Monte Carlo simulations were performed applying the obtained electron beam parameters from the electron acceleration process in laser-plasma interactions simulated with particle-in-cell (PIC) code for two laser energies of 20 J and 200 J. The corresponding electron spectra were imported into a Monte Carlo code FLUKA to simulate the production process of bremsstrahlung photons and positrons in Au converter. FLUKA simulations showed the record conversion of efficiency in MeV gammas can reach 10%, which reinforces the generation of positrons. The obtained results demonstrate the advantages of long-scale plasmas of near critical density (NCD) to increase the parameters of MeV particles and photon beams generated in relativistic laser-plasma interaction. The efficiency of the laser-driven generation of MeV electrons and photons by application of low-density polymer foams is essentially enhanced.
As one of the most widespread infectious diseases in the world, it is currently estimated that approximately 296 million people globally are chronically infected with Hepatitis B virus (HBV), the consequences of HBV infection cause more than 620,000 deaths each year. Although safe and effective HBV vaccines have reduced the incidence of new HBV infections in most countries, there are still around 1.5 million new infections each year. HBV remains a major health problem because there is no large-scale effective vaccination strategy in many countries with a high burden of disease, many people with chronic HBV infection are not receiving effective and timely treatment, and a complete cure for chronic infection is still far from being achieved.
Since its discovery, HBV has been identified as an enveloped DNA virus with a diameter of 42 nm. For efficient egress from host cells, HBV is thought to acquire the viral envelope by budding into multivesicular bodies (MVBs) and escape from infected cells via the exosome release pathway. It is clear that HBV hijacks the host vesicle system to complete self-assembly and propagation by interacting with factors that mediate exosome formation. Consequently, the overlap with exosome biogenesis, using MVBs as the release platform, raises the possibility for the release of exosomal HBV particles. Currently, virus containing exosomal vesicles have been described for several viruses. In light of this, this study explored whether intact HBV-virions wrapped in exosomes are released by HBV-producing cells.
First, this study established a robust method for efficient separation of exosomes from HBV virions by a combination of differential ultracentrifugation and iodixanol density gradient centrifugation. Fractionation of the density gradient revealed that two populations of infectious viral particles can be separated from the culture fluids of HBV-producing cells. The population present in the low-density peak co-migrates with the exosome markers. Whereas the population that appeared in the high-density fractions was the classical HBV virions, which are rcDNA-containing nucleocapsids encapsulated by the HBV envelope.
Subsequently, the characterization of this low-density population was performed, namely the highly purified exosome fraction was systematically investigated. Relying on the detergent sensitivity of the exosome membrane and the outer envelope of the HBV virus, disruption of the exosome structure by treatment with limited detergent revealed the presence of HBsAg in the exosomes. At the same time, mild and limited NP-40 treatment of highly purified exosomes and a further combination of density gradient centrifugation resulted in the stepwise release of intact HBV virions and naked capsids from the exosomes generated by HBV-producing cells. This implies the presence of intact HBV particles encapsulated by the host membrane.
The presence of exosome-encapsulated HBV particles was consequently also verified by suppressing the morphogenesis of MVBs or exosomes. Impairment of MVB- or exosome-generation with small molecule inhibitors has significantly inhibited the release of host membrane-encapsulated HBV particles as well. Likewise, silencing of exosome-related proteins caused a diminution of exosome output, which compromised the budding efficiency of wrapped HBV.
Moreover, electron microscopy images of ultra-thin sections combined with immunogold staining visualized the hidden virus in the exosomal structure. Additionally, the presence of LHBs on the surface of exosomes derived from HBV-expressing cells was also observed.
As expected, these exosomal membrane-wrapped HBV particles can spread productive infection in differentiated HepaRG cells. In HBV-susceptible cells, as LHBs on the membrane surface, this type of exosomal HBV appeared to be uptaken in an NTCP receptor-dependent manner.
Taken together these data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV. Exosomes hijacked by HBV act as a transporter impacting the dissemination of the virus.
Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway
(2022)
The human body is permanently exposed to its environment and thus to viruses and other pathogens, which require a flexible response and defense. Alongside to the innate immune system, the adaptive immune system provides highly specialized protection against these threats. The major histocompatibility complex class I (MHC I) antigen presentation system is a cornerstone of the adaptive immune system and a major constituent of cellular immunity. Pathogens such as viruses that invade a cell will leave traces in the form of proteins and peptides which are degraded and loaded onto MHC I molecules. MHC I peptide loading is performed by peptide loading complex (PLC) in the membrane of the endoplasmic reticulum as part of a multifaceted and comprehensive quality control machinery. Monitored by multiple layers of quality assurance, the MHC I molecules consequently display the immune status of the cell on its surface. In this context, the captured fragment of the virus serves as a call for help issued by the cell, alerting the adaptive immune system to the infection to mount an appropriate immune response.
