The Angolan population has to deal with a high child mortality rate and various deficiency syndromes due to malnutrition. The diet is mainly based on carbohydrate rich plants, lacking important macro- and micronutrients. Currently the majority of cultivated plants originate from regions outside Africa. Although indigenous edible plant species are incorporated in the African diet, they only play a minor role and are often neglected in agricultural systems. The introduction of nutritive, fast-growing American fruit species as ananas, avocado or guava displace natural growing crops. In recent years the potential of wild edible plants to combat malnutrition, face climate change and deal with existing soil conditions has become of special concern. Leavy vegetables as Amaranthus spec., legumes as cowpea (Vigna unguiculata (L.) Walp.) or fruits as marula (Sclerocarya birrea (A.Rich.) Hochst. or baobab (Adansonia digitata L.) have gained public and commercial interest. However, only a few African edible plant species are cultivated in small plantations (“Fazenda”) or home gardens in the surrounding of villages. Horticultural or nutritional data are often incomplete and not representative. The scientific knowledge even impairs for species which are not cultivated and regarded only as snack or child food by the local population. Such plants as from the genus of chocolate berries (Vitex L.), vine rubber (Landolphia P.Beauv.) or Parinari Aubl. were of special interest for this PhD thesis. Iron and zinc are widely deficient in the diet of the Angolan population and plant products as fruits from Canarium schweinfurthii Engl. or leaves of Crassocephalum rubens (Juss. ex Jacq.) S.Moore can help to combat resulting syndromes. However, the obtained contents of the 43 plant species have to be verified on additional plant material as contents are known to vary. Nevertheless, the results serve as a baseline and represent first insights into the nutritive profile of the respective wild edible plants. Those important comparative values direct future research, although the inconsistency of plant individuals impede exact statements. On the other hand, this fact also bears a great research potential for studies on the effects of plant varieties, localities or age of tissues. Yields of the studied plant species and incorporated volumes of the edible plant parts are still unknown, but important parameters. The forest tree Caloncoba welwitschii Oliv. has spiny fruits with a delicious, fatty orange pulp. Besides our studies no research focus was ever directed to its nutritional potential although the pulp is appreciated by the local population. A high number of seeds in respect to a comparatively low amount of pulp probably impede nutritional analysis and agricultural interest. However, an in-depth analysis of existing varieties and an appropriate selection could improve the yield as the pulp is of special nutritional value. It contains high amounts of calcium and magnesium, vitamin B1, B2 and E. The protein composition further revealed high amounts of indispensable amino acids (Thr, Val, Ile, Leu). The tree often remains in disturbed areas as it is usually protected against human placed fires. The rural population appreciate the ornamental flowers, its diverse medical applications and its ability to attract insects. This demonstrates the enormous potential wild edible plants can have protecting natural habitats and plant individuals. Useful species are safeguarded by locals and emphasizing the manifold utilizations and the nutritional potential of other species seem to be a promising opportunity protecting the natural environment. Benefits of indigenous food plants are therefore manifold. They already grow in surrounding areas and need no expensive cultivation techniques; they are likely more resistant to existing pests and cushion the effects of slash-and-burn farming on soil erosion and rising ground temperatures. Barren, acidic soils with high plant-available amounts of metals as aluminum and manganese impede the cultivation of commercial introduced crops. Application of fertilizers or soil amelioration techniques to enhance the yield are time and cost intensive. Otherwise, indigenous edible plant species are adapted to local growing conditions and grow without special