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Age is the single biggest risk factor for most major human diseases. As such, understanding the intricate molecular changes that drive biological aging holds great promise in attempting to slow
the onset of systemic diseases and thereby increase the effective health-span in modern societies.
This thesis explores several computational approaches to capture and analyze the molecular biological alterations triggered by intrinsic and extrinsic aging using skin as a model tissue to deliver genes and pathways as potential targets for intervention strategies.
Publication 1 demonstrates the utility of multi-omics data integration strategies for aging research, leading to the identification of four latent aging phases in skin tissue through an integrated cluster analysis of gene expression and DNA methylation data. The four phases improved the detection of molecular aging signals and were shown to be associated with sunbathing habits of the test subjects. Deeper analysis revealed extensive non-linear alterations in various biological pathways particularly at the transition into the fourth aging phase, coinciding with menopause, with potentially wide-reaching functional implications. Publication 2 describes the development of a novel type of age clock, that provides a new level of interpretability by embedding biological pathway information in the architecture of an artificial neural network. The clock not only generates meaningful biological age estimates from gene expression data, but further allows simultaneous monitoring of the aging states of various biological processes through the activations of intermediate neurons. Analyses of the inner workings of the clock revealed a wide-spread impact of aging on the global pathway landscape. Simulation experiments using the transcriptomic clock recapitulated known functional aging gene associations and allowed deciphering of the pathways by which accelerated aging conditions such as chronic sun exposure and Hutchinson-Gilford progeria syndrome exert their effects. Publication 3 further explores the molecular alterations caused by the pro-aging effector UV irradiation in the skin. The multi-omics data analysis of repetitively irradiated skin revealed signs of the immediate acquisition of aging- and cancer-related epigenetic signatures and concurrent wide-spread transcriptional changes across various biological processes. Investigations into the varying resilience to irradiation between subjects revealed prognostic biomarker signatures capable of predicting individual UV tolerances, with accuracies far surpassing the traditional Fitzpatrick classification scheme. Further analysis of the transcripts and pathways associated with UV tolerance identified a form of melanin-independent DNA damage protection in individuals with higher innate UV resilience.
Together, the approaches and findings described in this thesis explore several new angles to advance our understanding of aging processes and external drivers of aging such as UV irradiation in the human skin and deliver new insight on target genes and pathways involved.
The respiratory epithelium acts as both, a barrier of the respiratory tract to Nipah virus (NiV) entry and at the same time as a significant determinant of virus shedding. Both, for humans and pigs, replication in the respiratory tract epithelia is considered a major factor in transmission to other hosts. To understand why the virus constitutes a high-risk pathogen for livestock and humans, knowledge about
viral replication and host responses in relevant cells and tissues is crucial. Most in vitro studies, however, have been performed in conventional cell lines or non-differentiated lung cells. Only a few examples exist where Henipavirus infections have been investigated in fully-differentiated lung
epithelial cell models.
Thus, one aim of this thesis was to investigate infection, replication, spread and host protein dynamics of NiV in primary bronchial epithelial cells (BEC) cultivated at the air-liquid-interphase (ALI). By
immunofluorescence imaging, the NiV infection dynamics in BEC-ALI cultures were monitored over a 12 day time course, in order to provide detailed information about the infection process in the
respiratory epithelium of pigs and ferrets. Compared to undifferentiated primary BEC, the specific infectivity of NiV in BEC-ALI cultures was low. Infections remained focal and complete infection of the
cultures was not observed, even at 12 dpi. Analysis of viral titers and viral mRNA indicated a limited
virion release from the infected ALI-cultures while most of the newly synthesized NiV-RNA remained
cell associated. Immunofluorescence analysis of cross sections from infected ALI-cultures revealed
large infected areas that exhibited a strong cytopathic effect (CPE). Disruption of the epithelium
resulted in apical release of virus antigen-positive cell detritus while ciliated areas and basal cells were
less affected. From these data it was concluded, that NiV transmission could be supported by
exhalation of cell debris associated NiV and thus may contribute to rapid spread of infection in swine
populations.
A second aim was to explore the dynamics of host responses to NiV infection in differentiated BEC-ALI
culture and to assess whether this differs to conventional cell line data available from literature. Even
though strong CPE appeared in later phases of NiV infection, at least the porcine PBEC-ALI cultures
remained robust enough to allow protein sampling over 12 days infection course. Subsequent MS-based proteomics enabled unprecedent insight in complex cell culture response upon NiV infection.