The three-dimensional structure as well as the mechanistic details of parts of this complex machinery were characterized in the context of this dissertation. Among other tools, light-modulable nanotools were developed in this thesis, which permit external regulation of cellular processes in temporal and spatial resolution. Furthermore, methods and model systems for the biochemical characterization of cellular signaling cascades, proteins, as well as entire cell organelles were developed, which are likely to influence the field of cellular immunity and protein biochemistry in the future.
This cumulative work comprises a total of six publications whose scientific key advances will be briefly outlined in this abstract. In the introduction, the scientific background as well as the current state of research and methodological background knowledge are conveyed. The results section condenses the main aspects of the publications and links them to each other. Further details can be retrieved from the attached original publications.
In “Semisynthetic viral inhibitor for light control of the MHC I peptide loading complex, Winter, Domnick et al., Angew Chem Int Ed 2022” a photocleavable viral inhibitor of the peptide loading complex was produced by semi-synthesis. This nanotool was shown to be suitable for both purifying the PLC from human Raji cells as well as reactivating it in a light-controlled manner. Thus, this tool establishes the isolation of a fully intact and functional peptide loading complex for biochemical characterization. In addition, a novel flow cytometric analysis pipeline for microsomes was developed, allowing cellular vesicles to be characterized with single organelle resolution, similar to cells.
In “Molecular basis of MHC I quality control in the peptide loading complex, Domnick, Winter et al., Nat Commun 2022” the peptide loading complex was reconstituted into large nanodiscs, and a cryo-EM structural model of the editing module at 3.7 Å resolution was generated. By combining the structural model with in vitro glycan editing assays, an allosteric coupling between peptide-MHC I assembly and glycan processing was revealed, extending the known model of MHC I loading and dissociation from the PLC. These mechanisms provide a prototypical example for endoplasmic reticulum quality control.
In a related context, in “Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity, Müller, Winter et al., Nat Commun 2022” a recombinantly assembled editing module comprised of MHC I-tapasin-ERp57 was crystallized for X-ray structural biology. The resulting crystal structure at a resolution of 2.7 Å permitted the precise identification of characteristic features of the editing module and particularly of the peptide proofreading mechanism of tapasin. This study provided pivotal insights into the tapasin-mediated peptide editing of different MHC I allomorphs as well as similarities to TAPBPR-based MHC I peptide proofreading.
In “TAPBPR is necessary and sufficient for UGGT1-mediated quality control of MHC I, Sagert, Winter et al. (in preparation)” novel insights concerning the peptide proofreader TAPBPR and its close interplay with the folding sensor and glucosyltransferase UGGT1 were obtained. It was shown that TAPBPR is an integral part of the second level of endoplasmic quality control and is indispensable for effective MHC I coordination by UGGT1.
In “Light-guided intrabodies for on-demand in situ target recognition in human cells, Joest, Winter et al., Chem Sci 2021” intracellular nanobodies were equipped with a photocaged target recognition domain by genetic code expansion via amber suppression. These intrabodies, acting as high-affinity binding partners endowed with a fluorophore, could be used in a light-triggered approach to instantaneously visualize their target molecule...
Recent advances in artificial neural networks enabled the quick development of new learning algorithms, which, among other things, pave the way to novel robotic applications. Traditionally, robots are programmed by human experts so as to accomplish pre-defined tasks. Such robots must operate in a controlled environment to guarantee repeatability, are designed to solve one unique task and require costly hours of development. In developmental robotics, researchers try to artificially imitate the way living beings acquire their behavior by learning. Learning algorithms are key to conceive versatile and robust robots that can adapt to their environment and solve multiple tasks efficiently. In particular, Reinforcement Learning (RL) studies the acquisition of skills through teaching via rewards. In this thesis, we will introduce RL and present recent advances in RL applied to robotics. We will review Intrinsically Motivated (IM) learning, a special form of RL, and we will apply in particular the Active Efficient Coding (AEC) principle to the learning of active vision. We also propose an overview of Hierarchical Reinforcement Learning (HRL), an other special form of RL, and apply its principle to a robotic manipulation task.