care. Thus, the analysis of physical and chemical soil characteristics was conducted for eight traditionally utilized edible plants from the province Uíge. As plants evolved together with their parameters, they developed different strategies to deal with high amounts of plant-available metals. One example are accumulators, plants which contain extraordinarily high metal contents in their aboveground tissue. Anisophyllea quangensis Engl. ex Henriq., Landolphia lanceolata (K.Schum.) Pichon, Tristemma mauritianum J.F.Gmel., and Eugenia malangensis (O.Hoffm.) Nied. are first described to contain more than 1000 ppm aluminum in their dried leaves and can thus be categorized as Al-hyperaccumulator. These plants survive on marginal sides and bear additionally edible fruits with nutritional potential and small amounts of Al. For this reason, they are of special interest combating malnutrition and can at the same time cover and protect the ground. Diversifying the agricultural system with local growing plants seem to be a promising alternative to current monocultures of conventional crops. In course of the data evaluation, it became apparent that there is a huge lack of knowledge concerning wild edible plants in Africa. Comprehensive data about the nutritional profile, growing conditions, germination or yield only exist for model plants or common crops. Results of this PhD thesis therefore serve as first insight into the potential of a selection of wild edible plants from Angola. Exemplary a comprehensive literature review was conducted for the genus Landolphia P.Beauv. covering all research areas from rubber production over botanical investigations to mechanical studies. Striking are the close interactions of the different scientific disciplines. Ethnobotanical utilization patterns are closely linked to pharmacological studies or nutritional analysis. The subshrub Landolphia lanceolata, which is native to the D.R. Congo and Angola, was involved in all different analysis conducted in this thesis. Results and their complex interactions are briefly outlined. The rhizomatous species grows on fine sand in the savannah. The soil of the examined species from Uíge was extremely acidic and low in nutrients. However, high aluminum contents were found in the leaves indicating accumulation processes within the plant tissues. The aluminum content of the edible fruit pulp was further elevated, which, depending on quantity and frequency of consumption, can be a threat to human health. Other hazardous minerals were not found in the pulp. The delicious fruit contains further antioxidants, vitamin B1, C and E. Landolphia species can yield many fruits on one individuum, which are often sold on local markets and generate alternative income for the local population. Furthermore, leaves and roots of this species are known by traditional healers to relieve cough or rheumatism. Pharmacological studies already determined promising antiplasmodial activities of the root and leaf extract. The latex extraction from L. lanceolata roots is destructive and the obtained rubber of low quality. It is accordingly advisable to utilize other species of the genus Landolphia for rubber production. As illustrated by the many purposes of Landolphia lanceolata, indigenous plant species can help to overcome some of the major problems African populations have to face. Wild edible fruits diversify the local diet and are an important source of macro- and micronutrients. The protection of existing populations and their habitat as well as intensified cultivation can help to recover disturbed, open vegetation sites. Erosion can be prevented and the soil will be safeguarded without external support as power or fund requirements. Further, the economic situation of the rural population would be improved by the sale of products from the species. Another advantage of indigenous plants in contrast to conventional crops are their multipurpose applications. A lasting agriculture with minimum management requirements can be developed, render unnecessary fertilizers, herbicides or insecticides. Investigations on wild edible plants therefore bear a great potential directing future research activities, inform the local population and implement sustainable utilization forms protecting the natural environment.