Previous reports indicated a lack of efficient interferon type I induction in non-differentiated pig or
human BEC which were considered a prerequisite for efficient replication in the respiratory epithelium
and virusspread. In contrast to non-differentiated pig BEC (PBEC), in PBEC-ALI cultures multiple factors
involved in interferon responses were upregulated upon NiV infection. Thereby it was demonstrated
that NiV infection induced a robust innate immune response upon infection with elevated components of antigen processing and presentation resulting in the conversion from the constitutive proteasome to the immunoproteasome. In contrast to previous reports about NiV-infected non-differentiated
PBEC or endothelial cells, incomplete immunoproteasome formation and limitations in interferon
response could be excluded. Thus, a model is proposed in which NiV infection and spread in differentiated PBECs is slowed by potent innate immune responses to the virus infection. Overall, the
findings highlight the important role of the respiratory epithelium not only as a physical barrier to virus
infections but also indicate itsrole as a primary site of adaptive immune induction through NiV induced
antigen processing and MHC I presentation.
Finally, to allow functional studies of Henipaviruses at the BSL-2 biosafety level a recombinant CedPV
was generated and rescued. An imaging based screening and quantitative analysis pipeline was established to investigate the role of cellular factors and to screen for potential virus and host gene
directed inhibitory factors. Accordingly, different host and viral genes were targeted with a siRNA-pool
either targeting virus or selected cellular mRNAs followed by the infection with the CedPV and the
quantification of infected cells. With proof of concept of the siRNA screening pipeline, the recombinant
CedPV clone was used as a backbone to insert variousfluorescence reporter genesin order to optimize
the analysis workflow by allowing direct virus quantification in live, unstained samples. Consequently,
this thesis provides a valuable proof for future approaches related to the function of virus proteins,
influence of host-factors and virusreplication and Henipavirus-inhibitorscreens at low biosafety levels.
The role of cell-penetrating peptides in the induction of T cell responses by virus-like particles
(2023)
Many viral structural proteins can self-assemble into virus-like particles (VLPs). VLPs can serve as an effective vaccine or be used as a vaccine platform. One of these structural proteins is the hepatitis B virus core antigen (HBcAg), which appears to be suitable as an antigen carrier due to its high immunogenicity. HBcAg has a major immunodominant region (MIR) that is presented on the surface of the VLPs after self-assembly. Foreign antigens can be inserted into this region. Since HBcAg VLPs, unlike the Hepatitis B virus (HBV), do not have an envelope, they are not able to penetrate cell membranes efficiently. As an extracellular antigen, HBcAg VLPs primarily induce a strong humoral immune response.
In the present study, we investigated the extent to which HBcAg can be modified to also elicit an enhanced cellular, particularly a cytotoxic, immune response. A cytotoxic CD8+ T cell response is predominantly induced by intracellular antigens. Therefore, our goal was to increase the cell penetration capacity of VLPs. We aimed to achieve this by fusing cell-penetrating peptides (CPPs) to HBcAg. CPPs can spontaneously penetrate cell membranes to enter the cytoplasm of cells. To guarantee that the CCPs were localized to the surface of the VLPs, we fused CPPs to the N-terminus of HBcAg. The CCPs were followed by a tag to allow the purification of VLPs. The T cell epitopes, against which the induced CTL should be directed, were derived from the Large T antigen and inserted into the MIR of HBcAg. Finally, we fused fluorescent proteins to the C-terminus of HBcAg to track the entry of VLPs into cells.
Modifications of HBcAg may lead to reduced stability or altered structure of VLPs. To analyze the stability of VLPs, we used nanoscale differential scanning fluorimetry (nanoDSF) analysis. This revealed that the N-terminal fusion of CPPs or the tag to HBcAg does not reduce VLP stability. However, some peptides incorporated into the MIR had a significant effect on the structure and stability of the VLPs. While the incorporation of a Flag-tag or a peptide from ovalbumin had no negative effect on VLP stability, the incorporation of peptides representing T cell epitopes of Large T antigen interfered with VLP formation. Denaturation and reassembly of the aggregates significantly improved the homogeneity of the VLPs, and the C-terminal addition of arginine-rich domains enhanced stability.
Using live cell imaging and flow cytometry, we demonstrated that HBcAg VLPs functionalized with CPP exhibited up to 40% more efficient penetration into professional antigen-presenting cells (JAWS II) than HBcAg VLPs without CPP. This resulted in the increased presentation of integrated T cell epitopes by dendritic cells. In vivo, we detected significantly increased induction of SV40 Large T antigen-specific CTL in mice immunized with CPP-conjugated VLPs compared to unconjugated VLPs.