Many countries have restricted public life during the SARS-CoV2 pandemic. As related measures limited the access to sports facilities, this dissertation aimed (1) to examine changes in physical activity (PA) and well-being in affected countries, and (2) to determine the effectiveness of a digital home exercise program in this context.
Part 1 (PA/well-being) of the dissertation was a digital survey administered in 14 countries. Participants reported a 41 - 42% reduction of PA (NPAQ-SF) during restrictions (n=13,503 valid responses). Compliance with international PA guidelines decreased by nearly 19%. Mental well-being declined substantially (n=14,975 responses; 68.1 to 51.9 points on the WHO5 index) and the proportion of individuals at risk of depression tripled (14.2% to 45.2%). Physical well-being (SF-36 Pain) decreased slightly (85.8% to 81.3%). About two thirds (68.1%) of the respondents reported being interested in digital home exercise.
For Part 2 (digital home exercise) of the dissertation, an international multicenter randomized, controlled trial was performed allocating healthy adults (n=763; 33±12 years) to an intervention (IG) or control (CG) group. In contrast to the CG, the IG was offered live-streamed home exercise for four weeks. Subsequently, both groups had access to pre-recorded workouts for another four weeks. Outcomes were measured weekly using validated questionnaires. Mixed-models data analyses revealed an up to 1.65-fold (95% CI: 1.4-1.94; week 1) increase of PA relative to the CG. Moreover, small improvements in exercise motivation (SKK scale), psychological well-being (WHO-5 index), sleep quality (MOS Sleep Scale), and anxiety symptoms (GAD-7 Scale) were observed for IG.
The results of this dissertation suggest that public life restrictions associated with the pandemic had significant adverse effects on movement behavior and well-being. Digital home exercise can help to maintain and/or increase health- beneficial PA and well-being and may hence represent a supportive element of viral containment efforts.
The majority of B-cell precursor acute leukemias in infants are associated with the chromosomal translocation t(4;11)(q21;q23), resulting in the fusion of the mixed-lineage leukemia (MLL) and ALL1-fused gene of chromosome 4 (AF4) genes. While the fusion protein MLL-AF4 is expressed in all t(4;11) patients and essential for leukemia progression, the distinct role of the reciprocal fusion protein AF4-MLL, that is expressed in only 50-80% of t(4;11) leukemia patients (Meyer et al., 2018), remains unclear. In addition, t(4;11) leukemia could so far exclusively be generated in vivo in the presence of AF4-MLL and independent of the co-expression of MLL-AF4 (Bursen et al., 2010).
In a multifactorial approach inhibiting histone deacetylases (HDACs) and expressing the dominant negative mutation of Taspase1 (dnTASP1), both MLL fusion proteins were targeted simultaneously to evaluate a possible cooperative effect between MLL-AF4 and AF4-MLL during the progression of leukemia. Of note, neither HDACi nor dnTASP1 expression negatively affect endogenous MLL, but rather endorse its function hampered by the MLL fusion proteins (Ahmad et al., 2014; Bursen et al., 2004; Zhao et al., 2019). The mere expression of dnTASP1 failed to induce apoptosis, whereas dnTASP1 could elevate apoptosis levels significantly in HDACi-treated t(4;11) cells underlining the therapeutic potential of co-inhibiting both MLL fusion proteins.
Next, the impact of inhibiting either MLL-AF4 or AF4-MLL in vivo was resolved using whole transcriptome analysis. In PDX cells obtained by the Jeremias Laboratory (Völse, 2020) that co-expressed both t(4;11) fusion proteins, the knock-down of MLL-AF4 revealed the down-regulation of pivotal hemato-malignant factors. The expression of dnTASP1 led to massive deregulation of cell-cycle genes in vivo. Considering that the inhibition of particularly MLL-AF4 but not AF4-MLL impaired leukemic cell growth in vivo (Völse, 2020), the results of this work suggest a cooperative effect between both fusion proteins, while the loss of AF4-MLL during leukemia progression appears not essential.
Thereafter, a possible short-term role of AF4-MLL during the establishment of t(4;11) leukemia was analyzed. For this purpose, an in vitro t(4;11) model was constructed to investigate the transforming potential of transiently expressed AF4-MLL in cells constitutively expressing MLL-AF4, putatively reflecting the situation in vivo. Due to the lack of a leukemic background of the applied cell line, the aim was to investigate the long-term potential of AF4-MLL to significantly alter the epigenome rather than mimicking the development of leukemia. Strikingly, short-term-expressed AF4-MLL in cooperation with MLL-AF4 exerted durable epigenetic effects on gene transcription and chromatin accessibility. The here obtained in vitro data suggest a clonal evolutionary process initiated by AF4-MLL in a cooperative manner with MLL-AF4. Importantly, no long-term changes in chromatin accessibility could be observed by the transient expression of either MLL-AF4 or AF4-MLL alone.
