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Traditional species identification based on morphological characters is laborious
and requires expert knowledge. It is further complicated in the case of
species assemblages or degraded and processed material. DNA-barcoding,
species identification based on genetic data, has become a suitable alternative,
yet species assemblages are still difficult to study. In the past decade
meta-barcoding has widely been adopted for the study of species communities,
due to technological advances in modern sequencing platforms and
because manual separation of individual specimen is not required. Here,
meta-barcoding is put into context and applied to the study of bee-collected
pollen as well as bacterial communities. These studies provide the basis
for a critical evaluation of the powers and limitations of meta-barcoding. Advantages
identified include species identification without the need for expert
knowledge as well as the high throughput of samples and sequences. In
microbiology, meta-barcoding can facilitate directed cultivation of taxa of interest
identified with meta-barcoding data. Disadvantages include insufficient
species resolution due to short read lengths and incomplete reference
databases, as well as limitations in abundance estimation of taxa and functional
profiling. Despite these, meta-barcoding is a powerful method for the
analysis of species communities and holds high potential especially for automated
biomonitoring.
The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses.
This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type
phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors.
The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase.
The role of chronophin dimerization was studied by generating a constitutively monomeric variant,
which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization.
The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were
investigated by performing domain swapping experiments with chronophin and its paralog AUM and
subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure
determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context.
Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and
characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.
Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis
(2013)
Atherosclerosis is the underlying cause of cardiovascular diseases and a major threat to human health worldwide. It involves not only accumulation of lipids in the vessel wall but a chronic inflammatory response mediated by highly specific cellular and molecular responses. Macrophages and dendritic cells (DCs) play an essential role in taking up modified lipids and presenting them to T and B lymphocytes, which promote the immune response. Enhanced activation, migration and accumulation of inflammatory cells at the local site leads to formation of atherosclerotic plaques.
Atherosclerotic plaques become hypoxic due to reduced oxygen diffusion and high metabolic demand of accumulated cells. The various immune cells experience hypoxic conditions locally and inflammatory stimuli systemically, thus up-regulating Hypoxia-inducible factor 1α. Though the role of HIF1α in macrophages and lymphocytes has been elucidated, its role in DCs still remains controversial, especially with respect to atherosclerosis. In this project work, the role of HIF1α in DCs was investigated by using a cell specific knockout mouse model where HIF1α was deleted in CD11c+ cells.
Aortic root sections from atherosclerotic mice showed presence of hypoxia and up-regulation of HIF1α which co-localized with CD11c+ cells. Atherosclerotic splenic DCs also displayed enhanced expression of HIF1α, proving non-hypoxic stimulation of HIF1α due to systemic inflammation. Conditional knockout (CKO) mice lacking HIF1α in CD11c+ cells, under baseline conditions did not show changes in immune responses suggesting effects of HIF1α only under inflammatory conditions. When these mice were crossed to the Ldlr-/- line and placed on 8 weeks of high fat diet, they developed enhanced plaques with higher T-cell infiltration as compared to the wild-type (WT) controls. The plaques were of a complex phenotype, defined by increased percent of smooth muscle cells (SMCs) and necrotic core area and reduced percent of macrophages and DCs. The mice also displayed enhanced T-cell activation and a Th1 bias in the periphery.
The CKO DCs themselves exhibited increased expression of IL 12 and a higher capacity to proliferate and polarize naive T cells to the Th1 phenotype in vitro. The DCs also showed decreased expression of STAT3, in line with the inhibitory effects of STAT3 on DC activation seen in previous studies. When STAT3 was overexpressed in DCs in vitro, IL 12 was down-regulated, but its expression increased significantly on STAT3 inhibition using a mutant vector. In addition, when STAT3 was overexpressed in DCs in vivo using a Cre regulated lentiviral system, the mice showed decreased plaque formation compared to controls. Interestingly, the effects of STAT3 modulation were similar in WT and CKO mice, intending that STAT3 lies downstream of HIF1α. Finally, using a chromatin immunoprecipitation assay (ChIP), it was confirmed that HIF1α binds to hypoxia responsive elements (HREs) in the Stat3 gene promoter thus regulating its expression. When DCs lack HIF1α, STAT3 expression is not stimulated and hence IL 12 production by DCs is uninhibited. This excessive IL 12 can activate naive T cells and polarize them to the Th1 phenotype, thereby enhancing atherosclerotic plaque progression.
This project thus concludes that HIF1α restrains DC activation via STAT3 generation and prevents excessive production of IL 12 that helps to keep inflammation and atherosclerosis under check.
Attention-Deficit/Hyperactivity Disorder (ADHD) is characterized by symptoms of inattentiveness and hyperactivity/impulsivity. Besides, increasing evidence points to ADHD patients showing emotional dysfunctions and concomitant problems in social life. However, systematic research on emotional dysfunctions in ADHD is still rare, and to date most studies lack conceptual differentiation between emotion processing and emotion regulation. The aim of this thesis was to systematically investigate emotion processing and emotion regulation in adult ADHD in a virtual reality paradigm implementing social interaction. Emotional reactions were assessed on experiential, physiological, and behavioral levels.
Experiment 1 was conducted to develop a virtual penalty kicking paradigm implying social feedback and to test it in a healthy sample. This paradigm should then be applied in ADHD patients later on. Pleasant and unpleasant trials in this paradigm consisted of hits respectively misses and subsequent feedback from a virtual coach. In neutral trials, participants were teleported to different spots of the virtual stadium. Results indicated increased positive affectivity (higher valence and arousal ratings, higher zygomaticus activations, and higher expression rates of positive emotional behavior) in response to pleasant compared to neutral trials. Reactions to unpleasant trials were contradictory, indicating increased levels of both positive and negative affectivity, compared to neutral trials. Unpleasant vs. neutral trials revealed lower valence ratings, higher arousal ratings, higher zygomaticus activations, slightly lower corrugator activations, and higher expression rates of both positive and negative emotional behavior. The intensity of emotional reactions correlated with experienced presence in the virtual reality.
To better understand the impact of hits or misses per se vs. hits or misses with coach feedback healthy participants’ emotional reactions, only 50% of all shots were followed by coach feedback in experiment 2. Neutral trials consisted of shots over the free soccer field which were followed by coach feedback in 50 % of all trials. Shots and feedback evoked more extreme valence and arousal ratings, higher zygomaticus activations, lower corrugator activations, and higher skin conductance responses than shots alone across emotional conditions. Again, results speak for the induction of positive emotions in pleasant trials whereas the induction of negative emotions in unpleasant trials seems ambiguous. Technical improvements of the virtual reality were reflected in higher presence ratings than in experiment 1.
Experiment 3 investigated emotional reactions of adult ADHD patients and healthy controls after emotion processing and response-focused emotion regulation. Participants successively
went through an ostensible online ball-tossing game (cyber ball) inducing negative emotions, and an adapted version of the virtual penalty kicking game. Throughout cyber ball, participants were included or ostracized by two other players in different experimental blocks. Participants were instructed to explicitly show, not regulate, or hide their emotions in different experimental blocks. Results provided some evidence for deficient processing of positive emotions in ADHD. Patients reported slightly lower positive affect than controls during cyber ball, gave lower valence ratings than controls in response to pleasant penalty kicking trials, and showed lower zygomaticus activations than controls especially during penalty kicking. Patients in comparison with controls showed slightly increased processing of unpleasant events during cyber ball (higher ratings of negative affect, especially in response to ostracism), but not during penalty kicking. Patients showed lower baseline skin conductance levels than controls, and impaired skin conductance modulations. Compared to controls, patients showed slight over-expression of positive as well as negative emotional behavior. Emotion regulation analyses revealed no major difficulties of ADHD vs. controls in altering their emotional reactions through deliberate response modulation. Moreover, patients reported to habitually apply adaptive emotion regulation strategies even more frequently than controls. The analyses of genetic high-risk vs. low-risk groups for ADHD across the whole sample revealed similar results as analyses for patients vs. controls for zygomaticus modulations during emotion processing, and for modulations of emotional reactions due to emotion regulation.
To sum up, the virtual penalty kicking paradigm proved to be successful for the induction of positive, but not negative emotions. The importance of presence in virtual reality for the intensity of induced emotions could be replicated. ADHD patients showed impaired processing of primarily positive emotions. Aberrations in negative emotional responding were less clear and need further investigation. Results point to adult ADHD in comparison to healthy controls suffering from baseline deficits in autonomic arousal and deficits in arousal modulation. Deficits of ADHD in the deliberate application of response-focused emotion regulation could not be found.
Frontal asymmetry, a construct invented by Richard Davidson, linking positive and negative valence as well as approach and withdrawal motivation to lateralized frontal brain activation has been investigated for over thirty years. The frontal activation patterns described as relevant were measured via alpha-band frequency activity (8-13 Hz) as a measurement of deactivation in electroencephalography (EEG) for homologous electrode pairs, especially for the electrode position F4/ F3 to account for the frontal relative lateralized brain activation.
Three different theories about frontal activation patterns linked to motivational states were investigated in two studies. The valence theory of Davidson (1984; 1998a; 1998b) and its extension to the motivational direction theory by Harmon-Jones and Allen (1998) refers to the approach motivation with relative left frontal brain activity (indicated by relative right frontal alpha activity) and to withdrawal motivation with relative right frontal brain activation (indicated by relative left frontal alpha activity). The second theory proposed by Hewig and colleagues (2004; 2005; 2006) integrates the findings of Davidson and Harmon – Jones and Allen with the reinforcement sensitivity theory of Jeffrey A. Gray (1982, 1991). Hewig sees the lateralized frontal approach system and withdrawal system proposed by Davidson as subsystems of the behavioral activation system proposed by Gray and bilateral frontal activation as a biological marker for the behavioral activation system. The third theory investigated in the present studies is the theory from Wacker and colleagues (2003; 2008; 2010) where the frontal asymmetrical brain activation patterns are linked to the revised reinforcement sensitivity theory of Gray and McNaughton (2000). Here, right frontal brain activity (indicated by lower relative right frontal alpha activity) accounts for conflict, behavioral inhibition and activity of the revised behavioral inhibition system, while left frontal brain activation (indicated by lower relative left frontal alpha activity) stands for active behavior and the activity of the revised behavioral activation system as well as the activation of the revised flight fight freezing system. In order to investigate these three theories, a virtual reality T-maze paradigm was introduced to evoke motivational states in the participants, offering the opportunity to measure frontal brain activation patterns via EEG and behavior simultaneously in the first study. In the second study the virtual reality paradigm was additionally compared to mental imagery and a movie paradigm, two well-known state inducing paradigms in the research field of frontal asymmetry.