In this study, we demonstrated that a stronger cellular immune response can be induced by CPP-functionalized HBcAg VLPs than with the unmodified HBcAg VLPs in vitro as well as in vivo. This discovery may have positive implications for future vaccine development where an enhanced cellular component of the immune response is desirable.
As the animal-to-human interface becomes increasingly narrow, transmission events of zoonotic pathogens between animals and humans become more and more probable. While SARS-CoV-2 already accomplished a spillover infection to humans and is responsible for the current pandemic, the bat H9N2 IAV with so far unknown zoonotic potential was only recently discovered. In order to identify I) the role and potential of a newly discovered, potentially pre-pandemic virus, such as the bat H9N2, or II) possible future prevailing virus mutant variants of an already known pandemic virus, such as SARS-CoV-2, it is important to characterize these emerging viruses in vivo as soon and as good as possible.
The first objective in this dissertation (Publications I and II) therefore deals with the characterization of bat H9N2 and the estimation of its zoonotic or even pandemic potential.
In Publication I, a general susceptibility of directly inoculated Egyptian fruit bats to bat H9N2 was confirmed by successful seroconversion, although exhibiting only moderate viral shedding. All three contact animals remained seronegative, though one contact bat showed slight lesions in the histopathological analysis.
Publication II further addressed the question of the zoonotic potential of this virus. Inoculation of day-old turkey hatchlings demonstrated moderate susceptibility to bat H9N2 infection with a measurable seroconversion, while day-old chicken hatchlings were not susceptible to bat H9N2. Ferrets proved to be highly susceptible to bat H9N2 with high viral shedding, a transmission efficiency rate of 100% to direct contact animals at 2 days post contact, but with only minimal clinical signs. Importantly, the virus demonstrated the ability to evade the MxA-restriction factor and to replicate efficiently in human lung tissue explants. Furthermore, seasonal IAV- and standard IAV-vaccines showed no cross reactivity against the bat-N2 protein in humans. Therefore, further research on such viruses is urgently needed in order to prevent a renewed pandemic situation in the future as caused by SARS-CoV-2.
The second objective in this dissertation dealt with the identification and characterization of emerging SARS-CoV-2 Variants of Concern (VOCs).
Therefore, in Publication III, competitive infection experiments were performed using the Syrian golden hamster, the ferret, and transgenic mouse models (K18-hACE2 and hACE2-KI). These studies revealed replicative and transmissive predominance of Alpha VOC over Beta VOC, but not over SARS-CoV-2 WT in the hamster model, although Beta VOC substantially replicated in the lungs of donor animals. In contrast, the Alpha VOC had an unambiguous replication and transmission advantage over WT SARS-CoV-2 in the ferret and both mouse models. A recombinant SARS-CoV-2 WT-SAlpha virus helped to assign the fitness advantage of this variant particularly to the spike protein-associated mutations.
In Publication IV, in vitro results inferred an early replicative fitness advantage of Omicron BA.1 over Delta VOC, although the opposite was observed in competitively inoculated hamsters, ferrets and naive hACE2-KI mice. In addition, Publication IV demonstrated a disadvantage in transmission for the VOC Omicron BA.1 over the Delta VOC and a lack of susceptibility of ferrets after a single infection with the VOC Omicron BA.1. An mRNA vaccination of K18-hACE2 mice caused a drastic reduction of infectious virus particles in organ material following an infection with a recombinant SARS-CoV-2 WT-SDelta, but not when challenged with the SARS-CoV-2 SOmicron BA.1 clone.
This dissertation includes numerous, comprehensive experimental studies that are generally important for the characterization of emerging, potentially pre-pandemic viruses and may provide crucial information about the future dominance of certain virus variants in an ongoing pandemic. Here, the need for the use of a variety of animal models becomes apparent. By characterizing and classifying potentially zoonotic strains, these methods will help to better prepare for potentially upcoming pandemics and, in the case of a zoonotic or even pandemic event, to better detect and understand the circulating strains and their evolution.
Emerging infectious diseases are among the greatest threats to human, animal and plant health as well as to global biodiversity. They often arise following the human-mediated transport of a pathogen beyond its natural geographic range, where host species are typically not well adapted due to a lack of co-evolutionary host-pathogen dynamics. One such pathogen is the fungus Pseudogymnoascus destructans (Pd), which causes White-Nose disease in hibernating bats. While Pd was first observed in North America where it has led to mass-mortalities in some bat species, the pathogen originates from Eurasia where infection is not associated with mortality. Most of the Pd research has focused on the invasive North American range, which likely underestimated the genetic structure of the pathogen and the role it might play in the disease dynamics.