The vertebrate inner ear is a remarkable sensory organ, harboring two different senses: the auditory system, responsible for hearing, and the vestibular system, responsible for balance. Even though the anatomical structure of the vertebrate inner ear is very complex, only three different cell types are mainly involved on a cellular level in the perception of sound as well as balance and movement: sensory hair cells that are surrounded by supporting cells receive the stimulus and transfer it via sensory neurons to the brain. Worldwide, millions of people suffer from sensorineural hearing loss, caused by the loss of sensory hair cells and/or their innervating neurons within the inner ear. In mammals, including humans, both cell types are only produced during fetal stages making loss of these cells and the resulting consequences irreversible. In contrast, it is known that zebrafish produce sensory hair cells throughout life and additionally possess the remarkable capacity to regenerate them upon lesion. However, it is unknown whether new sensory neurons are also formed throughout life in the zebrafish statoacoustic ganglion (SAG), which transduces signals from the inner ear to the brain. Moreover, it is unknown whether sensory neurons are replaced upon loss. Hence, the first aim of this study was to investigate whether new sensory neurons are produced beyond larval stages. To this end, analysis of different transgenic lines combined with immunohistochemistry against known markers for neuronal stem and progenitor cells, neurons, glia and myelinating cells as well as markers for proliferation were used to identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In the juvenile SAG, a pool of highly proliferating Neurod/Nestin-positive neuronal progenitors produces large amounts of new sensory neurons. In contrast, at adult stages this neurogenic niche transitions to a quiescent state, in which Neurod/Nestin-positive neuronal progenitor cells are no longer proliferating and the neurogenesis rate is very low. Moreover, BrdU pulse chase experiments revealed the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestinpositive progenitor pool throughout life, indicating a neural stem cell-like cell population upstream of the neuronal progenitor cell pool. Additionally, expression of glia markers and a switch in the myelination pattern was found to mark the peripheral and central nervous system transitional zone (PCTZ) as a prominent landmark of the SAG. To further study the nature of the proliferating but otherwise unknown stem cell-like cell population replenishing the Neurod/Nestin-positive neuronal progenitor pool, the transcriptome of proliferating cells and their progeny of the juvenile and adult SAG was analyzed via single cell RNA-sequencing using the Smart-Seq2 technology. Therefore, a pipeline including preparation of the SAG as well as cell dissociation followed by fluorescence-activated cell sorting was established to obtain single cells from the SAG. The fluorescent reporters Tg(pcna:GFP) and Tg(nestin:mCherry-CreERT2) were used to label proliferating cells (GFP-only positive), proliferating progenitors (GFP/mCherry-double positive as well as nonproliferating progenitor cells (mCherry-positive). Additionally, based on the perdurance of the fluorophores in the progeny of the cells expressing the reporter constructs, this sorting strategy also enables to sort the progeny of proliferating cells differentiating into neuronal progenitor cells (GFP/mCherry-double positive but not expressing pcna) to trace back the putative stem cell-like cell population replenishing the Neurod/Nestin-positive progenitor population. Similar, the sorting strategy also included newborn neurons as the progeny of neuronal progenitors (mCherry-positive but not expressing nestin). In the transcriptome data obtained from the juvenile SAG, the majority of the analyzed cells could be assigned to the neuronal lineage, reflecting the neuronal differentiation trajectory from neuronal progenitor cells transitioning to newborn neurons and even further differentiating into mature neurons. Additionally, two different putative neuronal stem cell-like cell clusters were identified which are currently under validation. In contrast, in the adult transcriptome data the majority of cells were identified as cells from the sensory lineage, including cells expressing markers specific for hair cells and the sensory epithelium. Only a minority of cells came from the neuronal lineage, with the group of newborn and differentiating neurons clustering together in one cluster. Very few cells were identified as neuronal progenitor cells and did not cluster together, whereas both putative stem cell-like cell populations could be identified as distinct cluster. However, validation of the putative stem cell population remains subject to further studies. The second aim of this thesis was to investigate the regenerative capacity of the adult SAG and to study whether the neurogenic progenitor cell niche can be reactivated and to give rise to new sensory neurons upon damage. Therefore, a lesion paradigm using unilateral injections into the otic capsule was established. Upon lesion, mature SAG neurons undergo apoptosis and a massive infiltration with immune cells was found. Importantly, the Neurod-positive progenitor cells reentered the cell cycle displaying a peak in proliferation at 8 days post lesion before they returned to homeostatic levels at 57 days post lesion. In parallel to reactive proliferation, an increase in neurogenesis from the Neurod-positive progenitor pool was observed. Reactive neurogenesis started at around 4 days post lesion, peaked at 8 days post lesion decreased again to low homeostatic levels at 57 days post lesion. The administration of the thymidine analog BrdU to label proliferating cells and their progeny revealed the generation of new sensory neurons from proliferating neuronal progenitor cells within 19 days post lesion. Interestingly, reactive proliferation as well as an increased neurogenesis rate were also detected in the unlesioned SAG, revealing a systemic effect of the unilateral lesions. Taken together, this study is the first to show that neurogenesis in the zebrafish SAG persists way beyond larval stages. New neurons descend from a population of Neurod/Nestin-positive neuronal progenitor cells that is highly proliferative during juvenile stages but turn quiescent at adulthood. Nevertheless, this neuronal progenitor cell pool is replenished throughout life by a currently unknown neuronal stem cell-like cell population. Additional this study reveals the regenerative capacity of the adult SAG: upon lesion Neurod/Nestin-positive progenitor cells are reactivated to re-enter the cell cycle, proliferate and give rise to new neurons leading to an increased neurogenesis rate to replace lost mature neurons. Studying the underlying genes and pathways in zebrafish compared to mammalian species will hopefully provide valuable insights that will help developing cures for auditory and vestibular neuropathies in the future.