In the two studies, there was confirming evidence for the theory of Hewig and colleages (2004; 2005; 2006), showing higher bilateral frontal activation for active behavior and lateralized frontal activation patterns for approach (left frontal brain activation) and avoidance (right frontal brain activation) behavior. Additionally a limitation for the capability model of anterior brain asymmetry proposed by Coan and colleagues (2006), where the frontal asymmetry should be dependent on the relevant traits driving the frontal asymmetry pattern if a relevant situation occurs, could be found. As the very intense virtual reality paradigm did not lead to a difference of frontal brain activation patterns compared to the mental imagery paradigm or the movie paradigm for the traits of the participants, the trait dependency of the frontal asymmetry in a relevant situation might not be given, if the intensity of the situation exceeds a certain level. Nevertheless there was an influence of the traits in the virtual reality T-maze paradigm, because the shown behavior in the maze was trait-dependent.
The implications of the findings are multifarious, leading from possible objective personality testing via diversification of the virtual reality paradigm to even clinical implications for depression treatments based on changes in the lateralized frontal brain activation patterns for changes in the motivational aspects, but also for changes in bilateral frontal brain activation when it comes to the drive and preparedness for action in patients. Finally, with the limitation of the capability model, additional variance in the different findings about frontal asymmetry can be explained by taking the intensity of a state manipulation into account.
Polymorphonuclear neutrophils (PMNs) are phagocytic cells of the innate immune system that efficiently kill bacteria. However, they also have regulatory effects on other immune cells and contribute to immunosuppression in cancer, which worsens the outcome. In particular, this has been demonstrated for a subset of granulocytic cells called myeloid- derived suppressor cells (MDSCs), but its distinction from PMNs is controversial. Most authors have explored the suppressive effects of MDSCs on T cells, but recent data suggest that NK cells are also affected. NK cells are crucial for the combat of tumor cells, in particular leukemic cells. There is hardly data available on the interaction between NK cells and suppressive granulocytic cells. Therefore, the aim of this thesis was to explore the effects of MDSCs and PMNs on the NK cell function against the leukemia cell line K562.
In co-culture experiments, I demonstrate that granulocytic MDSCs and PMNs had similar effects on NK cell function and homeostasis. On the one hand, they positively influenced the survival and maturation of NK cells. On the other, they inhibited the activation, cytotoxicity and cytokine production of NK cells, both IFNγ and TNFα, in response to K562 target cells. Furthermore, I show a down-regulation of the activating receptor NKp30 on NK cells in the presence of MDSCs or PMNs, which may form part of the underlying suppressive mechanisms.
However, there is also evidence for the involvement of other molecules. Further investigations are needed to confirm a relevant suppression of NK cells by granulocytic cells in cancer patients, and to identify therapeutic targets. The recognition that regular PMNs have similar effects on NK cells as MDSCs could simplify future experiments, since MDSCs are heterogeneous and laborious to isolate and identify.
NKcells and granulocytes are among the first immune cells to reconstitute after hematopoietic stem cell transplantation, and NK cells may be particularly exposed to suppressive effects of granulocytes this scenario. Modulating these suppressive effects of granulocytes on NK cells therapeutically may yield a better NK cell function and an improved cancer prognosis.

The main function of the small intestine is the absorption of essential nutrients, water and vitamins. Moreover, it constitutes a barrier protecting us from toxic xenobiotics and pathogens. For a better understanding of these processes, the development of intestinal in vitro models is of great interest to the study of pharmacological and pathological issues such as transport mechanisms and barrier function. Depending on the scientific questions, models of different complexity can be applied.
In vitro Transwell® systems based on a porous PET-membrane enable the standardized study of transport mechanisms across the intestinal barrier as well as the investigation of the influence of target substances on barrier integrity. However, this artificial setup reflects only limited aspects of the physiology of the native small intestine and can pose an additional physical barrier. Hence, the applications of this model for tissue engineering are limited.
Previously, tissue models based on a biological decellularized scaffold derived from porcine gut tissue were demonstrated to be a good alternative to the commonly used Transwell® system. This study showed that preserved biological extracellular matrix components like collagen and elastin provide a natural environment for the epithelial cells, promoting cell adhesion and growth. Intestinal epithelial cells such as Caco-2 cultured on such a scaffold showed a confluent, tight monolayer on the apical surface. Additionally, myofibroblasts were able to migrate into the scaffold supporting intestinal barrier formation.
In this thesis, dendritic cells were additionally introduced to this model mimicking an important component of the immune system. This co-culture model was then successfully proven to be suitable for the screening of particle formulations developed as delivery system for cancer antigens in peroral vaccination studies. In particular, nanoparticles based on PLGA, PEG-PAGE-PLGA, Mannose-PEG-PAGE-PLGA and Chitosan were tested. Uptake studies revealed only slight differences in the transcellular transport rate among the different particles. Dendritic cells were shown to phagocytose the particles after they have passed the intestinal barrier. The particles demonstrated to be an effective carrier system to transport peptides across the intestinal barrier and therefore present a useful tool for the development of novel drugs.
Furthermore, to mimic the complex structure and physiology of the gut including the presence of multiple different cell types, the Caco-2 cell line was replaced by primary intestinal cells to set up a de novo tissue model. To that end, intestinal crypts including undifferentiated stem cells and progenitor cells were isolated from human small intestinal tissue samples (jejunum) and expanded in vitro in organoid cultures. Cells were cultured on the decellularized porcine gut matrix in co-culture with intestinal myofibroblasts. These novel tissue models were maintained under either static or dynamic conditions.
Primary intestinal epithelial cells formed a confluent monolayer including the major differentiated cell types positive for mucin (goblet cells), villin (enterocytes), chromogranin A (enteroendocrine cells) and lysozyme (paneth cells). Electron microscopy images depicted essential functional units of an intact epithelium, such as microvilli and tight junctions. FITC-dextran permeability and TEER measurements were used to assess tightness of the cell layer. Models showed characteristic transport activity for several reference substances. Mechanical stimulation of the cells by a dynamic culture system had a great impact on barrier integrity and transporter activity resulting in a tighter barrier and a higher efflux transporter activity.
In Summary, the use of primary human intestinal cells combined with a biological decellularized scaffold offers a new and promising way to setup more physiological intestinal in vitro models. Maintenance of primary intestinal stem cells with their proliferation and differentiation potential together with adjusted culture protocols might help further improve the models. In particular, dynamic culture systems and co culture models proofed to be a first crucial steps towards a more physiological model. Such tissue models might be useful to improve the predictive power of in vitro models and in vitro in vivo correlation (IVIVC) studies. Moreover, these tissue models will be useful tools in preclinical studies to test pharmaceutical substances, probiotic active organisms, human pathogenic germs and could even be used to build up patient-specific tissue model for personalized medicine.
The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively.
BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety.
The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target.
In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann’s reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann’s reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above.
We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB.
The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme.
Our structural analysis revealed that despite its low sequence identity of 28%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD.
The number of fungal infections is rising in Germany and worldwide. These infections are mainly caused by the opportunistic fungal pathogen C. albicans, which especially harms immunocompromised people. With increasing numbers of fungal infections, more frequent and longer lasting treatments are necessary and lead to an increase of drug resistances, for example against the clinically applied therapeutic fluconazole. Drug resistance in C. albicans can be mediated by the Multidrug resistance pump 1 (Mdr1), a membrane transporter belonging to the major facilitator family. However, Mdr1-mediated fluconazole drug resistance is caused by the pump’s regulator, the transcription factor Mrr1 (Multidrug resistance regulator 1). It was shown that Mrr1 is hyperactive without stimulation or further activation in resistant strains which is due to so called gain of function mutations in the MRR1 gene.
To understand the mechanism that lays behind this constitutive activity of Mrr1, the transcription factor should be structurally and functionally (in vitro) characterized which could provide a basis for successful drug development to target Mdr1-mediated drug resistance caused by Mrr1. Therefore, the entire 1108 amino acid protein was successfully expressed in Escherichia coli. However, further purification was compromised as the protein tended to form aggregates, unsuitable for crystallization trials or further characterization experiments. Expression trials in the eukaryote Pichia pastoris neither yielded full length nor truncated Mrr1 protein. In order to overcome the aggregation problem, a shortened variant, missing the N-terminal 249 amino acids named Mrr1 ‘250’, was successfully expressed in E. coli and could be purified without aggregation. Similar to the wild type Mrr1 ‘250’, selected gain of function variants were successfully cloned, expressed and purified with varying yields and with varying purity. The Mrr1 `250’ construct contains most of the described regulatory domains of Mrr1. It was used for crystallization and an initial comparative analysis between the wild type protein and the variants. The proposed dimeric form of the transcription factor, necessary for DNA binding, could be verified for both, the wild type and the mutant proteins. Secondary structure analysis by circular dichroism measurements revealed no significant differences in the overall fold of the wild type and variant proteins. In vitro, the gain of function variants seem to be less stable compared to the wild type protein, as they were more prone to degradation. Whether this observation holds true for the full length protein’s stability in vitro and in vivo remains to be determined. The crystallization experiments, performed with the Mrr1 ‘250’ constructs, led to few small needle shaped or cubic crystals, which did not diffract very well and were hardly reproducible. Therefore no structural information of the transcription factor could be gained so far.
Infections with M. tuberculosis, the causative agent of tuberculosis, are the leading cause of mortality among bacterial diseases. Especially long treatment times, an increasing number of resistant strains and the prevalence of for decades persisting bacteria create the necessity for new drugs against this disease. The cholesterol import and metabolism pathways were discovered as promising new targets and interestingly they seem to play an important role for the chronic stage of the tuberculosis infection and for persisting bacteria.
