Open Access

In Morocco, there are two well-recognised honey bee (Apis mellifera L.) subspecies: A. m. intermissa in the north and A. m. sahariensis in the south-east. The latter subspecies is found in the arid and semiarid climates of the Sahara Desert. In this study, we used honey bees from four areas of south-eastern Morocco which are, to some degree, isolated by arid zones. We analysed the shape and size of the forewings, using the method of geometric morphometrics. The bees from the four areas of south-eastern Morocco differed significantly in terms of wing shape. Moreover, bees from traditional hives were smaller than those from modern hives. The bees from south-eastern Morocco were clearly different from the reference samples obtained from the Morphometric Bee Data Bank in Oberursel, Germany, representing most of the global variation in honey bees. Surprisingly, the bees were also different from A. m. sahariensis, which should occur in the study area, according to earlier studies. This difference could have been caused by introgression with non-native subspecies imported by beekeepers. The distinct honey bees from south-eastern Morocco deserve to be protected. We provide a method for identifying them, which can help protect them.

Alzheimer’s Disease (AD) is a debilitating neurodegenerative disorder affecting millions of people globally. It is majorly characterised by the pathologic cerebral accumulation of protein aggregates, named senile plaques, consisting of amyloid- β (Aβ) peptides. These aggregates mediate toxic effects to the surrounding cells, contribute to the loss of neuronal tissue and downstream loss of cognitive capabilities that are characteristic for AD. Their formation in AD pathology is a multi-decade long gradual process of self- aggregation in which the equilibrium of formation and clearance is disturbed. Transvascular clearance is a major pathway to remove Aβ from the brain into the bloodstream for systemic clearance by the liver and kidney. Damage to the cerebral microvasculature is a common feature of AD pathology resulting in a weakened blood brain barrier (BBB) and consequently reduced capacity for transvascular Aβ clearance. Acute tissue damage leads to the recruitment of perivascular located mesenchymal stromal cells (MSCs). These, normally quiescent cells, then become active and start proliferating. They secrete extracellular matrix (ECM) proteins to stabilise the tissue and by cytokine secretion, they recruit and coordinate immune cells to aid in healing. In the brain, the heterogeneous pool of MSC consists of pericytes in the capillary bed and perivascular fibroblasts (PVFs) on larger descending arterioles. With recent developments in the understanding of MSC biology, their role in diseases of the central nervous system, especially AD, has been growing in relevance. Their involvement in BBB stabilisation, their involvement in Aβ clearance as well as their vulnerability in AD pathology are of interest but remain elusive. Hence, the aim of this study was to determine the effects of cerebral Aβ pathology on MSCs and elucidate their involvement in Aβ clearance. To this end, the Hic1CreERT2/Rosa26LSL- tdTomato- APPPS1 mouse model was used, which enables the observation of MSCs via a tdTomato label in context of Aβ pathology. This revealed pronounced vulnerability of specifically pericytes to Aβ pathology, characterised by a reduction in the amount of healthy pericytes and the adoption of a pro-inflammatory phenotype. Treatments of cultured primary MSCs with different Aβ species revealed that they were more vulnerable to Aβ42 than to Aβ40. PVFs in turn displayed no signs of vulnerability to Aβ pathology but displayed a higher abundance that was accompanied by the elevated expression of LRP1, the most prominent Aβ transporter. LRP1 expression was generally higher in PVFs than in pericytes as observed in situ and in vitro. This coincided with an increased capacity of PVFs to clear Aβ in vitro as compared to pericytes. In fact, Aβ clearance in cultured primary MSCs was observed to be vastly driven by LRP1 and consistent with this, LRP1 overexpression resulted in improved clearance. The use of the non-statin lipid modulating drug probucol yielded increased LRP1 expression and consequently improved Aβ clearance in MSCs in vitro as well as in vivo. Additionally, in vivo probucol treatment in Hic1CreERT2/Rosa26LSL- tdTomato- APPPS1 mice lead to a marked reduction in cortical Aβ load. As different forms of Aβ are known to be cleared with variable efficiency, the capacity of MSCs to clear distinct species of Aβ was tested, showing a higher propensity of MSCs to clear aggregated forms of Aβ than monomers. Taken together, the results presented in this thesis demonstrate the involvement of MSCs in Aβ clearance and highlight the possibility to improve clearance by targeting LRP1.

