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Remodeling of higher order chromatin structures
Get PDFIn fast allen Eukaryoten ist die DNA in einem dynamischen Polymer namens Chromatin organisiert. Das Nukleosom, der elementare Baustein des Chromatins, besteht aus einem Histonoktamer, um das 146 Basenpaare der DNA gewickelt werden. Zwischen den Nukleosomen befindet sich ein Stück freie DNA, die sogenannte Linker-DNA. Die Nukleosomen sind meist auf der DNA in sehr regelmäßigen Abstand angeordnet. Dieser regelmäßige Abstand ist wichtig, um kryptische Transkription zu verhindern und das Genom vor Doppelstrangbrüchen zu schützen. Die Position der Nukleosomen auf der DNA wird durch ATP-abhängige Nukleosomen-Remodeling-Enzyme beeinflusst. Diese Enzyme können die Nukleosomen von der DNA entfernen, sie auf der DNA zusammenbauen, umstrukturieren und entlang der DNA verschieben.
Chromatin faltet sich sowohl in vitro als auch in vivo zu Strukturen höherer Ordnung. Nukleosomen-Arrays unterliegen auch einer Phasentrennung und bilden dadurch dichte Chromatin-kondensate. Die Chromatinfaltung und die Phasentrennung stellen eine Herausforderung für Nukleosomen-Remodeling-Enzyme dar, da sie das Chromatin binden und darauf einwirken können müssen. In dieser Arbeit habe ich die Nukleosom Verschiebereaktion von Remodeling-Enzymen in unterschiedlich gefalteten Chromatinsubstraten charakterisiert.
Im ersten Kapitel verwendete ich die Spalthefe als Modellsystem, um das nucleosome sliding im Euchromatin und im kompakteren Heterochromatin zu vergleichen. Dafür überexprimierte ich Nukleosomen-Remodeling-Enzyme, deren Targeting zu Heterochromatin durch die Fusion mit einer Heterochromatin-bindenden Domäne erreicht wurde. Allerdings stellte sich heraus, dass die Überexpression des Remodelers Hrp3 für S. pombe toxisch war, unabhängig vom Targeting. Hrp3 Überexpression unterdrückte die Expression eines in Heterochromatin platzierten Reportergens und verursachte Defekte bei der Positionierung von Nukleosomen an den Genkörpern.
Obwohl die Informationen, die durch Short-Read-Sequenzierung für Heterochromatin-Regionen erhalten wurden, spärlich waren, ließ sich eine ATP –Hydrolyse-abhängige Zunahme der Regelmäßigkeit der Nukleosomenpositionierung über subtelomerischen Regionen feststellen. Darüber hinaus führten wir erfolgreich eine gene-by-gene Analyse durch, um die Regelmäßigkeit und Wiederholungslängen, die sog. Nucleosome Repeat Length (NRL) von Nukleosomen-Arrays in Wildtyp- und Remodeler-Deletionsstämmen zu messen. Die häufigste NRL beträgt 150 bp; sie ist damit um ein paar Basenpaare noch geringer als bislang angenommen.