All in all, considering endogenous MLL, MLL-AF4 and AF4-MLL in a targeted treatment is a promising approach for a more tailored therapy against t(4;11) leukemia, and AF4-MLL is suggested to act in a cooperative manner with MLL-AF4 especially during the development of a t(4;11) leukemia.
This work investigated the influence of the CRISPR/Cas9 mediated knockout of 5-lipoxygenase (5-LO) on different adherent tumour cell lines derived from solid tumours. For this, the 5-LO expressing tumour cell lines HCT-116, HT-29, and U-2 OS were transiently transfected using a plasmid carrying the CRISPR/Cas9 complex sequence to the ALOX5 gene. Subsequently, cells were selected using Puromycin and analysed via Western blotting and DNA Sanger sequencing. Cells that were transfected with a control plasmid missing the guide RNA sequence, were used as a control for all experiments.
Differential gene expression analysis, performed after next-generation RNA sequencing, revealed that the expression of various genes was altered after the knockout of 5-LO. In HCT-116 cells, 28 genes were expressed differentially in all 5-LO knockout single-cell clones, while in HT-29 cells the expression of 18 genes and in U-2 OS cells of 234 genes was influenced by the knockout of 5-LO. These findings were validated by real-time qPCR. A lot of the genes that were influenced by the 5-LO knockout are known to be connected to epithelial-mesenchymal-transition (EMT), a process necessary for tumour metastasis. The results from RNA sequencing were the starting point for further investigations. In the following, different aspects of the tumour cell lines were examined. In HT-29, as
well as in U-2 OS cells, it was shown that knockout of the 5-LO resulted in impaired cell proliferation. Also, the formation of three-dimensional tumour spheroids was altered. In HT-29 cells, the knockout of 5-LO increased the number of cells in spheroids. In contrast, in U-2 OS cells, the number of cells per spheroid was decreased, even though the diameter of the spheroids was increased, due to more loosely packed spheroids. The difference between 5-LO positive and negative U-2 OS cells became even more obvious after embedding the spheroids in an artificial extracellular matrix. In that scenario, cells lacking the 5-LO formed smaller spheroids that did not have the same ability to grow into the extracellular matrix as 5-LO positive cells did. Also, directed cell migration was strongly influenced by the knockout of 5-LO. In both, HCT-116 and U-2 OS cells, directed cell migration towards a serum gradient was increased in 5-LO knockout single-cell clones. Pharmacological inhibition of the enzyme was used to investigate, whether canonical or non-canonical functions were responsible for the previously mentioned effects.
Therefore, vector control cells were treated with the 5-LO inhibitors Zileuton and CJ-13610 in different concentrations. Interestingly, only some of the effects mediated by the complete knockout of 5-LO could be reproduced by inhibiting the enzyme, leading to the suggestion, that canonical, as well as non-canonical functions of 5-LO, play a role in these tumour cells.
To conclude, it was shown in this study, that 5-LO affects various cellular functions when expressed in adherent tumour cell lines. These cell line-dependent effects result in altered gene expression, enhanced proliferation, and spheroid formation, as well as impaired cell motility, and can be mediated by enzymatic activity as well as other non-canonical functions.
The aim of this thesis is to provide a complete and consistent derivation of second-order dissipative relativistic spin hydrodynamics from quantum field theory. We will proceed in two main steps. The first one is the formulation of spin kinetic theory from quantum field theory using the Wigner-function formalism and performing an expansion in powers of the Planck constant. The essential ingredient here is the nonlocal collision term. We will find that the nonlocality of the collision term arises at first order in the Planck constant and is responsible for the spin alignment with vorticity, as it allows for conversion between spin and orbital angular momentum.
In the second step, this kinetic theory is used as the starting point to derive hydrodynamics including spin degrees of freedom. The so-called canonical form of the conserved currents follows from Noether’s theorem.
Applying an HW pseudo-gauge transformation, we obtain a spin tensor and energy-momentum tensor with obvious physical interpretation. Promoting all components of the HW tensors to be dynamical, we derive
second-order dissipative spin hydrodynamics. The additional equations of motion for the dissipative currents are obtained from kinetic theory generalizing the method of moments to include spin degrees of freedom.