In this thesis, the 3-ketoacyl-CoA thiolase FadA5 from M. tuberculosis was characterized and the potential for specifically targeting this enzyme was investigated. FadA5 catalyzes the last step of the β-oxidation reaction in the side-chain degradation pathway of cholesterol. We solved the three dimensional structure of this enzyme by X-ray crystallography and obtained two different apo structures and three structures in complex with acetyl-CoA, CoA and a hydrolyzed steroid-CoA, which is the natural product of FadA5. Analysis of the FadA5 apo structures revealed a typical thiolase fold as it is common for biosynthetic and degradative enzymes of this class for one of the structures. The second apo structure showed deviations from the typical thiolase fold. All obtained structures show the enzyme as a dimer, which is consistent with the observed dimer formation in solution. Thus the dimer is likely to be the catalytically active form of the enzyme. Besides the characteristic structural fold, the catalytic triad, comprising two cysteines and one histidine, as well as the typical coenzyme A binding site of enzymes belonging to the thiolase class could be identified. The two obtained apo structures differed significantly from each other. One apo structure is in agreement with the characteristic thiolase fold and the well-known dimer interface could be identified in our structure. The same characteristics were observed in all complex structures. In contrast, the second apo structure followed the thiolase fold only partially. One subdomain, spanning 30 amino acids, was in a different orientation. This reorientation was caused by the formation of two disulfide bonds, including the active site cysteines, which rendered the enzyme inactive. The disulfide bonds together with the resulting domain swap still permitted dimer formation, yet with a significantly shifted dimer interface. The comparison of the apo structures together with the preliminary activity analysis performed by our collaborator suggest, that FadA5 can be inactivated by oxidation and reactivated by reduction. If this redox switch is of biological importance requires further evaluation, however, this would be the first reported example of a bacterial thiolase employing redox regulation.
Our obtained complex structures represent different stages of the thiolase reaction cycle. In some complex structures, FadA5 was found to be acetylated at the catalytic cysteine and it was in complex with acetyl-CoA or CoA. These structures, together with the FadA5 structure in complex with a hydrolyzed steroid-CoA, revealed important insights into enzyme dynamics upon ligand binding and release. The steroid-bound structure is as yet a unique example of a thiolase enzyme interacting with a complex ligand. The characterized enzyme was used as platform for modeling studies and for comparison with human thiolases. These studies permitted initial conclusions regarding the specific targetability of FadA5 as a drug target against M. tuberculosis infection, taking the closely related human enzymes into account. Additional analyses led to the proposal of a specific lead compound based on the steroid and ligand interactions within the active site of FadA5.
Human Vγ9Vδ2 T cells are the main γδ T cell subset in the circulation, accounting for up to 5% of the total peripheral blood lymphocyte population. They have been suggested to be important in response to tumors and infections. Their immune mechanisms encompass cell killing via cytotoxicity and secretion of pro-inflammatory cytokines such as IFNγ and tumor necrosis factor (TNF). The main stimulators of Vγ9Vδ2 T cells are isopentenyl pyrophosphate (IPP) and (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), denominated phosphoantigens (PAg).
A major advance in the understanding of PAg detection and Vγ9Vδ2 T cell activation has been the identification of the butyrophlin 3A (BTN3A) proteins as key mediators in these processes. In humans, three isoforms constitute the BTN3A family: BTN3A1, BTN3A2, and BTN3A3; and their genes are localized on the short arm of chromosome 6. The role of BTN3A1 has been highlighted by BTN3A-specific monoclonal antibody 20.1 (mAb 20.1), which has an agonist effect and causes proliferation, expansion, and activation of primary human Vγ9Vδ2 T cells. On the other hand, BTN3A-specific monoclonal antibody 103.2 (mAb 103.2) is antagonistic, inhibiting the Vγ9Vδ2 T cell response. The actual mechanism underlying both PAg- and mAb 20.1-mediated activation is not completely elucidated, but the importance of BTN3A1 is clear.
The main objective of this dissertation was to characterize the role of BTN3A1 in the PAg-dependent and PAg-independent Vγ9Vδ2 T cell activation and to evaluate its contribution in the response to influeza A virus infected cells. This research work demonstrated, by using Vγ9Vδ2 TCR MOP-transduced murine cells (reporter cells), that human chromosome 6 (Chr6) is mandatory for PAg-induced stimulation, but not for stimulation with mAb 20.1. The reporter cells responded to mAb 20.1 in cultures with BTN3A1-transduced Chinese hamster ovary cells (CHO BTN3A1) as antigen presenting cells. Nevertheless, for PAg-dependent activation the presence of Chr6 in CHO BTN3A1 was mandatory.
Although reporter cells expressing clonotypically different Vγ9Vδ2 TCRs showed similar PAg response, they clearly differed in the mAb 20.1 response. The reporter cell line transduced with Vγ9Vδ2 TCR D1C55 demonstrated essentially no response to mAb 20.1 compared to Vγ9Vδ2 TCR MOP cells. These findings were further supported by experiments performed with human PBMCs-derived Vγ9Vδ2 T cell clones. The results indicate heterogeneity in the PAg- and 20.1-dependent responses, in terms of CD25 and CD69 expression, among three different Vγ9Vδ2 T cells clones.
Co-cultures of reporter cells with Raji RT1BI and PAg plus mAb 20.1 or single chain antibody 20.1 (sc 20.1) revealed no additive or synergistic activating effects. In contrast, mAb 20.1 or sc 20.1 inhibited the PAg-mediated activation of the reporter cells.
The comparison of the relative contribution of the isoforms BTN3A2 and BTN3A3, in the activation of Vγ9Vδ2 T cells, was undertaken by overexpression of these isoforms in CHO cells. The results showed that BTN3A2 contributes to both PAg- and mAb-induced Vγ9Vδ2 T cell activation. On the contrary, BTN3A3 does not support PAg-mediated γδ T cell response.
Additionally, mutations in the proposed PAg- and mAb 20.1-binding sites of the extracellular BTN3A1 domain were generated by means of site-directed mutagenesis. These mutations revoked the mAb 20.1-induced Vγ9Vδ2 T cell activation, but not that induced by PAg.
Finally, co-cultures of Vγ9Vδ2 TCR MOP-transduced murine reporter cells with influenza A/PR/8/34-infected cells, or infection of PBMCs with this virus strain indicated that BTN3A1 might be dispensable for the Vγ9Vδ2 T cell response against influenza A.
The data of this research work points out that: i) in addition to BTN3A1, other Chr6-encoded genes are necessary for Vγ9Vδ2 T cell activation with PAg; ii) clonotypical (CDR3) differences influence the PAg- and mAb 20.1-mediated Vγ9Vδ2 T cell activation; iii) the PAg- and mAb 20.1-induced responses are not synergistic and interfere with each other; iv) BTN3A2 and BTN3A3 isoforms differ in the ability to support PAg- or mAb 20.1-dependent Vγ9Vδ2 T cell activation; v) the importance of the intracellular B30.2 domain of BTN3A1, in the Vγ9Vδ2 T cell activation, might be higher than that of the extracellular domain; and vi) in spite of the importance of BTN3A1 in the activation of Vγ9Vδ2 T cells, it is possible that many molecules with redundant functions are involved in the elimination of influenza virus infection by these cells.
In summary, it is possible to hypothesize a model in which BTN3A1 detects prenyl pyrophosphates in the cytoplasm via its B30.2 domain and in association with another protein(s). The binding of PAg to this domain induces a multimerization of BTN3A1 or a conformational change of its extracellular domain (mimicked by mAb 20.1). These modifications might be recognized by the Vγ9Vδ2 TCR or by an associated T cell protein. In the case that the TCR directly recognizes BTN3A1, the intensity of the response will depend on the Vγ9Vδ2 TCR clonotype. Future research will allow to gain a better understanding of BTN3A1, its interaction with other proteins, its actual role in the activation of Vγ9Vδ2 T cells, and its importance in specific models of cancer or infection. This knowledge will be necessary to transform these cells into effective tools in the clinic.
Anxiety is an affective state characterized by a sustained, long-lasting defensive response, induced by unpredictable, diffuse threat. In comparison, fear is a phasic response to predictable threat. Fear can be experimentally modeled with the help of cue conditioning. Context conditioning, in which the context serves as the best predictor of a threat due to the absence of any conditioned cues, is seen as an operationalization of sustained anxiety.
This thesis used a differential context conditioning paradigm to examine sustained attention processes in a threat context compared to a safety context for the first time. In three studies, the attention mechanisms during the processing of contextual anxiety were examined by measuring heart rate responses and steady-state-visually evoked potentials (ssVEPs). An additional focus was set on the processing of social cues (i.e. faces) and the influence of contextual information on these cues. In a last step, the correlates of sustained anxiety were compared to evoked responses by phasic fear, which was realized in a previously established paradigm combining predictable and unpredictable threat.
In the first study, a contextual stimulus was associated with an aversive loud noise, while a second context remained unpaired. This conditioning paradigm created an anxiety context (CTX+) and a safety context (CTX-). After acquisition, a social agent vs. an object was presented as a distractor in both contexts. Heart rate and cortical responses, with ssVEPs by using frequency tagging, to the contexts and the distractors were assessed. Results revealed enhanced ssVEP amplitudes for the CTX+ compared to the CTX− during acquisition and during presentation of distractor stimuli. Additionally, the heart rate was accelerated in the acquisition phase, followed by a heart rate deceleration as a psychophysiological marker of contextual anxiety.
Study 2 used the same context conditioning paradigm as Study 1. In contrast to the first study, persons with different emotional facial expressions were presented in the anxiety and safety contexts in order to compare the differential processing of these cues within periods of threat and safety. A similar anxiety response was found in the second study, although only participants who
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were aware of the contingency between contexts and aversive event showed a sensory amplification of the threat context, indicated by heart rate response and ssVEP activation. All faces irrespective of their emotional expression received increased attentional resources when presented within the anxiety context, which suggests a general hypervigilance in anxiety contexts.