Im zweiten Kapitel testete ich in vitro, ob die Chromatinfaltung und die Phasentrennung das nucleosome sliding behindern. Diese Studie wurde mit der D. melanogaster ATPase ISWI durchgeführt, die Nukleosomen verschieben kann. Nach der Rekonstitution von Nukleosomen-Arrays induzierte ich die intramolekulare Faltung und Phasentrennung durch Zugabe unterschiedlicher Salzmengen. Die gebildeten Chromatinkondensate enthielten Nukleosomenkonzentrationen wie sie auch im Zellkern zu finden sind. Erstaunlicherweise blieben die Kondensate für sehr voluminöse Komplexe zugänglich, was sie zu einem nützlichen Modellsubstrat macht, um die Herausforderungen zu untersuchen, denen Remodeler in einer dichten Chromatinumge-bung begegnen. ISWI reicherte sich in Chromatinkondensaten an und verlangsamte die Fusion der Kondensate in einer konzentrationsabhängigen Weise. Mit Hilfe eines neuartigen, bildgebenden nucleosome sliding Assays konnten wir die Remodeling-Raten innerhalb und außerhalb von Chromatinkondensaten vergleichen. Wir konnten bestätigen, dass das nucleosome sliding innerhalb von Chromatinkondensaten stattfindet. Die Anfangsgeschwindigkeit für das nucleosome sliding innerhalb der Kondensate war nur um das Zweifache niedriger als in Lösung. Zusammenfassend stellen die Kondensate keine starke Barriere für nucleosome sliding dar. Um die viskoelastischen Eigenschaften von Chromatinkondensaten zu charakterisieren, setzten wir optische Pinzetten ein, um die Kondensate kontrolliert fusionieren zu lassen. Der Verlust der ATP-Hydrolyse führte zu einer Verhärtung der Chromatinkondensate und einer verringerten Dynamik von ISWI. Wir erklären unsere Ergebnisse mit Hilfe eines monkey-bar Modells, in dem die beiden DNA-Bindungsdomänen von ISWI zwischen starken und schwachen Bindungsmodi wechseln. So stellt ISWI sicher, dass es auch im Zellkern, wo die hohe Nukleosomenkonzentration die Dissoziationskonstanten deutlich übersteigt, mobil bleibt. Unsere Ergebnisse deuten darauf hin, dass Pathologie-assoziierte Phänotypen auch zum Teil durch Veränderungen der Chromatindynamik und nicht ausschließlich durch eine Störung der kanonischen Remodeling-Funktionen verursacht werden könnten.
Im dritten Kapitel untersuchte ich die Wechselwirkung zwischen ISWI und dem acidic patch, der für seine Aktivierung wichtig ist. ISWI durchläuft während der Katalyse globale Konformationsänderungen, was die Strukturanalyse schwierig macht. Ich verwendete Mononukleosomen mit einem UV-aktivierten Crosslinker, der in acidic patch Nähe angebracht war, mit dem Ziel ISWI in einer seltenen Konformation anzureichern. In dieser vorläufigen Studie zeigen wir, dass die Affinität von ISWI zum acidic patch mit der Länge der Linker-DNA und im ADPBeFx-gebundenen Zustand zunimmt. Die im Rahmen dieser Dissertation entwickelten Assays und diskutierten Konzepte könnten in Zukunft dazu dienen, neue Wege für Therapeutika eröffnen.In almost all eukaryotes, the DNA is organized in a dynamic polymer called chromatin. The nucleosome, the smallest unit a building monomer of chromatin, is formed by wrapping 146 bp of DNA around an octamer composed of histone proteins. Nucleosomes are interspaced with a piece of free DNA, called linker DNA. Nucleosomes tend to be evenly spaced, and this regular spacing is important for preventing cryptic transcription and protecting the genome from double-strand breaks. Nucleosome positions on DNA are influenced by ATP-dependent chromatin remodeling complexes. These remodelers can evict or assemble nucleosomes, incorporate his-tone variants and slide nucleosomes along DNA.
Chromatin can fold into higher order structures, both in vitro and in vivo. Nucleosome arrays also undergo phase separation and form chromatin condensates. Chromatin folding and phase separation put challenges on nucleosome remodelers that must act on it. In this thesis, I characterized nucleosome sliding in differently folded chromatin substrates.
In the first chapter, I used fission yeast as a model system to compare nucleosome sliding in euchromatin and the generally more compact heterochromatin using overexpression and heterochromatin targeting approaches. Targeting was achieved by fusion of chromatin remodelers with a heterochromatin-binding domain. Overexpression of the remodeler Hrp3 was toxic to fission yeast, independent of targeting. Hrp3 overexpression derepressed expression of a reporter gene placed into heterochromatin and caused defects in nucleosome positioning over gene bodies. Although the information obtained by short read sequencing for heterochromatin regions was sparse, we have identified an ATP-dependent increase in a regularity over subtelomeric regions. Moreover, we have successfully performed a gene-by-gene analysis to measure the regularity and repeat lengths of nucleosome arrays in wild type and remodeler-deletion strains. The most prevalent NRL turned out to be 150 bp, even tighter than published before.