Background and Aim: Genome-wide association studies revealed a strong association between cardiovascular diseases (CVD) and clonal hematopoiesis of indeterminate potential (CHIP), highlighting one of its most common CHIP-driving mutations-TET2 (ten-eleven translocation 2), as a target for CHIP related CVD research. Our lab has established the generation of self-organizing cardiac organoids (SCO), which demonstrate the cellular composition and organization of the native human heart, and mimics human myocardial responses to stress stimulation. This project aims to examine whether SCOs would be an appropriate CHIP model and decipher promising drugs for cardiovascular CHIP treatment.
Methods: To study TET2-mutant cardiovascular CHIP, we set up the TET2 cardiac-CHIP model through a knockdown (KD) of TET2 in myeloid cells that infiltrated our lab-made SCO. Immunofluorescence and qPCR were performed to ascertain TET2-KD myeloid cell infiltration, SCO fibrosis, and apoptosis assessments. SCO fibrosis was further analyzed by immunofluorescence staining, and cardiac contractile frequency and amplitude were determined by calcium flux analysis. Finally, RNAseq was performed to analyze transcriptomic changes in drug/vehicle-treated TET2-KD myeloid cells and the TET2 cardiac-CHIP model.
Results: The TET2 cardiac-CHIP model resulted in significantly increased inflammation in SCO, accompanied by fibrosis and more cleaved Caspase-3, causing cardiomyocytes apoptosis and promoting the release of cTNT. The shortlisted drugs revealed a reduction of proliferation in TET2-KD myeloid cells, decreased pro-inflammatory cytokines, and a higher apoptosis level. Furthermore, the TET2 cardiac-CHIP model treated with selected drugs showed a remarkable decline in TET2-KD myeloid cell infiltration and pro-inflammation cytokines, cardiomyocyte apoptosis, fibrosis, and lowered cTNT levels, while drug control groups were not affected. Moreover, the drug treatment groups improved the heartbeat frequency and amplitude accessed by the calcium transient assay. RNAseq data also validated the above findings.
Conclusions & Discussion: Our results indicate that SCOs are an efficient pre-clinical model for studying and validating CHIP genes and drug interactions. Our data revealed that TET2-KD myeloid cells invade SCO and secrete pro-inflammatory cytokines, which promote apoptosis of cardiomyocytes and the release of cTNT. In this regard, our TET2 cardiac-CHIP model matches the inflammatory phenotype previously characterized in CHIP patients. Nevertheless, this phenotype could be rescued using positive drug candidates (Clopidogrel, R406, and Lanatoside C) selected by this project, emphasizing the significant value of our TET2 cardiac-CHIP model for drug screens and pre-clinical validation studies. Furthermore, among these three drug candidates, we found Lancatoside C, as proved by FDA/EMA, showed an unmet possibility for clinical therapeutic demand, insinuating potential benefit in repurposing Lanatoside C for the treatment of TET2-mutant cardiovascular CHIP.
As part of two drilling campaigns of the International Continental Scientific Drilling Program (ICDP), several geophysical borehole measurements were carried out by the Leibniz Institute for Applied Geophysics (LIAG) in two lakes. The acquired data was used to answer stratigraphic and paleoclimatic research questions, including the establishment of robust age-depth models and the construction of continuous lithological profiles.
Lake Towuti is located on Sulawesi (Indonesia), within the "Indo-Pacific Warm Pool" (IPWP), a globally important region for atmospheric heat and moisture budgets. The lake exists for approximately one million years, but its exact age is uncertain. We present the first agedepth model for the approximately 100 m continuous sediment sequence from the central part of the lake. The basis for this model is the magnetic susceptibility measured in the borehole and a tephra layer with an age of about 797 ka at 72 m depth. Our age-depth model is inferred from cyclostratigraphic analysis of borehole data and covers a period from 903 ± 11 to 131 ± 67 ka. We suggest that orbital eccentricity and/or changes between global cold and warm periods are responsible for hydroclimatic changes in the IPWP, that these changes affect sedimentation processes in Lake Towuti, and that we can measure and observe this effect in the sediment properties today. Additionally, we created a continuous artificial lithological profile from a series of different borehole data using cluster analysis. This provides information from parts of the borehole where no sediment is available due to core loss.
Lake Ohrid is 1.36 million years old and is located on the Balkan Peninsula on the border between Albania and North Macedonia. The primary hole 'DEEP' in the central part of the lake has been the subject of several investigations, but information about sediments of the marginal locations 'Pestani' and 'Cerava' have not been published yet. In our study, we use natural gamma radiation (GR) measured in the borehole to generate an age-depth model for DEEP. This is performed using the correlation of GR to the global LR04 reference record of Lisiecki and Raymo (2005).