In the third study, the differentiation of predictable and unpredictable threat as an operationalization of fear and anxiety was examined on a cortical and physiological level. In the predictable condition, a social cue was paired with an aversive event, while in the unpredictable condition the aversive event remained unpaired with the respective cue. A fear response to the predictable cue was found, indicated by increased oscillatory response and accelerated heart rate. Both predictable and unpredictable threat yielded increased ssVEP amplitudes evoked by the context stimuli, while the response in the unpredictable context showed longer-lasting ssVEP activation to the threat context.
To sum up, all three studies endorsed anxiety as a long-lasting defensive response. Due to the unpredictability of the aversive events, the individuals reacted with hypervigilance in the anxiety context, reflected in a facilitated processing of sensory information and an orienting response. This hypervigilance had an impact on the processing of novel cues, which appeared in the anxiety context. Considering the compared stimuli categories, the stimuli perceived in a state of anxiety received increased attentional resources, irrespective of the emotional arousal conveyed by the facial expression. Both predictable and unpredictable threat elicited sensory amplification of the contexts, while the response in the unpredictable context showed longer-lasting sensory facilitation of the threat context.
An essential step in eukaryotic gene expression is splicing, i.e. the excision of non-coding sequences from pre-mRNA and the ligation of coding-sequences. This reaction is carried out by the spliceosome, which is a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs) and a large number of proteins. Spliceosomal snRNPs are composed of one snRNA (or two in case of U4/6 snRNPs), seven common Sm proteins (SmD1, D2, D3, B, E, F, G) and several particle-specific proteins. The seven Sm proteins form a ring shaped structure on the snRNA, termed Sm core domain that forms a structural framework of all spliceosomal snRNPs. In the toroidal Sm core domain, the individual Sm proteins are arranged in the sequence SmE-SmG-SmD3-SmB- SmD1-SmD2-SmF from the first to the seventh nucleotide of the Sm site, respectively. The individual positions of Sm proteins in the Sm core domain are not interchangeable.
snRNPs are formed in vivo in a step-wise process, which starts with the export of newly transcribed snRNA to the cytoplasm. Within this compartment, Sm proteins are synthesized and subsequently transferred onto the snRNA. Upon formation of the Sm core and further modifications of snRNA, the snRNP is imported into the nucleus to join the spliceosome.
Prior to assembly into snRNPs, Sm proteins exist as specific hetero-oligomers in the cytoplasm. The association of these proteins with snRNA occurs spontaneously in vitro but requires the assistance of two major units, PRMT5- and SMN- complexes, in vivo. The early phase of assembly is critically influenced by the assembly chaperone pICln. This protein pre-organizes Sm proteins to functional building blocks and enables their recruitment onto the PRMT5 complex for methylation. Sm proteins are subsequently released from the PRMT5 complex as pICln bound entities and transferred onto the SMN-complex. The SMN complex then liberates the Sm proteins from the pICln-induced kinetic trap and allows their transfer onto the snRNA. Although the principal roles of SMN- and PRMT5 complexes in the assembly of snRNPs have been established, it is still not clear how newly translated Sm proteins are guided into the assembly line.
In this thesis, I have uncovered a new facet of pICln function in the assembly of snRNPs. I have shown that newly synthesized Sm proteins are retained at the ribosome upon termination of translation. Their release is facilitated by pICln, which interacts with the cognate Sm protein hetero-oligomers at their site of synthesis on the ribosome and recruits them into the assembly pathway. Additionally, I have been able to show that the early engagement of pICln with the Sm proteins ensures the flawless oligomerization of Sm proteins and prevents any non-chaperoned release and diffusion of Sm proteins in the cytoplasm.
In a second project, I have studied the mechanism of U7 snRNP assembly. This particle is a major component of the 3’ end processing machinery of replication dependent histone mRNAs. A biochemical hallmark of U7 is its unique Sm core in which the two canonical Sm proteins D1 and D2 are replaced by so-called “like Sm proteins”. The key question I addressed in my thesis was, how this “alternative” Sm core is assembled onto U7 snRNA. I have provided experimental evidence that the assembly route of U7 snRNPs and spliceosomal snRNPs are remarkably similar: The assembly of both particles depends on the same assembly factors and the mechanistic details are similar. It appears that formation of the U7- or spliceosomal- core specific 6S complex is the decisive step in assembly.
Motoneuron diseases form a heterogeneous group of pathologies characterized by the progressive degeneration of motoneurons. More and more genetic factors associated with motoneuron diseases encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of motoneuron diseases. Recent results suggest that SMN interacts with hnRNP R and TDP-43 in neuronal processes, which are not part of the classical SMN complex. This point to an additional function of SMN, which could contribute to the high vulnerability of spinal motoneurons in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The current study elucidates functional links between SMN, the causative factor of SMA (spinal muscular atrophy), hnRNP R, and TDP-43, a genetic factor in ALS (amyotrophic lateral sclerosis). In order to characterize the functional interaction of SMN with hnRNP R and TDP-43, we produced recombinant proteins and investigated their interaction by co-immunoprecipitation. These proteins bind directly to each other, indicating that no other co-factors are needed for this interaction. SMN potentiates the ability of hnRNP R and TDP-43 to bind to ß-actin mRNA. Depletion of SMN alters the subcellular distribution of hnRNP R in motoneurons both in SMN-knockdown motoneurons and SMA mutant mouse (delta7 SMA). These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis and ALS.
ALS and FTLD (frontotemporal lobar degeneration) are linked by several lines of evidence with respect to clinical and pathological characteristics. Both sporadic and familial forms are a feature of the ALS-FTLD spectrum, with numerous genes having been associated with these pathological conditions. Both diseases are characterized by the pathological cellular aggregation of proteins. Interestingly, some of these proteins such as TDP-43 and FUS have also common relations not only with ALS-FTLD but also with SMA. Intronic hexanucleotide expansions in C9ORF72 are common in ALS and FTLD but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non ATG-initiated translation is responsible for the pathophysiology. This study tries to characterize the cellular function of C9ORF72 protein. To address this, lentiviral based knockdown and overexpression of C9ORF72 was used in isolated mouse motoneurons. The results clearly show that survival of these motoneurons was not affected by altered C9ORF72 levels, whereas adverse effects on axon growth and growth cone size became apparent after C9ORF72 suppression. Determining the protein interactome revealed several proteins in complexes with C9ORF72. Interestingly, C9ORF72 is present in a complex with cofilin and other actin binding proteins that modulate actin dynamics. These interactions were confirmed both by co-precipitation analyses and in particular by functional studies showing altered actin dynamics in motoneurons with reduced levels of C9ORF72. Importantly, the phosphorylation of cofilin is enhanced in C9ORF72 depleted motoneurons and patient derived lymphoblastoid cells with reduced C9ORF72 levels. These findings indicate that C9ORF72 regulates axonal actin dynamics and the loss of this function could contribute to disease pathomechanisms in ALS and FTLD.
Due to the rotation of the earth in the solar system all inhabitants of our planet are exposed to regular environmental changes since more than 3.5 billion years. In order to anticipate these predictable changes in the environment, evolutionarily conserved biological rhythms have evolved in most organisms – ranging from ancient cyanobacteria up to human beings – and also at different levels of organization – from single cells up to behavior. These rhythms are endogenously generated by so called circadian clocks in our body and entrained to the 24 h cycle by external timing cues. In multi-cellular organisms the majority of the cells in the body is equipped with such an oscillator. In mammals, the circadian system is structured in a hierarchical fashion: A central pacemaker resides in the bilateral suprachiasmatic nucleus (SCN) of the hypothalamus, while subsidiary peripheral clocks exist in nearly every tissue and organ.
In contrast to the aforementioned recurrent environmental changes most organisms are also exposed to unpredictable changes in the environment. In order to adapt to these sudden alterations the acute activation of the stress response system, involving the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, displays a fundamental survival mechanism. However, if activation of the stress system becomes chronic, devastating somatic and affective disorders might be the consequence.
At first glance, the circadian and the stress system seem to represent two separate bodily control systems that are involved in adaptation to predictable and unpredictable stimuli, respectively. However, both systems are fundamental for survival, and thus, communicate with each other at various levels. Early studies already demonstrated that stressor exposure at different times of the diurnal cycle generates different stress effects, whereupon the type of stressor plays a pivotal role. Moreover, alterations in the SCN and peripheral circadian clocks could be shown following stressor exposure.
In cooperation with various co-workers, I investigated whether the stress responsiveness is modulated by the endogenous clock in a diurnal fashion and whether repeated psychosocial stress impacts the circadian clock depending on the time of day of stressor exposure. Therefore, male C57BL/6 mice were repeatedly exposed to a psychosocial stressor, either at the beginning of the inactive/light phase (SDL mice) or active/dark phase (SDD mice).
Subsequently, different behavioral, physiological/endocrine and immunological/ inflammatory consequences were assessed. It could be shown that the effects of repeated psychosocial stressor exposure strongly depend on the time of day of stressor exposure. The present results demonstrate that repeated daily stressor exposure has a more negative outcome when applied during the active/dark phase compared to the inactive/light phase. Stressor exposure during the active phase resulted in a loss of general activity, decreased interest in an unfamiliar conspecific, a shift towards a more pro-inflammatory body milieu, and rhythm disturbances in plasma hormones, all representing well-accepted hallmarks of depression. In contrast, C57BL/6 mice exposed to the stressor in their inactive phase exhibited minor physiological alterations that might prevent the formation of the maladaptive consequences mentioned above, thus representing beneficial adaptations.
The second focus of this thesis was put on the investigation of the effects of repeated psychosocial stressor exposure at different times of the light-dark cycle on various levels of the circadian system. An increased expression of the PERIOD2 (PER2) protein, which represents an essential core clock component, could be found in the SCN of mice repeatedly exposed to the stressor during their active phase. In consistence with the alterations in the central circadian pacemaker, the daily rhythm of different hormones and the activity rhythm were considerably affected by SDD. Mice exposed to the psychosocial stressor in their active phase showed a shifted, or absent, rhythm of the hormones corticosterone and leptin. Moreover, their activity was found to be phase-delayed, which seems to be attributable to the Period (Per) gene since Per1/Per2 double-mutants still exhibited their normal activity rhythm following 19 days of stressor exposure during the active phase. In contrast, a phase-advance in the peripheral adrenal gland clock could be seen in C57BL/6 mice subjected to the stressor during their inactive phase. This phase-shift might be required for maintaining the normal rhythmicity in hormonal release and activity.