The structure and dynamics of membranes are crucial to ensure the proper functioning of cells. There are some compounds used in therapeutics that show nonspecific interactions with membranes in addition to their specific molecular target. Among them, two compounds recently used in therapeutics against COVID-19, remdesivir and favipiravir, were subjected to molecular dynamics simulation assays. In these, we demonstrated that the compounds can spontaneously bind to model lipid membranes in the presence or absence of cholesterol. These findings correlate with the corresponding experimental results recently reported by our group. In conclusion, insertion of the compounds into the membrane is observed, with a mean position close to the phospholipid head groups.

Psychische Störungen beinhalten einerseits Störungen der Emotionsregulation und des Verhaltens, andererseits bedeutsames Leiden oder Einschränkungen in den sozialen Beziehungen. In dieser Arbeit wird die Verbindung zwischen diesen beiden Aspekten untersucht. In einer Metaanalyse (Studie 1) werden die Zusammenhänge zwischen (mal)adaptiver Emotionsregulation und den verschiedenen Facetten des Social Mind (Theory of Mind, affektive Empathie, empathischer Stress und Mitgefühl) genauer betrachtet. Ein besonderes Augenmerk wird danach auf die Zwangsstörung gelegt, als ein Beispiel für eine psychische Störung mit großem Effekt auf die sozialen Beziehungen. Mithilfe von Selbstberichten und des EmpaToms, eine videobasierte Aufgabe, wird die Ausprägung der Social Mind-Facetten zwischen Personen mit einer diagnostizierten Zwangsstörung und einer nicht-klinischen Stichprobe verglichen (Studie 2). Die dritte Studie fokussiert auf die Interaktion zwischen Patient:innen mit Zwangsstörung und deren Therapeut:innen während der Therapie (Studie 3). Dabei beleuchten die Ergebnisse die gegenseitige Beeinflussung in der sozialen Mimikry von Patient:innen und Therapeut:innen und den Einfluss der Mimikry auf die therapeutische Allianz.:1. Einleitung 1.1. Aufbau der Arbeit 1.2. Social Mind und Emotionsregulation 1.3. Soziale Beeinträchtigungen bei Zwangsstörungen 2. Methode 3. Studien 3.1. Studie 1: A Meta-Analysis of the Relationship Between Emotion Regulation and Different Facets of the Social Mind 3.2. Studie 2: Empathy, Compassion and Theory of Mind in Obsessive-Compulsive Disorder 3.3. Studie 3: Mimicry in Psychotherapy – an actor partner model of therapists’ and patients’ non-verbal behavior and its effects on the working alliance 4. Diskussion 4.1. Metaanalyse: Emotionsregulation und Social Mind 4.2. Social Mind und Zwangsstörung 4.3. Mimikry und Zwangsstörung 4.4. Praktische Implikationen der Arbeit 4.5. Limitationen der drei Studien 4.6. Über das Konzept der Empathie 5. Zusammenfassung

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Every day there are somatosensory stimuli on our skin that we perceive one moment and the next not, despite their unchanged physical presence (e.g., insects, wind, clothing). Yet, which are the physiological determinants and neural correlates that accompany external stimuli to enter consciousness or not? To address this question and inform theories of consciousness, this dissertation presents three empirical studies that used weak electrical finger-nerve stimulation which led - despite being physically identical - to subjective experiences of stimulus presence and absence. The first two studies investigated the interaction of tactile conscious perception with two dominant body rhythms: the cardiac and respiratory cycle. The third study investigated the configuration of neural networks being involved in this near-threshold phenomenon. Tactile conscious perception changed over the course of the cardiac cycle (increased detection during diastole) and respiration was tuned such that stimuli occurred more likely during late inspiration / early expiration, resulting in increased detection during early expiration. On the neural level, conscious perception was accompanied by global broadcasting of sensory content across the brain without substantial reconfiguration of the whole-brain functional network in terms of graph metrics. The cardiac cycle effect on conscious tactile perception is a result of cognitive processes which model and predict our body’s internal state to inform perception and guide behavior (e.g., tuning respiration). This perceptual integration of interoceptive and exteroceptive 'beliefs' is also an explanation for widely distributed brain activity differences without whole-brain functional network changes when a tactile stimulus is perceived.