In the second chapter, I tested if nucleosome array folding and phase separation impede nucleosome sliding in vitro. This study was performed with D. melanogaster ATPase ISWI, that slides nucleosomes on its own. I have reconstituted nucleosome arrays and induced intramolecular folding and phase separation by addition of varying amounts of salt. The chromatin condensates that formed contained nucleosome concentrations in the same range as the nucleus and were accessible to large complexes, making them a useful model substrate to study challenges encountered by remodelers in a crowded chromatin environment. ISWI was enriched inside chromatin condensates and it slowed down condensate fusion in a concentration-dependent manner. We have developed a novel, imaging-based nucleosome sliding assay, which allowed us to compare remodeling rates in- and outside of chromatin condensates. We confirmed that nucleosome sliding takes place inside chromatin condensates. The initial velocity for nucleosome sliding inside the condensates was only two-fold lower than in solution. Taken together, ISWI slides nucleosomes inside chromatin condensates and condensates do not pose a strong barrier for sliding. To characterize viscoelastic properties of chromatin condensates, we employed optical tweezers to fuse them in a controlled manner. Loss of ATP hydrolysis led to hardening of chromatin condensates and decreased dynamics of the remodeler. We rationalize our results with the help of a ‘monkey-bar’ model in which ISWI’s two DNA binding domains cycle between strong and weak binding modes, thereby ensuring mobility through the nucleus, where the high nucleosomes concentration well exceeds the dissociation constants. Our findings suggest that pathologies-associated phenotypes might be caused in part by changes in chromatin dynamics, and not exclusively by disruption of canonical remodeler functions.
In the third chapter, I investigated the interaction of ISWI and the acidic patch, which is important for its activation. Nucleosome remodelers are going through global conformational changes during nucleosome sliding, making structural analysis challenging. I used mononuclesomes with a UV-activating crosslinker close to the acidic patch that will covalently bind molecules nearby. In this preliminary study, we show that the affinity of ISWI towards the acidic patch increases with linker DNA length and in ADPBeFx bound state. Developed assays and discussed concepts in this dissertation might open new avenues for therapeutics
Autologe Chondrozytenimplantation
Get PDFIn this document I present my two publications. Both publications are about autologous chondrocyte implantation of the third generation (ACI). This technique of cartilage regeneration has become very popular and showed excellent results in the past years. However, this complex therapy method involves immense costs. In this regard, it is necessary to know all the influencing factors of the ACI, which can be at the indication making benefited of. We concentrated us on the influence of defect localization, defect size and the effect of previous microfracture therapy on the ACI afterwards. In our two studies, we analysed our knee patients with ACI NOVOCART® 3D done between 2004 and 2018. A matched pair analysis was carried out in both of our studies. Thanks to this method, it was possible to eliminate the influencing factors, which we did not look at. The goal of matching was to find for every patient from one group another patient from the second group with similar observable characteristics. The criteria for matching were in both paper similar. The criteria were age, defect localization, body mass index, number of defects treated or in the first paper also the intraoperatively measured absolute defect size. The exact surgical and then the rehabilitative procedures are explained in the publications under "surgical technique and rehabilitation". Clinical data were gathered by a standardized scheme. The preoperative clinical state was carried out together with the indication. Subsequently, the gathering of the clinical data was performed by our questionnaire 6,12,24 and 36 months after the surgery. The questionnaires included the subjective evaluation by the IKDC score as well as the visual analog scale for pain (VAS). Furthermore, the patient-specific and the defect-specific data were documented. The imaging examinations were done by the MRI and for the statistical analysis was SPSS program used. The specific statistical tests and models that brought us to the corresponding results are mentioned individually in the paper.