In my work, I therefore evaluated the genetic structure of Pd in its native range with the aim of uncovering cryptic diversity and further use population genetic data to address some key ecological aspects of the disease dynamics. With an extensive reference collection of more than 5,000 isolates from 27 countries I first demonstrated strong differentiation between two monophyletic clades across several genetic measures (multi-locus genotypes, full genome long-read sequencing and Illumina NovaSeq on isolate pools). These findings are consistent with the presence of two cryptic species which are both causative agents of bat White-Nose disease (‘Pd-1’, which corresponds to P. destructans sensu stricto, and ‘Pd-2’). Both species exist in the same geographic range and co-occur in the same hibernacula (i.e., in sympatry), though with specialised host preferences. I further described the fine-scale population structure in Eurasia which revealed that most genotypes are unique to single hibernacula (more than 95% of genotypes). The associated differences in microsatellite allele frequencies among hibernacula allowed the use of assignment methods to assign the North American isolates (exclusively Pd-1) to regions in Eurasia. Hence, a region in Ukraine (Podilia) is the most likely origin of the North American introduction.
To gain further insights into the spatial and temporal dynamics of White-Nose disease on a localised scale, several hibernacula were sampled with high intensity (artificial hibernaculum in Germany and natural karst caves in Bulgaria). Low rates of Pd gene flow were observed even among closely situated hibernacula. This indicates that Pd does not remain viable on bats over summer or it would be frequently exchanged among bats (and hence hibernacula) resulting in a homogenous distribution of genotypes. Instead, bats need to become re-infected each hibernation season to explain the yearly re-occurrence of White-Nose disease. Given the distribution and richness of Pd genotypes on hibrnacula walls and infected bats of the same hibernacula, bats become infected from the hibernacula walls when they return after summer. This means that environmental reservoirs exist within hibernacula (i.e., the walls) on which Pd spores persist during bat absence and which drive the yearly re-occurrence of White-Nose disease. In an experimental setup, I confirmed the long-term viability of Pd spores on abiotic substrate for at least two years and furthermore discovered temporal variations in Pd spores’ ability to germinate. In fact, these variations followed a seasonal pattern consistent with the timing of bats absence (reduced germination) and presence (increased germination) and could indicate adaptations of Pd to the bats’ life-cycle. The infection of bats from environmental reservoirs hence seems to be a central aspect of White-Nose disease dynamics and Pd biology.
Pds ability to remain viable for extended periods outside the host increases its risk of being anthropogenically transported and might have played a role in the emergence of White-Nose disease in North America. The existence of a second species (Pd-2) poses a great additional danger to North American bats considering that its introduction there could lead to deaths and associated population declines in so-far unaffected species given what is known about differing host species preferences in Eurasian bats. Even within the native range of Pd, the movement of Pd between differentiated fungal populations could facilitate genetic exchanges (e.g., through sexual reproduction) between genetically distant genotypes. Such genetic exchanges could lead to phenotypic jumps in pathogenicity or host-species preferences and should hence be prevented.
The native range of a pathogen holds great potential to better understand the genetic and ecological basis of a (wildlife) disease. My work informs about the dangers associated with the accidental transport of Pd (and other pathogens) and highlights the need for ‘prezootic’ biosecurity-oriented strategies to prevent disease outbreaks globally. Once a pathogen has arrived in a new geographic range, and particularly if it has environmentally durable spores (as demonstrated for Pd), it will be difficult/impossible to eradicate. Furthermore, a pathogen’s ability to remain viable outside the host and infect them from environmental reservoirs has been associated with an increased risk of species extinctions and needs to be considered when designing management strategies to mitigate disease impact.