It has previously been suggested that activation of the HPA axis upon stressor exposure at different times of the light-dark cycle is depending on whether the stressor is of physical or psychological nature. Data from the HPA axis analysis now refine previous findings, indicating that psychosocial stressors also modulate HPA axis responses based on the time of day of stressor presentation. The present results demonstrate that HPA axis activity was reduced following repeated stressor exposure during the active phase. It is reasonable to speculate that this reduced basal activity of the stress system represents a failure in HPA axis adjustment, which could contribute to the negative consequences of repeated psychosocial stressor exposure during the dark phase.
Taken together, it can be concluded that the endogenous clock in mice modulates the stress responsiveness in a circadian fashion and that repeated psychosocial stressor exposure affects the biological clock depending on the time of day of stressor presentation. Thereby, stressor exposure during the active phase results in a more negative outcome as compared to stressor experience during the inactive phase. It is assumed that the interaction between the circadian clock and the stress system is a complex issue that might ensure that the endogenous clock does not get out of synchrony in any order.
Fulminant myocarditis is rare but a potentially life-threatening disease. Acute or mild myocarditis following acute ischemia is generally associated with a profound activation of the host’s immune system. On one hand this is mandatory to protect the host’s heart by fighting the invading agents (i.e., bacteria, viruses or other microbial agents) and/or to induce healing and repair processes in the damaged myocardium. On other hand, uncontrolled activation of the immune system may result in the generation of auto-reactive (not always beneficial) immune cells.
Myocarditis or inflammatory cardiomyopathy is characterized by focal or diffuse infiltrates, myocyte necrosis and/or apoptosis and subsequent fibrotic replacement of the heart muscle. In humans, about 30% of the myocarditis-patients develop dilated cardiomyopathy. As the clinical picture of myocarditis is multifaceted, it is difficult to diagnose the disease. Therefore, the main goal of the present work was to test and further develop novel non-invasive methods for the detection of myocardial inflammation by employing both contrast enhanced MRI techniques as well as novel nuclear tracers that are suitable for in vivo PET/ SPECT imaging.
As a part of this thesis, a pre-clinical animal model was successfully established by immunizing female Lewis rats with whole-porcine cardiac myosin (CM). Induction of Experimental Autoimmune Myocarditis (EAM) is considered successful when anti-myosin antibody titers are increased more than 100-fold over control animals and pericardial effusion develops. In addition, cardiac tissues from EAM-rats versus controls were analyzed for the expression of various pro-inflammatory and fibrosis markers. To further exploit non-invasive MRI techniques for the detection of myocarditis, our EAM-rats were injected either with (1) ultra-small Paramagnetic iron oxide particles (USPIO’s; Feraheme®), allowing for in vivo imaging , (2) micron sized paramagnetic iron oxide particles (MPIO) for ex vivo inflammatory cell-tracking by cMRI, or (3) with different radioactive nuclear tracers (67gallium citrate, 68gallium-labeled somatostatin analogue, and 68gallium-labeled cyclic RGD-peptide) which in the present work have been employed for autoradiographic imaging, but in principle are also suitable for in vivo nuclear imaging (PET/SPECT). In order to compare imaging results with histology, consecutive heart sections were stained with hematoxylin & eosin (HE, for cell infiltrates) and Masson Goldner trichrome (MGT, for fibrosis); in addition, immuno-stainings were performed with anti-CD68 (macrophages), anti-SSRT2A (somatostatin receptor type 2A), anti-CD61 (β3-integrins) and anti-CD31 (platelet endothelial cell adhesion molecule 1).
Sera from immunized rats strongly reacted with cardiac myosin. In immunized rats, echocardiography and subsequent MRI revealed huge amounts of pericardial effusion (days 18-21). Analysis of the kinetics of myocardial infiltrates revealed maximal macrophage invasion between days 14 and 28. Disappearance of macrophages resulted in replacement-fibrosis in formerly cell-infiltrated myocardial areas. This finding was confirmed by the time-dependent differential expression of corresponding cytokines in the myocardium. Immunized animals reacted either with an early or a late pattern of post-inflammation fibrosis. Areas with massive cellular infiltrates were easily detectible in autoradiograms showing a high focal uptake of 67gallium-citrate and 68gallium labeled somatostatin analogues (68Ga DOTA-TATE). Myocardium with a loss of cardiomyocytes presented a high uptake of 68gallium labeled cyclic RGD-peptide (68Ga NOTA-RGD). MRI cell tracking experiments with Feraheme® as the contrast-agent were inconclusive; however, strikingly better results were obtained when MPIOs were used as a contrast-agent: histological findings correlated well with in vivo and ex vivo MPIO-enhanced MRI images.
Imaging of myocardial inflammatory processes including the kinetics of macrophage invasion after microbial or ischemic damage is still a major challenge in, both animal models and in human patients. By applying a broad panel of biochemical, histological, molecular and imaging methods, we show here that different patterns of reactivity may occur upon induction of myocarditis using one and the same rat strain. In particular, immunized Lewis rats may react either with an early or a late pattern of macrophage invasion and subsequent post-inflammation fibrosis. Imaging results achieved in the acute inflammatory phase of the myocarditis with MPIOs, 67gallium citrate and 68gallium linked to somatostatin will stimulate further development of contrast agents and radioactive-nuclear tracers for the non-invasive detection of acute myocarditis and in the near future perhaps even in human patients.
The impact of acquired severe motor impairments is pervasive and may lead to a complete loss of communication and voluntary motor control, rendering the patient behaviourally unresponsive. In routine clinical care it may thus be unclear, whether some of these patients are even conscious. Given that finding a cure is unlikely, care focuses on providing the best possible quality of life (QoL), and knowing its predictors might contribute to that aim. Patients who still can communicate often report a high QoL, and several predictors have been identified. However, many instruments used to assess QoL require at least residual verbal and motor abilities. Thus, a method to assess QoL independent of these requirements is desirable. In addition, many instruments assume QoL to be temporarily stable, and little information is available on predictors of instantaneous QoL, i.e. QoL as it fluctuates from moment to moment throughout the day.
The correct regulation of cell growth and proliferation is essential during normal animal development. Myc proteins function as transcription factors, being involved in the con-trol of many growth- and proliferation-associated genes and deregulation of Myc is one of the main driving factors of human malignancies.
The first part of this thesis focuses on the identification of directly regulated Myc target genes in Drosophila melanogaster, by combining ChIPseq and RNAseq approaches. The analysis results in a core set of Myc target genes of less than 300 genes which are mainly involved in ribosome biogenesis. Among these genes we identify a novel class of Myc targets, the non-coding small nucleolar RNAs (snoRNAs). In vivo studies show that loss of snoRNAs not only impairs growth during normal development, but that overexpression of several snoRNAs can also enhance tumor development in a neu-ronal tumor model. Together the data show that Myc acts as a master regulator of ribo-some biogenesis and that Myc’s transforming effects in tumor development are at least partially mediated by the snoRNAs.
In the second part of the thesis, the interaction of Myc and the Zf-protein Chinmo is described. Co-immunoprecipitations of the two proteins performed under endogenous and exogenous conditions show that they interact physically and that neither the two Zf-domains nor the BTB/POZ-domain of Chinmo are important for this interaction. Fur-thermore ChIP experiments and Myc dependent luciferase assays show that Chinmo and Myc share common target genes, and that Chinmo is presumably also involved in their regulation. While the exact way of how Myc and Chinmo genetically interact with each other still has to be investigated, we show that their interaction is important in a tumor model. Overexpression of the tumor-suppressors Ras and Chinmo leads to tu-mor formation in Drosophila larvae, which is drastically impaired upon loss of Myc.
The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression.
Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability.
The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis.
ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process.
DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing.
Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets.
ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines.
Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.
The enteric nervous system (ENS) innervates the gastrointestinal (GI) tract and controls central aspects of GI physiology including contractility of the intestinal musculature, glandular secretion and intestinal blood flow. The ENS is composed of neurons that conduct electrical signals and of enteric glial cells (EGCs). EGCs resemble central nervous system (CNS) astrocytes in their morphology and in the expression of shared markers such as the intermediate filament protein glial fibrillary acidic protein (GFAP). They are strategically located at the interface of ENS neurons and their effector cells to modulate intestinal motility, epithelial barrier stability and inflammatory processes. The specific contributions of EGCs to the maintenance of intestinal homeostasis are subject of current research.
From a clinical point of view EGC involvement in pathophysiological processes such as intestinal inflammation is highly relevant. Like CNS astrocytes ECGs can acquire a reactive, tissue-protective phenotype in response to intestinal injury. In patients with chronic inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, alterations in the EGC network are well known, particularly a differential expression of GFAP, which is a hallmark of reactive gliosis in the CNS.
With increasing recognition of the role of EGCs in intestinal health and disease comes the need to study the glial population in its complexity. The overall aim of this thesis was to comprehensively study EGCs with focus on the reactive GFAP-expressing subpopulation under inflammatory conditions in vivo and in vitro. In a first step, a novel in vivo rat model of acute systemic inflammation mimicking sepsis was employed to investigate rapidly occuring responses of EGCs to inflammation. This study revealed that within a short time frame of a few hours, EGCs responded to the inflammation with an upregulation of Gfap gene expression. This inflammation-induced upregulation was confined to the myenteric plexus and varied in intensity along the intestinal rostro-caudal axis. This highly responsive myenteric GFAP-expressing EGC population was further characterized in vivo andin vitro using a transgenic mouse model (hGFAP-eGFP mice). Primary purified murine GFAP-EGC cultures in vitro were established and it was assessed how the transcriptomic and proteomic profiles of these cells change upon inflammatory stimulation. Here, myenteric GFAP-EGCs were found to undergo a shift in gene expression profile that predominantly affects expression of genes associated with inflammatory responses. Further, a secretion of inflammatory mediators was validated on protein level. The GFAP+ subpopulation is hence an active participant in inflammatory pathophysiology. In an acute murine IBD model in vivo, GFAP-EGCs were found to express components of the major histocompatibility complex (MHC) class II in inflamed tissue, which also indicates a crosstalk of EGCs with the innate and the adaptive lamina propria immune system in acute inflammation.