6.2. Results of Paper 1 ”Effect of defect size and localization of third generation autologous chondrocyte implantation in the knee joint"
The hypothesis of our first paper ”Effect of defect size and localization of third generation autologous chondrocyte implantation in the knee joint" was that defects at the patella and defects with a higher relative defect size lead to worst results. A matched pair analysis was carried out. There were 25 patellar and 25 femoral defects. The follow-up period lasted 3 years.
The mean age in the femoral group was 34.6 years (15-53). The group consisted of 11 men and 14 women. The mean intraoperative absolute defect size was 4.8 cm2 (2-15). The average body mass index (BMI) of 27.3 kg/m2 (20-36) was recorded. In the other group of patellar ACI was it 33.3 years (13-56), BMI of 26.3 kg/m2 (19-35), 10 men and 15 women and intraoperative defect size of 4.6 cm2 (2-12). Initially, MRI images were used to do the computer-assisted segmentation of the defect and of the whole cartilage layer. The clinical result was measured before the surgery and 6, 12, 24 and 36 months after the surgery. As for the clinical assessment there were used the IKDC and VAS scores. In both groups could IKDC and VAS provide a significant difference compared to the preoperative condition. After 3 years we noticed in the femoral group an improvement from 33.9 (SD 18.1) preoperatively to 71.5 (SD 17.4) in IKDC and from 6.9 (SD 2.9) to 2.4 (SD 2.5) in VAS. The second group of the patellar defects showed after 3 years an increase in the IKDC from 36.1 (SD 12.6) to 54.7 (SD 20.3) and an improvement in the VAS from 6.7 (SD 2.8) to 3.4 (SD 2). After 1-3 years postoperatively the femoral IKDC score was significant better (p <0.05) than the patellar group, which confirmed part of our hypothesis. With the data from the MRI segmentation we could calculate the relative defect size. The calculation was done by the ratio between the absolute defect size and the whole cartilage layer of the relevant knee. The result was calculated in percentage. A comparison between the relative defects revealed a significant difference between femoral (6.7%) and patellar group (18.9%). Consequently, it implies that although the absolute defect size in both groups was the same, the share of the defect on the cartilage layer of the patella was higher than femoral. However, according to our data neither the absolute nor the relative defect size has a significant impact on the outcome.
6.3. Conclusion of Paper 1 ”Effect of defect size and localization of third generation autologous chondrocyte implantation in the knee joint"
Thanks to our work, we were able to confirm that third generation of ACI offers benefits to the patients with cartilage defects. The patellar defects lead to a worse clinical result compared to the femoral defects. In terms of influence on the outcome the absolute and relative defect size showed itself as irrelevant. Despite this fact, our study was the first to look at the issue of the relative defect size and it would be worth further exploring this topic.
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6.4. Results of Paper 2 “Third-generation autologous chondrocyte implantation after failed bone marrow stimulation leads to inferior clinical results”
In our second published paper “Third‑generation autologous chondrocyte implantation after failed bone marrow stimulation leads to inferior clinical results” we observed the outcome of patients of the second line matrix-based ACI after failed microfracture therapy. The observation period was three years postoperatively. The hypothesis was that the second line ACI after unsuccessful microfracture provides inferior results compared to the first line ACI. Similar to the first paper, we did a matched pair analysis. We matched two groups with together 40 ACI patients. The first group of 20 patients represented first line ACI without pre-surgery or pre- treatment of the cartilage. The second matched group represented the second line ACI. It means the patients with a previous unsuccessful microfracture therapy. Clinical data collecting was carried out using IKDC and VAS scores. The first group had preoperatively an IKDC score of 37.0, which increased to 77.7 after two years. The postoperative IKDC increase was always statistically significant, comparing to preoperative values. Analysing the VAS at rest and in motion we noticed a significant improvement at all follow ups as well. In the second group was the subjective IKDC score preoperatively at 29.9 and then after six months at 44.3. After 12 months was it 50.1, as the further increase continued. A significant improvement compared to preoperative findings was observed at all times. The VAS begun at 6.8 in motion and 4.4 at rest. Both VAS scores improved significantly after 6 and 12 months, while the VAS at rest reached significant difference also after 3 years compared to preoperative results. When comparing our two groups in terms of IKDC and VAS in motion, a significant difference in favour of first line ACI was observed in all follow-ups. Similar was it in VAS at rest with only one exception after 6 months. The first line group of patients without previous microfracture treatment was always superior to the group of second line ACI. This confirmed our hypothesis.