In den letzten Jahren gewannen ω-Transaminasen zunehmend an Bedeutung. Ihr breites Substratspektrum, das sowohl Aminosäuren als auch Amine umfasst, macht sie interessant für biotechnologische Anwendungen. Im Gegensatz zu α-Aminotransferasen sind ω-Aminotransferasen nicht auf α-Aminosäuren als Aminodonor bzw. α-Ketosäuren als Aminoakzeptoren beschränkt. Auch sind einige ω-Transaminasen in der Lage, Aldehyde oder Ketone zu aminieren. Dadurch sind sie vielseitig einsetzbar. Seit ihrer Entdeckung wurden ω-Transaminasen in einer Vielzahl von Organismen nachgewiesen. Viele dieser Enzyme stammen aus Pilzen und Bakterien. Da es ständig Bedarf an neuen Transaminasen gibt, wurden verschiedene Organismen auf das Vorhandensein solcher Enzyme untersucht. Die Hefe Blastobotrys raffinosifermentans LS3 ist einer dieser Organismen. Für diese Hefe existiert bereits eine Vielzahl biotechnologischer Anwendungen, was unter anderem an ihren vielseitigen physiologischen Möglichkeiten liegt. Um das Spektrum dieses Stammes noch zu erweitern, wurde sein Genom auf ORFs gescannt. Die ermittelten ORFs wurden translatiert und die so erhaltenen, theoretischen Proteine in einer Proteindatenbank gespeichert. Die Einträge dieser Datenbank wurden einem „hmmerscan“ (hmm ist kurz für „hidden Markov model“) unterzogen. Dabei werden die Proteine in sogenannte Pfams, kurz für Proteinfamilien, eingeteilt. Drei Proteine wurden der Familie PF00202.21 zugeordnet. Das ist die sogenannte Aminotran_3 Familie. In dieser Familie befinden sich eukaryotische ω-Transaminasen. Die Gene brota1, brota2 und brota3 codieren für diese Enzyme. Jeweils eins der Gene wurde in den Vektor XPLOR®3 kloniert, damit die potentiellen ω-Transaminasen in B. raffinosifermentans G1212 [aleu2 atrp1:ALEU2] [1] überexprimiert werden können. Eine Besonderheit von BroTA1 ist, dass es neben der Aminotran_3 Domäne noch eine AAA Domäne aufweist. Deshalb ist es mit etwa 85 kDa auch deutlich größer als die meisten ω-Transaminasen, die meist zwischen 45 und 50 kDa liegen. BroTA2 und BroTA3 beinhalten nur die Aminotran_3 Domäne. Alle drei Enzyme zeigen niedrige Aktivität bei der kinetischen Auflösung racemischer β-Aminosäuren.
Neben den eukaryotischen ω-Transaminasen wurden auch einige bakterielle Enzyme untersucht. Literatursuche und das Screenen der Stammsammlung der Arbeitsgruppe Hefegenetik des Leibniz-Instituts für Pflanzengenetik und Kulturpflanzenforschung führten zu mehreren potentiellen bakteriellen ω-Transaminasen. Das zu Beginn dieser Arbeit noch als hypothetisches Protein bezeichnete Enzym von Variovorax boronicumulans hat sich als ω-Transaminase herausgestellt. Das Enzym wurde detailliert hinsichtlich des Substratspektrums und seiner biochemischen Eigenschaften charakterisiert. Es handelt sich hierbei um eine ω-Transaminase mit β-Aktivität. Diese Transaminase akzeptiert sowohl aromatische als auch aliphatische β-Aminosäuren als Substrat. Sequenzvergleiche dieses Enzyms mit anderen ω-Transaminasen, die nur aliphatische Aminosäuren akzeptieren, führten zu tieferen Einblicken in konservierte Bereiche dieser beiden Gruppen von ω-Transaminasen.
Der dritte Ansatz war das Anpassen einer bekannten ω-Transaminase des thermophilen Bakteriums Sphaerobacter thermophilus an ein potentielles Motiv für aromatische ω-Transaminasen. Dadurch sollte die Aktivität des Enzyms erhöht werden. Dieser Ansatz führte zu 7 Varianten des Enzyms mit höherer Aktivität als der Wildtyp. Durch diese Versuche wurden einige für die Transferaseaktivität wichtige Aminosäurereste offenbart. So hat sich zum Beispiel herausgestellt, dass N70 offenbar wichtig für den Umsatz von γ-Aminosäuren ist, da ein Austausch gegen Glutamat zu einer verminderten Aktivität mit γ-Aminopentansäure führte.