The age information is then transferred via prominent seismic marker horizons to the other two sites, Pestani and Cerava, where it provides the first age-control points for the construction of age-depth models from correlation of GR to LR04. The generated age-depth models are tested using cyclostratigraphic methods, but the limits of this approach are revealed. At DEEP, sedimentation rates (SR) from the cyclostratigraphic method and the correlative approach differ by 2.8 %, at Pestani this difference is 16.7 %, and at Cerava the quality of the data does not allow a reliable evaluation of SR using the cyclostratigraphic approach. We used cluster analysis to construct artificial lithological profiles at all three sites and integrated them into the respective age-depth models. This enables us to determine which sediment types were deposited at what time, and we recognize the change between warm and cold periods in the sediment properties at all three locations. The analyses in this study were all performed on borehole and seismic data and thus do not involve sediment core data. Especially at Pestani and Cerava, new insights into the sedimentological history of Lake Ohrid could be obtained.
In the last part we discuss the occurrence of the half-precession (HP) signal in the European region during the last one million years. The focus is on Lake Ohrid, but a range of other proxies, from the eastern Mediterranean, across the European continent, up to Greenland are analyzed in regards to HP. Applying filters, we focus on the frequency range with a period of 13-8.5 ka and only HP remains in the records. We use correlative methods to determine the clarity of the HP signal in proxies distributed across the European realm. Additionally, we determined the development of HP over time. The HP signal is clearest in the southeast and decreases toward the north. It is further more pronounced in interglacial periods and in the younger part (<621 ka) of most proxies. We suggest that there are mechanisms that transmit the HP signal from its origin near the equator to higher latitudes via different processes. In this context, for instance, the African monsoon, the Nile River and the Mediterranean outflow via the Strait of Gibraltar can be important factors.
Die Emergenz digitaler Netzwerke ist auf die ständige Entwicklung und Transformation neuer Informationstechnologien zurückzuführen.
Dieser Strukturwandel führt zu äußerst komplexen Systemen in vielen verschiedenen Lebensbereichen.
Es besteht daher verstärkt die Notwendigkeit, die zugrunde liegenden wesentlichen Eigenschaften von realen Netzwerken zu untersuchen und zu verstehen.
In diesem Zusammenhang wird die Netzwerkanalyse als Mittel für die Untersuchung von Netzwerken herangezogen und stellt beobachtete Strukturen mithilfe mathematischer Modelle dar.
Hierbei, werden in der Regel parametrisierbare Zufallsgraphen verwendet, um eine systematische experimentelle Evaluation von Algorithmen und Datenstrukturen zu ermöglichen.
Angesichts der zunehmenden Menge an Informationen, sind viele Aspekte der Netzwerkanalyse datengesteuert und zur Interpretation auf effiziente Algorithmen angewiesen.
Algorithmische Lösungen müssen daher sowohl die strukturellen Eigenschaften der Eingabe als auch die Besonderheiten der zugrunde liegenden Maschinen, die sie ausführen, sorgfältig berücksichtigen.
Die Generierung und Analyse massiver Netzwerke ist dementsprechend eine anspruchsvolle Aufgabe für sich.
Die vorliegende Arbeit bietet daher algorithmische Lösungen für die Generierung und Analyse massiver Graphen.
Zu diesem Zweck entwickeln wir Algorithmen für das Generieren von Graphen mit vorgegebenen Knotengraden, die Berechnung von Zusammenhangskomponenten massiver Graphen und zertifizierende Grapherkennung für Instanzen, die die Größe des Hauptspeichers überschreiten.
Unsere Algorithmen und Implementierungen sind praktisch effizient für verschiedene Maschinenmodelle und bieten sequentielle, Shared-Memory parallele und/oder I/O-effiziente Lösungen.
Redox homeostasis must be kept in balance for an intact redox signaling, which is necessary to control neuronal pathways such as growth cone pathfinding, synaptic plasticity and transmission (Oswald, Garnham, Sweeney, & Landgraf, 2018).