6.5. Conclusion of Paper 2 “Third-generation autologous chondrocyte implantation after failed bone marrow stimulation leads to inferior clinical results”
The matrix-based ACI as the third generation of this procedure confirmed itself as an appropriate approach in the therapy of full cartilage defects. The benefits of this method were significantly proven for the patient with and without previous microfracture surgery. However, the ACI therapy after previous failed microfracture procedure was clearly inferior to first line ACI. It implies, that the ACI should be initially preferred instead of microfracture in the larger defects
Click chemistry for enhanced and emerging biological applications: from novel dyes for cell proliferation assays to mRNA-based vaccination
Get PDFThe present dissertation represents a focused endeavor aimed at broadening click-chemistry-based applications in the field of nucleic acids. The principal objective is to extend the synthetic utility of bioorthogonal click chemistry by delving into the following diverse, yet equally crucial areas of research: DNA fluorescent-labeling for bioimaging purposes and ligand development for nucleic acid cell targeting in emerging therapeutics. We aimed to develop new fluorescent probes to improve detection sensitivity and quality of imaging methods. To achieve this, we established a divergent synthetic pathway that produced ready-to-click, pH insensitive rhodamine dyes with outstanding brightness, which were proven to be a superior alternative in EdU cell proliferation assays. Furthermore, we prepared a group of fluorescein- and rhodamine-based multivalent dendrons by branching click chemistry, which exhibited self-quenching effects and demonstrated potential application as FRET quenchers. Additionally, in an effort to develop emerging therapeutics using click chemistry, particularly in vaccination and cancer immunotherapies, we synthesized three distinct ready-to-click mannose functionalized ligands with the aim of targeting dendritic cells (DCs). Our analyses confirmed the uptake of the ligands’ fluorescent conjugates into DCs through endocytosis. Finally, spurred by the COVID-19 pandemic, baseclick GmbH took on the task of developing a SARS-CoV-2 vaccine candidate. To this aim, we utilized our DC targeting ligands and baseclick’s mRNA labeling technology to successfully synthesize a mannose mRNA conjugate. At present, pharmacokinetics and pharmacodynamics of the vaccine are under investigation. The promising in vitro results, nevertheless, instills optimism in our efforts to establish a framework for future exploration of cell targeted, click-chemistry-based nucleic acid therapies
Essential properties for safe behaviour of a perception function in automated driving
Get PDFThe recent success of Machine Learning (ML) has led to the widespread application of ML in various domains, such as household products, natural language processing, and recommendation programs. Technological and computational advances and the increasing availability of data have fueled this trend. In this context, machine learning is expected to enhance the safety of autonomous safety-critical systems due to its compelling performance.
In a safety case, it shall be shown that a function is sufficiently safe, i.e., an acceptable residual risk is not exceeded. To this end, it should be demonstrated that certain properties that ensure safety are satisfied and that causes of failures are adequately mitigated. For this reason, the goal of the work is to extract and confirm properties of the ML-based function and the data that are essential to the safety argument. Thus, the work addresses the research question, what properties are necessary for a safe behaviour of an ML-based perception function, and how can we acquire them.
Thereby, the use of ML in safety-critical systems, such as perception functions in automated driving, comes with the challenge of providing a convincing safety argument. For example, ML-based perception functions process sensor data and extract information about objects and drivable areas. When perception fails, major damage and fatalities can result. Therefore, in complex environments that evolve over time, the risk of ML functions failing must be reduced to an acceptable level.
Established standards in the automotive industry such as functional safety ISO 26262 and safety of intended functionality ISO 21448 do not explicitly state how to ensure the safety of ML-based functions. In addition, established approaches recommended in the standards cannot be directly applied to ML components.