Zerebrale kavernöse Malformationen (CCMs) sind Gefäßfehlbildungen im Gehirn oder Rückenmark und können sich klinisch aufgrund einer erhöhten Blutungsbereitschaft mit Kopfschmerzen, Gefühls- und Sprachstörungen bis hin zu Krampfanfällen äußern. Sie treten sporadisch oder im Rahmen einer autosomal-dominant erblichen Form auf. Kausale Sequenzveränderungen sind dabei in den drei Genen CCM1, CCM2 und CCM3 bekannt. Die Detektionsrate für pathogene Varianten ist mit bis zu 60 % für sporadische Fälle und mit weit über 90 % für familiäre Fälle sehr hoch. Während Genpanel-Analysen sehr verlässlich Einzelnukleotidveränderungen, kleine Insertions- und Deletionsvarianten sowie Kopienzahlveränderungen detektieren können, werden komplexe Strukturvarianten oder Veränderungen in nicht-kodierenden Regionen kaum erfasst. Diese rücken jedoch für die bisher genetisch unaufgeklärten Fälle immer mehr in den Fokus des Interesses. Diese Arbeit adressiert daher zum einen die Identifizierung neuer Strukturvarianten und deren funktionale Interpretation im Kontext der CCM-Erkrankung.
Im Rahmen der vorliegenden Arbeit ist der erstmalige Nachweis einer interchromosomalen Insertion bei einem CCM-Patienten gelungen. Die unbalancierte Insertion genomischen Materials von Chromosom 1 in die kodierende Region des CCM2-Gens konnte durch die Verbindung von bioinformatischen Auswertestrategien der Next Generation Sequencing-Genpanel-Daten, molekularzytogenetischen Analysen und einer molekularen Bruchpunktkartierung genau charakterisiert werden. Die Identifikation einer weiteren Strukturvariante, einer Deletion des Transkriptionsstarts von CCM1, verdeutlichte die Herausforderungen bei der Bewertung von Veränderungen in nicht-kodierenden Genbereichen. Für eine eindeutige Klassifikation der Variante wurden daher funktionale Analysen durchgeführt, die auf einer CRISPR/Cas9-vermittelten Nachbildung der Deletion in iPSCs und der anschließenden Differenzierung in Endothelzellen beruhte. Damit konnte gezeigt werden, dass die Deletion zu einem Verlust der CCM1 mRNA- und Proteinexpression führt. Zudem wurde in den differenzierten Endothelzellen eine für die CCM-Pathogenese charakteristische Deregulation von KLF2, THBS1, NOS3 und HEY2 beobachtet. Schließlich war es auf Basis dieser in vitro-Analysen möglich, die Variante entsprechend den ACMG-Richtlinien als wahrscheinlich pathogen zu bewerten und somit die molekulare CCM-Diagnose zu sichern.
Die Verbindung des CRISPR/Cas9-Systems mit iPSCs ist nicht nur für die Variantenbewertung von großem Nutzen, sondern bietet auch das Potential zum besseren Verständnis von Krankheitsmechanismen. Ein weiterer Fokus der vorliegenden Arbeit lag daher auf der Etablierung und Verwendung iPSC-basierter Zellkulturmodelle für die CCM-Modellierung. Zunächst ist es gelungen, mehrere iPSC-Linien mit einer kompletten CRISPR/Cas9-vermittelten CCM1-, CCM2- oder CCM3-Inaktivierung zu generieren. Diese wurden anschließend für die Differenzierung in hBMEC-ähnliche Zellen und innovative dreidimensionale vaskuläre Organoide verwendet. In diesen Systemen konnte beispielsweise eindrücklich eine tumorähnliche Proliferation CCM3-defizienter Endothelzellen nachvollzogen werden, die nur in Kontakt mit Wildtyp-Zellen auftrat. RNA-Sequenzierungen in einem CCM1-basierten Knockout-Modell konnten darüber hinaus die Rolle von CCM1 als Endothel-spezifisches Suppressorgen stärken. Die im Rahmen der Arbeit etablierten Systeme werden zukünftig für weitere Fragestellungen der CCM-Pathogenese wie der endothelialen Barrierestörung eingesetzt und stellen darüber hinaus sehr gut geeignete Plattformen für die effektive Entwicklung dringend benötigter therapeutischer Ansätze dar.
Posttranslational modifications are involved in the regulation of virtually all cellular processes, including immune response, nevertheless, they are also targets manipulated by invading pathogens. The first investigated example is protein citrullination which is an important posttranslational modification that acts on a multitude of processes like supervision of cell pluripotency and rheumatoid arthritis. Citrullination of targeted arginine residues is performed by the Peptidylarginine deiminase. Within the first published manuscript, being part of this thesis, it was possible to show the use of this posttranslational modification by the human pathogen Porphyromonas gingivalis to facilitate innate immune evasion at three distinct level. P. gingivalis was demonstrated to citrullinate proteins by Porphyromonas peptidylarginine deiminase resulting in diminished phagocytosis and subsequent killing by neutrophils. Furthermore, it was shown that citrullination of histone H3 enables P. gingivalis to survive in neutrophil extracellular traps and incapacitate the lysozyme-derived peptide LP9.