When analyzing genomic data, one of the key challenges is the annotation of new genes. The toolkit for incorporating newly discovered proteins into a comprehensive evolutionary and functional network is diverse. It includes search heuristics based on sequence similarity to identify significantly similar sequences. Additionally, it involves identifying orthologs, which are also used for preliminary functional annotation. However, since the function of gene can change if given enough time, it is necessary to consider other information to identify functional divergence between proteins. As one complementary form of information, it is possible to annotate protein sequences with features such as functional domains, transmembrane domains, low complexity regions, secondary structure elements, or compositional properties. The sum of all features annotated onto a sequence, the feature architecture, can provide further information on a proteins function. To perform this task effectively, tools that can compare and classify feature architectures on a large scale are necessary However, multiple challenges arise when dealing with feature architecture. Many existing schemes for comparing feature architectures cannot cope with features arising from multiple annotation sources. Those that do, fall short in the resolution of overlapping and redundant feature annotations. In this thesis, I present different approaches to leveraging feature architectures as a complementary information source for evolutionary and functional studies. First, we introduce the Feature Architecture Similarity, a tool to perform pairwise scoring of the similarity between two feature architectures. It uses a scoring method that considers the presence/absence of features, as well as positional information. It also allows the integration of features from multiple annotation sources into one feature architecture, while resolving overlapping feature annotations. A benchmark on more than 10,000 human-yeast ortholog pairs, architecture similarities assessed with FAS are consistently more plausible than those obtained using e-values to resolve overlaps or leaving overlaps unresolved. We then demonstrate the utility of FAS on feature architecture comparison tasks in three case studies. In the second work package, we apply FAS in the assessment of the functional impact of alternative splicing. Here, we present SPICE, a tool that aids in understanding the functional variations within a proteome resulting from different protein isoforms expressed through alternative splicing. In this pipeline, we introduce a new measure, the Expression Weighted Feature Disturbance (EWFD), that combines the FAS score between protein isoforms with their relative transcript expression values. We demonstrate the use of SPICE with two datasets: First, we do an exemplary analysis using long-read sequencing data from the Long-read RNA-seq Genome Annotation Assessment Project and demonstrate how the results can be explored within the SPICE dashboard. Secondly, we explore how SPICE performs with the inclusion of novel, unannotated transcripts using a larger, more diverse dataset provided by the ENCODE consortium. In the last part, we move away from pairwise comparisons of feature architectures and instead consider groups of functional equivalent proteins for machine learning. Here, we train AI models based on the feature architectures of a functional group of proteins to identify novel members. We propose a novel approach to encode feature architectures for later machine learning applications in two variants. In the first, we dynamically define, for each model, a feature space out of the features annotated with FAS. In the second, we consider a static feature space based on the DSSP 8-state secondary structure representation and disordered regions. In a test on 59 orthologous groups of the tricarboxylic acid cycle annotated in the Kyoto Encyclopedia of Genes and Genomes we confirm that both variants have a high recall rate. With the three work packages introduced in this thesis, we extend the toolbox for studies on protein function with tools that effectively leverage feature architecture information on large-scale datasets and provide ideas for the future development of further methods for protein comparison and annotation.

Distress calls are a vocalization type widespread across the animal kingdom, emitted when the animals are under duress, e.g. when captured by a predator. Here, we report on an observation we came across serendipitously while recording distress calls from the bat species Carollia perspicillata, i.e. the existence of sex difference in the distress calling behaviour of this species. We show that in C. perspicillata bats, males are more likely to produce distress vocalizations than females when hand-held. Male bats call more, their calls are louder, harsher (faster amplitude modulated) and cover lower carrier frequencies than female vocalizations. We discuss our results within a framework of potential hormonal, neurobiological and behavioural differences that could explain our findings, and open multiple paths to continue the study of sex-related differences in vocal behaviour in bats.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphospha-tase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Site-directed mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.