Taken together, this work advances our knowledge on EGC (patho-)physiology by identifying and characterizing an EGC subpopulation rapidly responsive to inflammation. This study further provides the transcriptomic profile of this population in vivo and in vitro, which can be used to identify targets for therapeutic intervention. Due to the modulating influence of EGCs on the intestinal microenvironment, the study further underlines the importance of integrating EGCs into in vitro test systems that aim to model intestinal tissues in vitro and presents an outlook on a potential strategy.
The microbial communities that live inside the human gastrointestinal tract -the human gut
microbiome- are important for host health and wellbeing. Characterizing this new “organ”,
made up of as many cells as the human body itself, has recently become possible through
technological advances. Metagenomics, the high-throughput sequencing of DNA directly from
microbial communities, enables us to take genomic snapshots of thousands of microbes living
together in this complex ecosystem, without the need for isolating and growing them.
Quantifying the composition of the human gut microbiome allows us to investigate its
properties and connect it to host physiology and disease. The wealth of such connections was
unexpected and is probably still underestimated. Due to the fact that most of our dietary as well
as medicinal intake affects the microbiome and that the microbiome itself interacts with our
immune system through a multitude of pathways, many mechanisms have been proposed to
explain the observed correlations, though most have yet to be understood in depth.
An obvious prerequisite to characterizing the microbiome and its interactions with the host is
the accurate quantification of its composition, i.e. determining which microbes are present and
in what numbers they occur. Historically, standard practices have existed for sample handling,
DNA extraction and data analysis for many years. However, these were generally developed for
single microbe cultures and it is not always feasible to implement them in large scale
metagenomic studies. Partly because of this and partly because of the excitement that new
technology brings about, the first metagenomic studies each took the liberty to define their own
approach and protocols. From early meta-analysis of these studies it became clear that the
differences in sample handling, as well as differences in computational approaches, made
comparisons across studies very difficult. This restricts our ability to cross-validate findings of
individual studies and to pool samples from larger cohorts. To address the pressing need for
standardization, we undertook an extensive comparison of 21 different DNA extraction methods
as well as a series of other sample manipulations that affect quantification. We developed a
number of criteria for determining the measurement quality in the absence of a mock
community and used these to propose best practices for sampling, DNA extraction and library
preparation. If these were to be accepted as standards in the field, it would greatly improve
comparability across studies, which would dramatically increase the power of our inferences
and our ability to draw general conclusions about the microbiome.
Most metagenomics studies involve comparisons between microbial communities, for example
between fecal samples from cases and controls. A multitude of approaches have been proposed
to calculate community dissimilarities (beta diversity) and they are often combined with
various preprocessing techniques. Direct metagenomics quantification usually counts
sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to
technology-inherent differences in sampling depth, normalizing counts is necessary, for
instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by
subsampling. To derive a single value for community (dis-)similarity, multiple distance
measures have been proposed. Although it is theoretically difficult to benchmark these
approaches, we developed a biologically motivated framework in which distance measures can
be evaluated. This highlights the importance of data transformations and their impact on the
measured distances.
Building on our experience with accurate abundance estimation and data preprocessing
techniques, we can now try and understand some of the basic properties of microbial
communities. In 2011, it was proposed that the space of genus level variation of the human gut
microbial community is structured into three basic types, termed enterotypes. These were
described in a multi-country cohort, so as to be independent of geography, age and other host
properties. Operationally defined through a clustering approach, they are “densely populated
areas in a multidimensional space of community composition”(source) and were proposed as a
general stratifier for the human population. Later studies that applied this concept to other
datasets raised concerns about the optimum number of clusters and robustness of the
clustering approach. This heralded a long standing debate about the existence of structure and
the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in
the context of the vastly increased amounts of available data. We propose a refined framework
in which the different types should be thought of as weak attractors in compositional space and
we try to implement an approach to determining which attractor a sample is closest to. To this
end, we train a classifier on a reference dataset to assign membership to new samples. This way,
enterotypes assignment is no longer dataset dependent and effects due to biased sampling are
minimized. Using a model in which we assume the existence of three enterotypes characterized
by the same driver genera, as originally postulated, we show the relevance of this stratification
and propose it to be used in a clinical setting as a potential marker for disease development.
Moreover, we believe that these attractors underline different rules of community assembly and
we recommend they be accounted for when analyzing gut microbiome samples.
While enterotypes describe structure in the community at genus level, metagenomic sequencing
can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide
polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis
methodology for this level of resolution has only recently been developed and little exploration
has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the
landscape of genomic variation seems highly structured even beyond species resolution,
indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several
cases, these subspecies exhibit geo-stratification, with some subspecies only found in the
Chinese population. Generally however, they present only minor dispersion limitations and are
seen across most of our study populations. Within one individual, one subspecies is commonly
found to dominate and only rarely are several subspecies observed to co-occur in the same
ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable
over periods of more than three years. When interrogating their functional properties we find
many differences, with specific ones appearing relevant to the host. For example, we identify a
subspecies of E. rectale that is lacking the flagellum operon and find its presence to be
significantly associated with lower body mass index and lower insulin resistance of their hosts;
it also correlates with higher microbial community diversity. These associations could not be
seen at the species level (where multiple subspecies are convoluted), which illustrates the
importance of this increased resolution for a more comprehensive understanding of microbial
interactions within the microbiome and with the host.
Taken together, our results provide a rigorous basis for performing comparative metagenomics
of the human gut, encompassing recommendations for both experimental sample processing
and computational analysis. We furthermore refine the concept of community stratification into
enterotypes, develop a reference-based approach for enterotype assignment and provide
compelling evidence for their relevance. Lastly, by harnessing the full resolution of
metagenomics, we discover a highly structured genomic variation landscape below the
microbial species level and identify common subspecies of the human gut microbiome. By
developing these high-precision metagenomics analysis tools, we thus hope to contribute to a
greatly improved understanding of the properties and dynamics of the human gut microbiome.
Gambling is a popular activity in Germany, with 40% of a representative sample reporting having gambled at least once in the past year (Bundeszentrale für gesundheitliche Aufklärung, 2014). While the majority of gamblers show harmless gambling behavior, a subset develops serious problems due to their gambling, affecting their psychological well-being, social life and work. According to recent estimates, up to 0.8% of the German population are affected by such pathological gambling. People in general and pathological gamblers in particular show several cognitive distortions, that is, misconceptions about the chances of winning and skill involvement, in gambling. The current work aimed at elucidating the biopsychological basis of two such kinds of cognitive distortions, the illusion of control and the gambler’s and hot hand fallacies, and their modulation by gambling problems. Therefore, four studies were conducted assessing the processing of near outcomes (used as a proxy for the illusion of control) and outcome sequences (used as a proxy for the gambler’s and hot hand fallacies) in samples of varying degrees of gambling problems, using a multimethod approach.
The first study analyzed the processing and evaluation of near outcomes as well as choice behavior in a wheel of fortune paradigm using electroencephalography (EEG). To assess the influence of gambling problems, a group of problem gamblers was compared to a group of controls. The results showed that there were no differences in the processing of near outcomes between the two groups. Near compared to full outcomes elicited smaller P300 amplitudes. Furthermore, at a trend level, the choice behavior of participants showed signs of a pattern opposite to the gambler’s fallacy, with longer runs of an outcome color leading to increased probabilities of choosing this color again on the subsequent trial. Finally, problem gamblers showed smaller feedback-related negativity (FRN) amplitudes relative to controls.
The second study also targeted the processing of near outcomes in a wheel of fortune paradigm, this time using functional magnetic resonance imaging and a group of participants with varying degrees of gambling problems. The results showed increased activity in the bilateral superior parietal cortex following near compared to full outcomes.
The third study examined the peripheral physiology reactions to near outcomes in the wheel of fortune. Heart period and skin conductance were measured while participants with varying degrees of gambling problems played on the wheel of fortune. Near compared to full outcomes led to increased heart period duration shortly after the outcome. Furthermore, heart period reactions and skin conductance responses (SCRs) were modulated by gambling problems. Participants with high relative to low levels of gambling problems showed increased SCRs to near outcomes and similar heart period reactions to near outcomes and full wins.
The fourth study analyzed choice behavior and sequence effects in the processing of outcomes in a coin toss paradigm using EEG in a group of problem gamblers and controls. Again, problem gamblers showed generally smaller FRN amplitudes compared to controls. There were no differences between groups in the processing of outcome sequences. The break of an outcome streak led to increased power in the theta frequency band. Furthermore, the P300 amplitude was increased after a sequence of previous wins. Finally, problem gamblers compared to controls showed a trend of switching the outcome symbol relative to the previous outcome symbol more often.
In sum, the results point towards differences in the processing of near compared to full outcomes in brain areas and measures implicated in attentional and salience processes. The processing of outcome sequences involves processes of salience attribution and violation of expectations. Furthermore, problem gamblers seem to process near outcomes as more win-like compared to controls. The results and their implications for problem gambling as well as further possible lines of research are discussed.
Adjuvants are compounds added to an agrochemical spray formulation to improve or modify the action of an active ingredient (AI) or the physico-chemical characteristics of the spray liquid. Adjuvants can have more than only one distinct mode of action (MoA) during the foliar spray application process and they are generally known to be the best tools to improve agrochemical formulations. The main objective for this work was to elucidate the basic MoA of adjuvants by uncoupling different aspects of the spray application. Laboratory experiments, beginning from retention and spreading characteristics, followed by humectant effects concerning the spray deposit on the leaf surface and ultimately the cuticular penetration of an AI, were figured out to evaluate overall in vivo effects of adjuvants which were also obtained in a greenhouse spray test. For this comprehensive study, the surfactant classes of non-ionic sorbitan esters (Span), polysorbates (Tween) and oleyl alcohol polyglycol ether (Genapol O) were generally considered because of their common promoting potential in agrochemical formulations and their structural diversity.