The second investigated posttranslational modification is ubiquitination and its role in respiratory tract infections. Ubiquitination is the covalent attachment of a small protein that consisting of only 76 amino acids to the ε-amino group of lysine residues to posttranslational modify proteins. Acute infections of the lower respiratory tract such as viral and bacterial co-infections are among the most prevalent reasons of fatal casualties worldwide. Therefore, the interactions between host and pathogens resulting in the impairment of the hosts immune response and immune evasion of the pathogens, need to be elucidated. To get new insights in the infection driven changes in protein polyubiquitination and alterations in the abundance of ubiquitin E3 ligases involved in ubiquitination, cellular proteomes were monitored in detail by high resolution mass spectrometry. Therefore, the epithelial cell lines 16HBE14o- (Manuscript II) and A549 (Manuscript III) were co-infected with influenza A virus H1N1 and Streptococcus pyogenes or Staphylococcus aureus or with influenza A virus H1N1 and Streptococcus pneumoniae, respectively. Here, it could be shown in 16HBE14o- cells that co-infection of epithelial cells is not characterized by decreased cell survival and that observable effects on the proteome and ubiquitinome are mostly additive rather than synergistic. S. pyogenes infection affected the mitochondrial function, cell-cell adhesion, endocytosis and actin organization. Viral infection affected mRNA processing and Rho signaling. Viral and bacterial co-infection was detected to affect processes that were already affected by both of the corresponding single infections. No further pathways were strongly affected by the co-infection. A similar result has been observed in A549 cells co-infected IAV and S. pneumoniae. Overrepresented gene ontology terms depict the sum of those observed in the viral and bacterial single infection. Moreover, no significant change in cell survival upon co-infection compared to single bacterial infection was noticed for A549 cells either. This led to the suggestion that co-infection of investigated epithelial cells under examined conditions possesses additive rather than synergistic effect and thus, may not worsen the outcome of the infection within the studied conditions. Infections in other systems, may provide varying results and thus should be examined in future studies.
Amid the current global biodiversity crisis, being able to accurately monitor the changing state of biodiversity is essential for successful conservation actions and policy. Despite the pressing need for reliable and cost-effective monitoring methods, collecting such data remains extremely difficult for elusive species, such as temperate zone bats. Although bats are important indicators of environmental changes, monitoring bat populations is challenging because they are nocturnal, volant, small, and highly sensitive to human activities and disturbance. Thus far, population trends of temperate zone bats have been mainly based on visual surveys, including winter hibernation counts at underground sites. However, as bats may not always be roosting in visible locations within the hibernacula, it is currently unknown how these estimates relate to actual population sizes.
Infrared light barriers combined with camera traps are a novel method to monitor bats at underground sites. When installed at the entrance of hibernacula, infrared light barriers have the potential to estimate site-level population sizes more accurately than visual surveys, by counting all bats flying in and out of the site. Moreover, camera traps, consisting of a digital camera and white flash, can be used for species-level identification. However, for this new method to be applicable as a large-scale bat monitoring technique, it is important to characterize it with regard to three main criteria: is the method minimally invasive, is it accurate, and is it scalable in terms of spatial and temporal resolution? Therefore, the purpose of this thesis was to investigate the invasiveness and accuracy of this novel bat monitoring method, and to develop standardized and automated data analysis pipelines, both for the light barrier and camera trap data, to support the deployment of this method at scale.
In Publication I, we used light barrier data, infrared video recordings and acoustic data from an experimental field study to investigate whether the white flash of the camera trap has any measurable short- or long-term effect on bat activity and behavior. The flash of the camera trap was turned on and off every week at each site, which allowed us to compare the activity and behavior of bats between flash-on and flash-off nights. We found that despite the high sensitivity of bats to disturbance, they did not change their nightly activity patterns, flight direction, echolocation behavior, or long-term site use in response to the white flash of the camera trap. Based on these results, we concluded that camera traps using a white flash are a minimally invasive method for monitoring bat populations at hibernacula, providing high quality images that allows species-level identification.