The ability to combine single words to more complex meanings is the building block of the expressive power of human language. Semantic composition enables us to understand new concepts that we have not encountered before by combining the representations of the underlying individual concepts. The neural correlates of semantic composition have been at the heart of many research agendas in the past but the neural mechanisms at the most basic level still need to be explored consistently. This thesis aimed to advance our knowledge about the spatio-temporal network of brain regions involved in and necessary for basic semantic composition and presents work from three studies applying the same experimental paradigm across three different neuroscientific methodologies. Study I investigated which brain regions support basic semantic composition and how the regions interact via functional magnetic resonance imaging (fMRI). We found differential involvement of left-hemispheric brain regions for a more general combinatorial and a more specific plausibility evaluation process. We furthermore found evidence for an enhanced coupling between two key nodes of the semantic network during successful composition. In Study II, we probed the relevance of the involved brain regions by studying a cohort of people with aphasia (PWA) and conducting voxel-based lesion-symptom mapping (VLSM). We found a functional dissociation of frontal and temporal brain regions necessary for accurate and efficient semantic decisions, respectively. Finally, in Study III we investigated the temporal dynamics of basic semantic composition in PWA and healthy age-matched controls via electroencephalography (EEG). This study revealed deviant event-related potentials in PWA, where a lack of an early semantic component might be compensated by a stronger late component. Overall, this thesis provides novel insights into the spatial, causal and temporal underpinnings of basic semantic composition making use of three complementary methodologies of neurocognitive research.

The present thesis elucidates facts about a prominent steppe plant’s evolutionary history (i.e., geographic origin, migration route, distribution of genealogical lineages and polyploidization events) and contemporary population divergence (i.e., genetic diversity and differentiation, impacts of abiotic and biotic factors). Artemisia frigida has been chosen as the target species, with Mongolia as the focus study region. Because A. frigida is widely distributed in the both New and Old Worlds, it was a suitable candidate for the phylogeographic study. Moreover, because of its dominance in many communities in Mongolian steppes and tolerance for cold, drought and mechanical disturbances (grazing), evaluating the effect of environmental factors and grazing pressures on its population genetics was profitable. The overall goal of this thesis was to assess the effects of paleo- and current climate, and land use changes on the distribution of A. frigida’s genealogical lineages and genetic variations. The thesis is divided into two main parts: (i) Chapter 3 focuses on Phylogeography. Within this, section 3.3 depicts a study on the phylogeography of A. frigida, covering samples from its distributional range across the northern hemisphere. The study resulted in Asia being the species’ main origination and diversification center, and the species spread northwards to the Russian Far East and eventually crossed the Bering Strait to North America. Among four geographical regions sampled, seven genetic lineages were found, with Middle Asia having the most diverse populations. According to our phylogenetic analysis, two populations of Kazakhstan in Middle Asia represented the most likely ancestral diploids, and subsequent polyploidization events have occurred on several occasions independently. The observed phylogeographic patterns of the species showed that paleoclimate, especially glaciation events of the Quaternary has predominantly affected species’ current distribution, along with the expansion and contraction of the Eurasian steppe. The second part (Chapter 4) is dedicated to Population genetics to reveal the effects of the current climate and land use on population genetic variation. Three studies were conducted at local and regional levels, focusing on Mongolia. The first study (Section 4.1- review of local literature) was done to offer background information about Mongolian steppes, and the effects of climate and grazing on the steppe vegetation. As a result, steppe vegetation responded to grazing in different ways, depending on the interplay of local environmental factors. In particular, an overall negative effect of grazing was found in desert, dry and high mountain steppes, but no or even positive effects in meadow and mountain steppes. The study