For this reason, we elaborate the challenges in validating perception functions.
On the one hand, the input space of an automated system and the intended functionality is very complex. On the other hand, formulating the specification of a perception function is a challenge in itself. Moreover, it is difficult to ensure the required properties hold over the entire input space and to verify and validate the implemented function with respect to the intended functionality.
Our focus is on the specification of the essential properties of a perception function for automated safety-critical systems and its realization, starting with design up to verification and validation. Only the two together, specification and realization, make it possible to develop a comprehensive safety argument. Specifically, the use case of a safety-critical pedestrian detection function for automated driving is investigated. For this purpose, a Deep Neural Network (DNN) for pedestrian detection is trained and its properties are investigated. In addition, novel methods are developed to satisfy its essential properties.
First, a set of safety requirements is derived and examined for their impact on the activities of the ML lifecycle.
In addition, functional insufficiencies are investigated as they might lead to hazards~\cite{ISSREW2020}. To this end, relevant data characteristics are extracted. Error categories are identified and remedial actions are proposed, focusing on the suitability of the training data. In addition to the approach to improve the training data, other measures are taken. When input data strongly differs from that used in training and test, its impact on the performance should also be analysed. In this case, we propose to complement data suitability with online anomaly detection that monitors the behaviour of the DNN.
To this end, we present two recent publications on anomaly detection. While FACER is trained to detect different types of noise that can distort the data, ReverseVAE is able to detect anomalies outside the distribution of training data. Both of these anomalies can have a large impact on the safe behaviour of an ML-based function. Another capability of ReverseVAE is the ability to manipulate the data with certain visual attributes. Thus, data can be generated, with defined visual attributes, which could later be used for training or testing of an ML-based function.
Since testing of an ML function cannot be guided only by the specification alone, we present different approaches to specifying a test oracle and testing approaches from different domains and application areas and novel test setups developed.
In order to address the challenges outlined above, an iterative and continuous specification of requirements in interaction with the development is proposed. To compensate for the traceability that is missing between the requirements and the lines of code in an ML-based function, we propose explicit artefact links and illustrate this with examples.
All in all, this work provides a holistic view on the research question of what properties are required for a safe behaviour of a ML-based perceptual function and how we can acquire them. This is intended to bridge the gap between already established safety practices applied to non ML-based systems and scientific knowedge in ML development
Strong-field physics in tailored light
Get PDFThis thesis reports on three distinct scenarios of strong tailored light fields interacting with matter in its gaseous and two-dimensional solid phases. Tailored light fields in the near- and mid-infrared (NIR, MIR) region facilitate precise control over electronic motion on an atomic level at atto- to femtosecond timescales.
The first scenario deals with high-harmonic generation (HHG), a source of coherent and ultra-short XUV light pulses. The obtained XUV spectra often require modification for various applications which are primarily carried out using highly absorptive filters or custom-designed dielectric mirrors which drastically reduce the flux of the XUV light. This thesis reports on a novel scheme enabling the suppression of individual harmonics of such an extreme ultraviolet (XUV) comb, simultaneously affecting specific even and odd orders in the high-harmonic spectra generated by strongly tailored, two-colour (ω-2ω), multi-cycle laser pulses in neon. Realistic macroscopic strong-field approximation calculations confirm the experimental observations and correlate the effect to the use of symmetry-broken laser fields. Semi-classical calculations further corroborate the effect and reveal their underlying mechanism, where a nontrivial spectral interference between subsequent asymmetric half-cycles is found to be responsible for the suppression. This scheme particularly benefits future molecular time-resolved spectroscopy studies relying on narrow-band XUV light excitation around different photon energies with high flux.