In Publication II, we used infrared video surveillance to quantify the accuracy of infrared light barriers, and we described a standardized methodology to estimate population sizes and trends of hibernating bat assemblages using light barrier data. We showed that light barrier accuracy varies based on the model and location of the installation relative to the entrance, with the best combination achieving nearly perfect accuracy over the spring emergence phase. When compared to light barrier-based estimates, we found that visual counts markedly underestimated population sizes, recovering less than 10% of the bats at the most complex hibernacula. Moreover, light barrier-based population trends showed regional patterns of growth and decline that were not detectable using the visual count data. Overall, we established that the light barrier data can be used to estimate the population size and trends of hibernating bat assemblages with unprecedented accuracy and in a standardized way.
In Publication III, we described a deep learning-based tool, BatNet, that can accurately and efficiently identify bat species from camera trap images. The baseline model was trained to identify 13 European bat species or species complexes using camera trap images collected at 32 hibernation sites (i.e., trained sites). We showed that the baseline model performance was very high across all 13 bat species on trained sites, as well as on untrained sites when the camera angle and distance from the entrance were comparable to the training images. At untrained sites with more atypical camera placements, we demonstrated the ability to retrain the baseline model and achieve an accuracy comparable to the trained sites. Additionally, we showed that the model can learn to identify a new species, while maintaining high classification accuracy for all original species. Finally, we established that BatNet can be used to accurately describe ecological metrics from camera trap images (i.e., species diversity, relative abundance, and species-specific phenology) that are relevant for bat conservation.
We conclude that infrared light barriers and camera traps offer a minimally invasive and accurate method to monitor site-level bat population trends and species-specific phenological estimates at underground sites. Such remote data collection approaches are particularly relevant for monitoring large, complex hibernation sites, where traditional visual surveys are not feasible or account only for a small fraction of the actual population. Combining this automated monitoring method with a deep learning-based species identification tool, BatNet, allows us quickly and accurately analyze millions of camera trap images resulting from large-scale, long-term camera trap studies. As a result, we can gain unprecedented insights into the behavior and population dynamics of these enigmatic species, drastically improving our ability to support data-driven bat conservation.
Until today, more than 100 years after its first description in Italy, the highly pathogenic avian influenza virus (HPAIV) has not lost its fearsome character for wild birds, poultry and humans. On the contrary, the number of outbreaks with high casualty rates in wild birds and poultry has multiplied in recent years and cases of zoonotic infections are also increasingly reported from HPAI endemic areas. The epidemiology of these infections is complex and also involves surface water and possibly sediments of shallow standing waters, which could play a role as a vector medium and/or virus reservoir. The goal of this project was to expand current knowledge of the influence of water on the spread of AIV. As part of this project, we were able to ...
1. ...improve AIV detection methods using real time RT-PCR in terms of sensitivity and breadth of viruses detected. In addition, we succeeded in economizing the procedure so that fewer resources are required and results are obtained faster (publication I: [173]).
2. ...develop an ultrafiltration-based enrichment method for AIV from surface water and evaluate it with field samples from HPAI outbreak areas in wild bird habitats (Wadden Sea coast of Schleswig-Holstein) and previously unaffected regions (Antarctic Weddell Sea) (publication II: [174]). Furthermore, protocols for testing different environmental sample matrices for AIV screening were tested and compared to results of passive monitoring by dabbing diseased or dead wild birds. AIV was detected in more than half (61%) of 44 water samples. We received additional sediment samples from 36 of the 44 water samples. In 18 of 36 of the sediments tested, as well as in 4.16% of 1705 fecal samples tested AIV was detected. However, the studies of the environmental samples mostly yielded only generic AIV detections, with viral loads in the range of the detection limit. This massively hampered further investigations for sub- and pathotyping. In contrast, 79.41% of 68 samples from passive monitoring showed high to very high HPAIV viral loads which also allowed sub- and pathotyping.
3. ...demonstrate in animal experiments that even very low titers (0.1 TCID50 ml-1) of HPAI viral infectivity in water can induce productive infection in susceptible but clinically largely resistant mallard ducks (publication III: [175]). Furthermore, we were able to develop evidence that there is a difference in virus spread that depends on the type of (contaminated) water source. This means that infections on poultry farms with inverted or nipple drinkers may follow a different course than infections in the wild, which are mediated via larger surface waters.
Overall, the results of this project highlight the important role of surface and drinking water, as well as aquatic sediments, in the spread of AIV. The methods developed here for AIV detection extend the possibilities for surveillance of AIV infections; however, passive remains superior to active surveillance of HPAIV infections in several aspects. Examination of various environmental samples did not yield a significant advantage in terms of an early warning system that would indicate the presence or spread of HPAIV in wild bird habitats prior to the occurrence of lethal infections in wild birds.