Nucleoredoxin (NXN) is an oxidoreductase and thioredoxin-like protein holding two conserved cysteine residues in its structure (Funato & Miki, 2007), which are essential for its redox-regulating functionality. The function of NXN in neurons is still less well studied. But the expression of NXN in neurons, which was confirmed through analyzing adult NXN-LacZ reporter mice, suggested a dominant functional role in neuronal pathways. Initial experiments revealed calcium-calmodulin-dependent kinase 2 a (Camk2a) as a potential interaction partner through a Yeast-2-Hybrid screen (not shown) which is the major protein to induce synaptic plasticity during neuronal activity. Therefore, neuronal expression of NXN and the potential interaction with Camk2a prompted us to investigate deeper into the neuronal pathway. The goal of this work was to confirm the interaction of Camk2a and NXN with further experiments and to characterize behavior of mice carrying a neuronal NXN deletion. To achieve a pan-neuronal depletion of NXN expression in our mouse model, we used the Cre/loxP system with a NestinCre driver. We did not achieve the expected complete deletion of NXN due to unknown compensatory mechanisms. Nevertheless, the partial deletion of NXN in our transgenic mouse model prevented embryonic lethality as occurring in complete NXN knockout mice (Funato et al., 2010). The interaction of Camk2a and NXN was confirmed through proximity ligation assay (PLA) and immunofluorescence staining of primary cortical neurons.
Investigations of the functional interaction revealed a lower redox-sensitivity of Camk2a activity in NXN-deficient brain samples. Additionally, the respiratory activity was significantly reduced in mitochondria of NXN deficient mouse brain pointing to possible dysfunctional mitochondria which is also observed in various neurodegenerative diseases, e.g.: Alzheimer, Parkinson, and Huntington disease (Norat et al., 2020). Unexpectedly, behavioral studies revealed only a subtle effect of the pan-neuronal NXN-deficiency. Significant differences between genotypes were found at the reduction of exploratory behavior and a reduced motivation for the voluntary wheel running in NesNXN-/- mice, which is normally seen as a joyful and rewarding activity. The observed behavior of NesNXN-/- mice potentially results from interaction mechanisms of NXN with Camk2a, as well as decreased oxidation of
Camk2a and further unidentified target proteins of NXN.
Conclusively, function of NXN was revealed as a non-essential redox modulator of Camk2a in neurons. The behavioral phenotype of NesNXN-/- mice is probably compensated through unknown mechanisms. Redox signaling of Camk2a in neurons is regulated through various components such as TXN or GSH, which can backup each other (Branco et al., 2017; Ren et al., 2017). NXN is an additional but not essential regulator.
Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). TAA-producing bacteria are the causative agent of many human diseases and TAA targeted anti-adhesive compounds might counteract such bacterial infections. The modularly structured Bartonella adhesin A (BadA) is one of the best characterised TAAs and serves as an attractive adhesin to study the domain-function relationship of TAAs during infection. BadA is a major virulence factor of B. henselae and is essential for the initial attachment to host cells via adhesion to extracellular matrix proteins. B. henselae is the causative agent of cat scratch disease and adheres to fibronectin using its long BadA fibres. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations.
Human, feline, and laboratory adapted B. henselae isolates display genomic and phenotypic differences. By analysing the genomes of eight B. henselae strains using long-read sequencing, a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes was identified. Moreover, numerous conserved flanking genes were characterised, however, their influence on the regulation of badA expression and modification remains to be explored. It seems that B. henselae G 5436 is the evolutionary ancestor of the other B. henselae strains analysed in this work. The diversity of the badA island among the B. henselae strains indicates that the downstream badA-like domain region might be used as a ‘toolbox’ for rearrangements in the badA gene. Overall, it is suggested that badA-domain duplications, insertions, and/or deletions are the result of active phase variation via site-specific recombination and contribute to rapid host adaptation in the scope of pathogenicity, immune evasion, and/or enhanced long-term colonisation.
The model strain B. henselae Marseille expresses a badA gene that includes 30 repetitive neck/stalk domains, each consisting of several predicted structural motifs. To further elucidate the motif sequences that mediate fibronectin binding, various modified badA constructs were generated. Their ability to bind fibronectin was assessed via whole-cell ELISA and fluorescence microscopy. In conclusion, it is suggested that BadA adheres to fibronectin in a cumulative fashion with quick saturation via unpaired β-strands appearing in structural motifs present in BadA neck/stalk domains 19, 27, and other homologous domains. Furthermore, antibodies targeting a 15-mer amino acid sequence in the DALL motif of BadA neck/stalk domain 27 were able to reduce fibronectin binding of the B. henselae mutant strain S27. Moreover, this DALL motif sequence is conserved in the genome of all analysed B. henselae strains. The identification of common binding motifs between BadA and fibronectin supports the development of new anti-adhesive compounds that might inhibit the initial adherence of B. henselae and other TAA-producing pathogens during infection.