The reduction of interfacial tension is one of the most crucial physico-chemical properties of surfactants. The dynamic surface tension (DST) was monitored to characterise the surface tension lowering behaviour which is known to influence the droplet formation and retention characteristics. The DST is a function of time and the critical time frame of droplet impact might be at about 100 ms. None of the selected surfactants were found to lower the surface tension sufficiently during this short timeframe (chapter I). At ca. 100 ms, Tween 20 resulted in the lowest DST value. When surfactant monomers are fully saturated at the droplet-air-interface, an equilibrium surface tension (STeq) value can be determined which may be used to predict spreading or run-off effects. The majority of selected surfactants resulted in a narrow distribution of STeq values, ranging between 30 and 45 mN m- 1. Nevertheless, all surfactants were able to decrease the surface tension considerably compared to pure water (72 mN m- 1). The influence of different surfactants on the wetting process was evaluated by studying time-dependent static contact angles on different surfaces and the droplet spread area on Triticum aestivum leaves after water evaporation. The spreading potential was observed to be better for Spans than for Tweens. Especially Span 20 showed maximum spreading results. To transfer laboratory findings to spray application, related to field conditions, retention and leaf coverage was measured quantitatively on wheat leaves by using a variable track sprayer. Since the retention process involves short time dynamics, it is well-known that the spray retention on a plant surface is not correlated to STeq but to DST values. The relationship between DST at ca. 100 ms and results from the track sprayer showed increasing retention results with decreasing DST, whereas at DST values below ca. 60 mN m- 1 no further retention improvement could be observed.
Under field conditions, water evaporates from the droplet within a few seconds to minutes after droplet deposition on the leaf surface. Since precipitation of the AI must essentially being avoided by holding the AI in solution, so-called humectants are used as tank-mix adjuvants. The ability of pure surfactants to absorb water from the surrounding atmosphere was investigated comprehensively by analysing water sorption isotherms (chapter II). These isotherms showed an exponential shape with a steep water sorption increase starting at 60% to 70% RH. Water sorption was low for Spans and much more distinct for the polyethoxylated surfactants (Tweens and Genapol O series). The relationship between the water sorption behaviour and the molecular structure of surfactants was considered as the so-called humectant activity. With an increasing ethylene oxide (EO) content, the humectant activity increased concerning the particular class of Genapol O. However, it could be shown that the moisture absorption across all classes of selected surfactants correlates rather better with their hydrophilic-lipophilic balance values with the EO content.
All aboveground organs of plants are covered by the cuticular membrane which is therefore the first rate limiting barrier for AI uptake. In vitro penetration experiments through an astomatous model cuticle were performed to study the effects of adjuvants on the penetration of the lipophilic herbicide Pinoxaden (PXD) (chapter III). In order to understand the influence of different adjuvant MoA like humectancy, experiments were performed under three different humidity levels. No explicit relationship could be found between humidity levels and the PXD penetration which might be explained by the fact that humidity effects would rather affect hydrophilic AIs than lipophilic ones. Especially for Tween 20, it became obvious that a complex balance between multiple MoA like spreading, humectancy and plasticising effects have to be considered.
Greenhouse trials, focussing the adjuvant impact on in vivo action of PXD, were evaluated on five different grass-weed species (chapter III). Since agrochemical spray application and its following action on living plants also includes translocation processes in planta and species dependent physiological effects, this investigation may help to simulate the situation on the field. Even though the absolute weed damage was different, depending both on plant species and also on PXD rates, adjuvant effects in greenhouse experiments displayed the same ranking as in cuticular penetration studies: Tween 20 > Tween 80 > Span 20 ≥ Span 80.
Thus, the present work shows for the first time that findings obtained in laboratory experiments can be successfully transferred to spray application studies on living plants concerning adjuvant MoA. A comparative analysis, using radar charts, could demonstrate systematic derivations from structural similarities of adjuvants to their MoA (summarising discussion and outlook). Exemplarily, Tween 20 and Tween 80 cover a wide range of selected variables by having no outstanding MoA improving one distinct process during foliar application, compared to non-ethoxylated Span 20 and Span 80 which primarily revealed a surface active action. Most adjuvants used in this study represent polydisperse mixtures bearing a complex distribution of EO and aliphatic chains. From this study it seems alike that adjuvants having a wide EO distribution offer broader potential than adjuvants with a small EO distribution. It might be a speculation that due to this broad distribution of single molecules, all bearing their individual specific physico-chemical nature, a wide range of properties concerning their MoA is covered.
Bacterial small non-coding RNAs (sRNAs) play fundamental roles in controlling and finetuning gene expression in a wide variety of cellular processes, including stress responses, environmental signaling and virulence in pathogens. Despite the identification of hundreds of sRNA candidates in diverse bacteria by genomics approaches, the mechanisms and regulatory capabilities of these posttranscriptional regulators have most intensively been studied in Gram-negative Gammaproteobacteria such as Escherichia coli and Salmonella. So far, almost nothing is known about sRNA-mediated regulation (riboregulation) in Epsilonproteobacteria, including the major human pathogen Helicobacter pylori. H. pylori was even thought to be deficient for riboregulation as none of the sRNAs known from enterobacteria are conserved in Helicobacter and since it lacks the major RNA chaperone Hfq, which is crucial for sRNA function as well as stability in many bacteria. Nonetheless, more than 60 cis- and trans-acting sRNA candidates were recently identified in H. pylori by a global RNA sequencing approach, indicating that this pathogen, in principle, has the capability to use riboregulation for its gene expression control. However, the functions and underlying mechanisms of H. pylori sRNAs remained unclear.
This thesis focused on the first functional characterization and target gene identification of a trans-acting sRNA, RepG (Regulator of polymeric G-repeats), in H. pylori. Using in-vitro and in-vivo approaches, RepG was shown to directly base-pair with its C/Urich terminator loop to a variable homopolymeric G-repeat in the 5’ untranslated region (UTR) of the tlpB mRNA, thereby regulating expression of the chemotaxis receptor TlpB. While the RepG sRNA is highly conserved, the length of the G-repeat in the tlpB mRNA leader varies among different H. pylori isolates, resulting in a strain-specific tlpB regulation. The modification of the number of guanines within the G-stretch in H. pylori strain 26695 demonstrated that the length of the homopolymeric G-repeat determines the outcome of posttranscriptional control (repression or activation) of tlpB by RepG. This lengthdependent targeting of a simple sequence repeat by a trans-acting sRNA represents a new twist in sRNA-mediated regulation and a novel mechanism of gene expression control, since it uniquely links phase variation by simple sequence repeats to posttranscriptional regulation.
In almost all sequenced H. pylori strains, tlpB is encoded in a two gene operon upstream of HP0102, a gene of previously unknown function. This study provided evidence that HP0102 encodes a glycosyltransferase involved in LPS O-chain and Lewis x antigen production. Accordingly, this glycosyltransferase was shown to be essential for mice colonization by H. pylori. The coordinated posttranscriptional regulation of the tlpB-HP0102 operon by antisense base-pairing of RepG to the phase-variable G-repeat in the 5’ UTR of the tlpB mRNA allows for a gradual, rather than ON/OFF, control of HP0102 expression, thereby affecting LPS biosynthesis in H. pylori. This fine-tuning of O-chain and Lewis x antigen expression modulates H. pylori antibiotics sensitivity and thus, might be advantageous for Helicobacter colonization and persistence.
Whole transcriptome analysis based on microarray and RNA sequencing was used to identify additional RepG target mRNAs and uncover the physiological role of this riboregulator in H. pylori. Altogether, repG deletion affected expression of more than 40 target gene candidates involved various cellular processes, including membrane transport and adhesion, LPS modification, amino acid metabolism, oxidative and nitrosative stress, and nucleic acid modification. The presence of homopolymeric G-repeats/G-rich sequences in almost all target mRNA candidates indicated that RepG hijacks a conserved motif to
recognize and regulate multiple target mRNAs in H. pylori.
Overall, this study demonstrates that H. pylori employs riboregulation in stress response and virulence control. In addition, this thesis has successfully established Helicobacter as a new model organism for investigating general concepts of gene expression control by Hfq-independent sRNAs and sRNAs in bacterial pathogens.
Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach.
This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching.
Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect.
The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature.
Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.
Early life stress, including exposure to prenatal stress (PS), has been shown to affect the developing brain and induce severe effects on emotional health in later life, concomitant with an increased risk for psychopathology. However, some individuals are more vulnerable to early-life stress, while others adapt successfully, i.e. they are resilient and do not succumb to adversity. The molecular substrates promoting resilience in some individuals and vulnerability in other individuals are as yet poorly investigated. A polymorphism in the serotonin transporter gene (5HTT/SLC6A4) has been suggested to play a modulatory role in mediating the effects of early-life adversity on psychopathology, thereby rendering carriers of the lower-expressing short (s)-allele more vulnerable to developmental adversity, while long (l)-allele carriers are relatively resilient. The molecular mechanisms underlying this gene x environment interaction (GxE) are not well understood, however, epigenetic mechanisms such as DNA methylation and histone modifications have been discussed to contribute as they are at the interface of environment and the genome. Moreover, developmental epigenetic programming has also been postulated to underlie differential vulnerability/resilience independent of genetic variation.
The present work comprises two projects investigating the effects of prenatal maternal restraint stress in 5-HTT deficient mice. In the first study, we examined to which extent previously observed changes in behavior and hippocampal gene expression of female 5-Htt+/- prenatally stressed (PS) offspring were associated with changes in DNA methylation patterns. Additionally, we investigated the expression of genes involved in myelination in hippocampus and amygdala of those animals using RT-qPCR. The genome-wide hippocampal DNA methylation screening was performed using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip® Mouse Promoter 1.0R arrays. In order to correlate individual gene-specific DNA methylation, mRNA expression and behavior, we used hippocampal DNA from the same mice as assessed before. 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a part of which were also differentially expressed. More specifically, we identified a differentially methylated region in the Myelin basic protein (Mbp) gene, which was associated with Mbp expression in a 5-Htt-, PS- and 5-Htt x PS-dependent manner. Subsequent fine-mapping linked the methylation status of two specific CpG sites in this region to Mbp expression and anxiety-related behavior. We furthermore found that not only the expression of Mbp but of large gene set associated with myelination was affected by a 5-Htt x PS interaction in a brain-region specific manner. In conclusion, hippocampal DNA methylation patterns and expression profiles of female PS 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are associated with changes in gene promoter methylation, and processes associated with myelination contribute to the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction.