highlighted the importance of the interaction effect of local environmental conditions and grazing in Mongolian steppe vegetation. The second study (Section 4.2) employed large scale climatic gradient and local scale grazing gradients to assess the effects of grazing and environmental factors on the population genetics of A. frigida. Precipitation gradient covered 110 – 300mm difference of mean annual precipitation from central to southern Mongolia. While three levels of grazing gradient, such as heavy, moderate, and least grazed sites were examined. According to the study, grazing in overall, had no substantial effect on the genetic diversity of A. frigida, while environmental factors, i.e., summer precipitation and soil phosphorous content, promoted high genetic diversity. Genetic differentiation among populations across large climatic gradients was extremely low, suggesting the existence of considerable gene flow among populations across the steppes of Mongolia. The third study (Section 4.3) employed grazing exclosures to evaluate the genuine effects of grazing. Because Mongolia has a long-term nomadic pastoralism history, and grazing of large herbivores is already an integral part of the steppe vegetation. Thus, we utilized reference site fences along the Trans-Mongolian Railway (TMR), where fences have been built and maintained since 1955, resulting in over 60 years of grazing exclusion. In addition, we supplemented this with data from Hustai National Park (HNP), where three fences were established in 2003. As a result, we found a significant positive impact of grazing on the genetic diversity of A. frigida, implying that a certain level of grazing is beneficial for the species. While no grazing effect on the population genetic differentiation was detected, but climatic and soil variables strongly influenced population genetic structure. In summary, this thesis provided an in-depth investigation of the phylogeography and population genetics of the species A. frigida, which can stand as an exemplar for other Eurasian steppe species. Paleoclimate had largely shaped the current distribution pattern of the species, while contemporary climate and environmental heterogeneity promoted species’ polyploidization and genetic variation. Grazing by large herbivores showed no detrimental effect, or even a positive impact on the genetic diversity of A. frigida. Artemisia frigida populations in Mongolia are thus apparently genetically ‘healthy’, in spite of pervasive grazing in the region. Climate variables and environmental heterogeneity had a substantial impact on the species’ both genetic diversity and differentiation, indicating its higher sensibility to climate change than to land use change. The findings of the thesis could be valuable in understanding species genetic variation under global land use and climate changes.:Summary 4 List of Abbreviations 6 Chapter 1. Introduction 8 Chapter 2. Material and methods 10 2.1. Study region: Mongolian steppe 10 2.2. Focus species: Artemisia frigida Willd. 11 2.3. Molecular markers 12 Chapter 3. Phylogeography 13 3.1. Eurasian steppe and its evolutionary history 13 3.2. Artemisia L. (Asteraceae) as model plant for phylogeography 16 3.3. A case study: Phylogeography of Artemisia frigida Willd. 19 Chapter 4. Plant population genetics under changing climate and grazing……. 40 4.1. Climate – grazing interactions in Mongolian steppe 41 4.2. Climate – grazing interactions on plant population genetics 59 4.3. Effect of grazing exclusion on plant populations genetics 83 Chapter 5. Overarching synthesis and discussion 101 5.1. Molecular markers: pros and cons 102 5.2. A review of phylogeographical studies on Eurasian steppe plants 104 5.3. A review on plant population genetic studies in Mongolia 109 5.4. Outlook 113 Acknowledgements 114 References 115 Curriculum vitae 137 Confirmation 143

Fungi and bacteria co-inhabit a wide variety of habitats, and their interactions are significant drivers of many ecosystem services and functions. Creating unique microenvironments, fungal mycelia and their surroundings (defined here as the mycosphere) allow for spatially distinct fungal bacterial activities and interactions at the microscale. Fungi in particular modulate the mycosphere pH and oxygen as the drivers and/or the results of various fungal processes. However, due to the microscopic diameters of hyphae (typically 2-10 μm), it is experimentally difficult to non-invasively access themycosphere to thereby analyze the local pH and oxygen on hyphae or around mycelia. Hence, in this thesis, I aimed to develop and deploy microscale techniques to analyzethe mycosphere pH and oxygen in vitroand thereby to further resolve their influences on the local microbial life for a better understanding of mycosphere habitat properties and functioning.