Individualization can be defined as the adaptation of instructional parameters to relevant characteristics of a specific learner. This definition raises several questions, however: Which characteristics are actually relevant? Which parameters of instruction need to be adjusted, and in which way, to positively interact with those characteristics? In a classroom context, additional questions arise: how can information about the relevant learner characteristics be delivered to the teacher? How can individualized instruction be delivered to each learner in a context that has originally been designed for whole-class instruction? By focusing on the measurement and modelling of learner characteristics and instructional adaptations, this dissertation aims to provide an insight into each of these issues.
This dissertation is divided into two parts. The first part is concerned with the theoretical (Paper 1) and statistical (Paper 2) modeling of learner characteristics in the context of individualized instruction. The second part is concerned with the measurement (Paper 3) and implementation (Paper 4) of individualized instruction in the classroom context.
Paper 1 summarizes existing research on individualization from different research traditions. From this summary I derive the need for a dynamic conceptualization of learner characteristics (acknowledging that learners change during and in interaction with the learning process) and synthesize a dynamic framework that details the opportunities for individualization on three different timescales. Paper 2 reports results from an exploratory study that investigated the potential benefits of utilizing person-centered analysis for the assessment of multivariate learner prerequisites and their interaction with instruction. We found that latent profiles over several reading related abilities could explain differential effectiveness of self-reported teaching foci in German third grade reading lessons. These findings indicate not just a need for stronger individualization of teaching but also an advantage of multivariate conceptualizations of learner characteristics. Additionally, they show the utility of person-centered approaches for the investigation of such multivariate learner characteristics and their interaction with instruction.
In the second part, I investigate possible approaches to the implementation and measurement of individualization in a classroom context. Paper 3 investigates whether teacher-, student- and observer perspectives converge when rating the amount of individualization present in regular classroom instruction. We found considerable agreement between the perspectives, indicating a common understanding of the construct at the classroom level as well as providing some evidence for the validity of the used measurement instruments. Paper 4 replicates findings concerning the effectiveness of formative assessment procedures for fostering reading education, supplemented by a moderator analysis showing that only children with low performance at the beginning of the school-year profited from its implementation. This indicates that the information provided by formative assessment procedures helps teachers to identify struggling readers but does not seem to be utilized for adapting instruction to specific deficits of average or high performing children.
In sum, this dissertation contributes to research on individualized instruction by demonstrating necessary conditions for its effectiveness. It posits the need for a dynamic conceptualization of learner characteristics, demonstrates the advantage of multivariate learner profiles, and points out ways towards the successful implementation of individualized instruction in the classroom.
Antimicrobial resistant infections arise as a consequential response to evolutionary mechanisms within microbes which cause them to be protected from the effects of antimicrobials. The frequent occurrence of resistant infections poses a global public health threat as their control has become challenging despite many efforts. The dynamics of such infections are driven by processes at multiple levels. For a long time, mathematical models have proved valuable for unravelling complex mechanisms in the dynamics of infections. In this thesis, we focus on mathematical approaches to modelling the development and spread of resistant infections at between-host (population-wide) and within-host (individual) levels.
Within an individual host, switching between treatments has been identified as one of the methods that can be employed for the gradual eradication of resistant strains on the long term. With this as motivation, we study the problem using dynamical systems and notions from control theory. We present a model based on deterministic logistic differential equations which capture the general dynamics of microbial resistance inside an individual host. Fundamentally, this model describes the spread of resistant infections whilst accounting for evolutionary mutations observed in resistant pathogens and capturing them in mutation matrices. We extend this model to explore the implications of therapy switching from a control theoretic perspective by using switched systems and developing control strategies with the goal of reducing the appearance of drug resistant pathogens within the host.
At the between-host level, we use compartmental models to describe the transmission of infection between multiple individuals in a population. In particular, we make a case study of the evolution and spread of the novel coronavirus (SARS-CoV-2) pandemic. So far, vaccination remains a critical component in the eventual solution to this public health crisis. However, as with many other pathogens, vaccine resistant variants of the virus have been a major concern in control efforts by governments and all stakeholders. Using network theory, we investigate the spread and transmission of the disease on social networks by compartmentalising and studying the progression of the disease in each compartment, considering both the original virus strain and one of its highly transmissible vaccine-resistant mutant strains. We investigate these dynamics in the presence of vaccinations and other interventions. Although vaccinations are of absolute importance during viral outbreaks, resistant variants coupled with population hesitancy towards vaccination can lead to further spread of the virus.