The second scenario deals with isolated soft-X-ray attosecond pulses generated via HHG in neon and driven by polarization-tailored few-cycle MIR pulses, commonly referred to as polarization gating. Accurate estimation of the attosecond soft-X-ray pulses in such experiments remains challenging given their higher photon energies and broad spectral bandwidth. Numerical results, based on strong-field approximation, are presented in this thesis to estimate the soft-X-ray pulse durations achievable using realistic polarization-gated MIR laser pulse parameters including macroscopic propagation effects. The intrinsic dispersion (attochirp) of such a pulse in the calculations is compensated by traversing it through a 90 cm plasma column after which a pulse duration of 110 as is obtained while the Fourier limit lay at 31 as. This
theoretical estimate serves as a reference in judging the accuracy of new retrieval techniques while also highlighting the need for new compression schemes which compensate for higher-order dispersion in the soft-X-ray region.
The third and final scenario presented in this thesis deals with using strong and far-off-resonant trefoil-shaped tailored light fields to alter time-reversal symmetry and induce valley selective bandgap modification (K and K' points in momentum space). The material chosen is monolayer hBN, an inversion-symmetry broken two-dimensional insulator. Such a scheme has only been predicted theoretically but not yet experimentally realized. Here, a novel apparatus is developed to produce trefoil light fields by combining two-colour (ω-2ω) counter-rotating pulses. Further, a controlled delay between the colours induces a rotation w.r.t. the hBN lattice, dynamically altering the band-gap and thereby electron population at each valley. A third linearly polarized pulse is then used to probe the induced valley polarization through a correlated change in the detected helicity of its intraband harmonics (third harmonic). The results presented here open further avenues in the direction of ultra-fast band engineering at petahertz frequencies which could revolutionize the future of electronics
Metagenomic analysis of ancient human and microbial DNA preserved under unusual taphonomic conditions
Get PDFIn this dissertation, I analyzed different types of ancient organic and inorganic materials preserved under different taphonomic conditions from different time points (~2900-235 BP). During the analysis, I tried to improve the currently used in situ, in vitro, and in silico methods of ancient DNA analysis. The results of this dissertation are summarized and published in three research articles.
In the first article, we analyzed skeletal remains of a late Bronze Age individual found in the Wimsener water caves in Germany. In addition to the skeletal remains, we analyzed the calcite deposits, found surrounding the bones of the individuals, from which we were able to retrieve ancient human DNA fragments, enough to reconstruct the full mitochondrial genome and to assign molecular sex to the individual. We demonstrated the ancient human and microbial DNA fragments diffused from the bones to the calcite stone deposits in the same direction of gravity. This study exemplifies using alternative source for obtaining ancient human and/or microbial DNA without causing destruction to the valuable archeological finding.
In the second article, we analyzed different paleofeces specimens from Hallstatt mines, Austria (dated to the Bronze Age – the Baroque times). We subjected them to microscopic, proteomic, and metagenomic analyses. The collective analysis allowed unveiling the following: i) the molecular sex and the mitochondrial haplogroups of the individuals; ii) consumption of fibrous plant-based diet as well as animal components; iii) non-Westernized gut microbiome composition until the Baroque times; iv) presence of gut parasites; and finally, v) consumption of fermented food (cheese-like) and beverages (beer). During this study, we reconstructed, for the first time, complete ancient fungal genomes of Saccharomyces cerevisiae and Penicillium roqueforti, and by comparative genomic analyses, we presented different lines of evidence on their being used in beer and cheese fermentation, respectively. The study presented a comprehensive interdisciplinary workflow for the analysis of such precious archeological materials.
In the third article, we analyzed different tissue specimens from the mummy of Anna Catharina Bischoff (ACB), from Basel, Switzerland. Initially, we aimed to find any molecular proof of presence of the syphilis-causing bacterium Treponema pallidum, which was not successful. However, by employing de novo metagenomic assembly, we were able to reconstruct a complete genome of a pathogen from the brain sample, belonging to the Mycobacteriaceae family. The genome analysis of the de novo detected pathogen supports the assumption of its pathogenicity and was very congruent with the radiological symptoms and its survival in the brain under high concentrations of mercury. The study presented a proof-of-concept on using metagenomic assembly to detect extinct or previously undescribed pathogens.
Overall, throughout this dissertation, I tried to use different analytical methods beyond what is already known and commonly used in the field of ancient DNA