Intracellular transport is fundamental to many cellular processes- from capturing and destroying pathogens to the propagation of nerve impulses. This transport is mediated by specialized enzymes that convert the free energy of ATP hydrolysis to ‘walk’ on polymeric filaments. The microtubule filament and its associated motors- dynein and kinesin - are responsible for the long-range transport of various cellular cargoes. While these two motors move in the opposite directions, they are often simultaneously present on individual cargoes leading to bidirectional motility characterized by frequency directional reversals. How this process is regulated and what determines the direction of cargo transport remains poorly understood. Addressing these questions requires a systematic analysis where the contribution of various factors in regulating/determining the transport direction of a well-defined cargo can be elucidated. This project establishes an in vitro assay where we reconstitute bidirectional motility of large unilamellar vesicles driven by purified dynein and kinesin-3 motors. Vesicles exhibit fast runs in either direction, with a subset exhibiting directional reversals. The transport features of these vesicles are remarkably similar to that of cargoes in vivo and do not require any additional regulatory proteins/complexes. The simultaneous opposing activity of dynein and kinesin-3 leads to tugs-of-war for a finite period. Finally, we use this assay to determine how microtubule-associated proteins that have differential activity towards dynein and kinesin affect the transport direction of vesicles. MAP9 biases the direction of vesicles towards the plus-end by limiting the ability of dynein to land on microtubules. Our approach can be extended to investigate the potential biasing activities of cellular factors such as posttranslational modifications of tubulin, motor-adaptors, etc. and physical factors, such as fluidity and tension of the vesicle membrane.:1 Introduction 1.1 Microtubules, dynein, kinesin, and microtubule-associated proteins (MAPs) 1.2 Bidirectional transport: In vivo observations and models 1.3 Bidirectional transport: In vitro reconstitution 1.4 Role of membranes in modulating motor activities 1.5 Aims of the thesis 2 Purification of dynein and dynactin 2.1 Purification of dynein and dynactin from adherent mammalian cell cultures 2.2 Purification of dynein and dynactin from suspension cultures using BacMam system 2.3 Validation of dynein and dynactin function using single-molecule motility assays 3 Reconstitution and characterization of bidirectional vesicle motility 3.1 DDB-KIF16B-vesicles exhibit directional reversals in vitro 3.2 Transport direction of vesicles is dependent on the relative concentration of DDB and KIF16B 3.3 Opposing motors do not affect the velocity of the driving motor 3.4 DDB and KIF16B engage in a tug-of-war before directional reversals 3.5 Discussion 4 Characterization of bidirectional motility in the presence of MAPs 4.1 Tau does not differentially affect DDB versus KIF16B at the single-molecule level 4.2 MAP9 affects landing of DDB but not KIF16B 4.3 Motor ensembles can circumnavigate the inhibitory effects of MAPs 4.4 Tau does not bias the transport direction of DDB-KIF16B-vesicles 4.5 MAP9 biases the transport direction of DDB-KIF16B-vesicles towards the plus-end 4.6 Discussion 5 Conclusion and outlook 6 Materials and Methods 6.1 Molecular Biology 6.2 Culture of Flp-In 293 cells 6.3 Protein biochemistry 6.4 In vitro motility assays 6.5 Data processing and analysis