In the second study, we aimed at investing the molecular substrates underlying resilience to PS. For this purpose, we exposed 5-Htt+/+ dams to the same restraint stress paradigm and investigated the effects of PS on depression- and anxiety-like behavior and corticosterone (CORT) secretion at baseline and after acute restraint stress in female 5-Htt+/+ and 5-Htt+/- offspring. We found that PS affected the offspring’s social behavior in a negative manner. When specifically examining those PS animals, we grouped the PS offspring of each genotype into a social, resilient and an unsocial, vulnerable group. While anxiety-like behavior in the EPM was reduced in unsocial, but not social, PS 5-Htt+/+ animals when compared to controls, this pattern could not be found in animals of the other genotype, indicating that social anxiety and state anxiety in the EPM were independent of each other. We then assessed genome-wide hippocampal gene expression profiles using mRNA sequencing in order to identify pathways and gene ontology (GO) terms enriched due to 5-Htt genotype (G), PS exposure (E) and their interaction (GxE) as well as enriched in social, but not unsocial, PS offspring, and vice versa. Numerous genes were affected by 5-Htt genotype, PS and most of all a GxE-interaction. Enrichment analysis using enrichr identified that the genotype affected mitochondrial respiration, while GxE-interaction-affected processes associated primarily with myelination and chromatin remodeling. We furthermore found that 5-Htt+/- mice showed profound expression changes of numerous genes in a genomic region located 10 mio kb upstream of the 5 Htt locus on the same chromosome. When looking at social vs. unsocial mice, we found that a much higher number of genes was regulated in 5 Htt+/- animals than in 5-Htt+/+ animals, reflecting the impact of GxE-interaction. Double the number of genes was regulated in social PS vs. control mice when compared to unsocial PS vs. control in both genotypes, suggesting that the successful adaption to PS might have required more active processes from the social group than the reaction to PS from the unsocial group. This notion is supported by the up-regulation of mitochondrial respiration in social, but not in unsocial, PS 5-Htt+/- mice when compared to controls, as those animals might have been able to raise energy resources the unsocial group was not. Next to this, processes associated with myelination seemed to be down-regulated in social 5-Htt+/- mice, but not in unsocial animals, when compared to controls. Taken together, PS exposure affected sociability and anxiety-like behavior dependent on the 5-Htt genotype in female offspring. Processes associated with myelination and epigenetic mechanisms involved in chromatin remodeling seemed be affected in a GxE-dependent manner in the hippocampus of these offspring. Our transcriptome data furthermore suggest that mitochondrial respiration and, with this, energy metabolism might be altered in 5-Htt+/- offspring when compared to 5-Htt+/+ offspring. Moreover, myelination and mitochondrial respiration might contribute to resilience towards PS exposure in 5-Htt+/- offspring, possibly by affecting brain connectivity and energy capabilities.
Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature.
Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMA.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of enzymes with important functions in physiology and disease. HAD phosphatases can target diverse metabolites, lipids, DNA, and serine/threonine or tyrosine phosphorylated proteins with often high specificity (Seifried et al., 2013). These enzymes thus markedly enlarge the repertoire and substrate spectrum of mammalian phosphatases. However, the basis of HAD phosphatase substrate specificity is still elusive and a number of mammalian HAD phosphatases remain uncharacterized to date. This study characterizes the biochemical and structural properties of AUM (aspartate-based, ubiquitous, Mg2+-dependent phosphatase), a previously unexplored mammalian HAD phosphatase.
In vitro phosphatase assays of purified, recombinant AUM showed phosphatase activity towards para-nitrophenyl phosphate and adenine and guanine nucleotide di- and triphosphates. Inhibitor studies indicated that similar to other HAD superfamily members, the AUM-catalyzed dephosphorylation reaction proceeds via a pentacovalent phosphoaspartate intermediate. In line with an aspartate-based catalytic mechanism, AUM was insensitive to inhibitors of serine/threonine phosphatases. The characterization of the purified recombinant murine enzyme also revealed that AUM exists in equilibrium between dimers and tetramers.
AUM was identified as the closest, yet functionally distinct relative of chronophin, a pyridoxal 5’-phosphate and serine/threonine-directed phosphatase. Phylogenetic analyses showed that AUM and chronophin evolved via duplication of an ancestral gene at the origin of the vertebrates. In contrast to chronophin, AUM acts as a tyrosine-specific HAD-type phosphatase in vitro and in cells. To elucidate how AUM and chronophin achieve these distinct substrate preferences, comparative evolutionary analyses, biochemical approaches and structural analyses were combined. Swapping experiments of less homologous regions between AUM and chronophin were performed. The mutational analysis revealed residues important for AUM catalysis and specificity. A single differently conserved residue in the cap domain of AUM or chronophin is crucial for phosphatase specificity (AUML204, chronophinH182). The X-ray crystal structure of the AUM cap fused to the catalytic core of chronophin (CAC, PDB: 4BKM) was solved to 2.65 Å resolution. It presents the first crystal structure of the murine AUM capping domain. The detailed view of the catalytic clefts of AUM and chronophin reveals the structural basis of the divergent substrate specificities. These presented findings provide insights into the design principles of capped HAD phosphatases and show that their substrate specificity can be encoded by a small number of predictable residues. In addition, the catalytic properties of AUM were investigated, identifying a mechanism of reversible oxidation regulating the activity of AUM in vitro. AUM phosphatase activity is inhibited by oxidation and can be recovered by reduction. The underlying molecular mechanism was revealed by mutational analyses. The cysteines C35, C104 and C243, located in the AUM core domain, are responsible for the inhibition of AUM by oxidation. C293 mediates the redox-dependent tetramerization of AUM in vitro. Based on the chronophin and CAC structure, a direct impact of the oxidation of C35 on the nucleophile D34 is proposed. In addition, a redox-dependent disulfide bridge (C104, C243), connecting the core and cap domain of AUM may be important for an open/close-mechanism. This hypothesis is supported by CD spectroscopy experiments that demonstrate a structural change in AUM upon reduction. These data present the first evidence for the regulation of AUM catalysis by reversible oxidation. This finding is so far unique in the field of HAD phosphatases.
In this context, the first cell-based AUM activity assay was developed. For this, the artificial substrate pNPP was combined with the reducing agent DTT to create a specific AUM activity readout. This fractionation-based assay is the first tool to differentiate between cell lines or tissues with different AUM concentrations or activities.
Taken together, the presented biochemical characterization reveals the specificity determinants and catalytic properties of AUM. General insights into structural determinants of mammalian HAD phosphatase substrate recognition are provided and reversible oxidation as possible regulatory mechanism for AUM is proposed. These findings constitute a framework for further functional analyses to elucidate the biomedical importance of AUM.
Several cohort studies showed that obesity increases the risk of chronic disease such as T2DM, hypertension and non-alcoholic fatty liver disease and various types of cancer. Different factors were described that might be involving in these diseases in obesity. Some of these suggested factors were chronic infection, elevated free fatty acids, increased ROS formation, mitochondrial dysfunction and raised NAPDH oxidase activity. Obesity is a multifactorial disease and it is very hard to distinguish between all of these factors. In this study, we wanted to focus on the association between obesity, oxidative stress and genomic damage in kidney, liver and colon, which are the most relevant organs for cancer risk according to the cohort studies. Our findings indicated elevated oxidative stress in kidney, liver and colon together with elevated lipid, RNA and DNA oxidation in the whole body. Additionally, we were able to show increased DNA damage in kidney, liver and colon.
Since obesity has become an epidemic all over the world, possible therapeutic applications such as life style changes (diet and sport), pharmacological supplements and various type of surgeries are increasing. As a second question, we focused on the effect of weight loss, which is supplied either by Roux-en-Y gastric bypass surgery or by caloric restriction designed in a way to provide the same extent of weight loss, on oxidative stress and genomic damage. Our results indicated that weight loss either by gastric bypass surgery or by caloric restriction led to reduced oxidative stress and genomic damage in kidney, liver and colon. We could not find any difference between the weight loss methods, except the DNA oxidation and repair marker urinary 8-oxodG, which was still elevated after RYGB, but not after caloric restriction.
It is known that hyperinsulinemia and in the long term T2DM are among the biggest concerns in obese individuals. Since we know the mutagenic potential of elevated insulin levels from previous data in our working group, the correlation between the highly mutagenic DNA DBSs marker, γ-H2AX and the plasma insulin level was tested and the findings indicated a positive correlation. In order to demonstrate the association between insulin-related oxidative stress and genomic damage, we used in vitro and in vivo models with Pten deficiency. In this part of study, the work was focused on liver.
Pten is a known negative regulator of the PI3K/Akt pathway, which is responsible for the elevated NADPH oxidase activity and mitochondrial dysfunction through elevated insulin levels. Pten inhibition or deficiency were used to sensitize the system to insulin. Non-transformed immortalized human hepatocytes were used to show the mutagenic potential of elevated insulin and these in vitro data revealed once more the link between insulin signaling, elevated oxidative stress and genomic damage. Since the metabolic function of the liver is not only due to the extent of the hepatic insulin response but is also affected by systemic interactions, a whole-body Pten haplodeficient mouse model with an additional Pten+/-/Akt2-/- group was utilized for in vivo investigation of insulin-mediated toxicity. Our findings in this model suggested that Pten deficiency alone can cause an increase in oxidative stress. HFD alone was sufficient to increase the expression of HO-1 and genomic damage significantly. Moreover, the combination (whole-body Pten haplodeficient mice fed with HFD) showed significantly elevated oxidative stress and genomic damage in mouse liver. However, Akt2 knockout could only reduce the oxidative stress and DNA damage in high fat diet fed mice significantly.
All these findings demonstrated that obesity can induce oxidative stress and genomic damage. Elevated insulin levels are associated with obesity-mediated oxidative stress and genomic damage. However, the underlying mechanisms are surely multifaceted and complicated. For example, Pten as oncogene might also induce other mechanisms besides the elevation of the PI3K/Akt pathway activity.
In conclusion, it is clear that oxidative stress and DNA damage are linked to obesity and that weight loss can reduce these two factors. Since DNA-damage is associated with an elevated cancer risk, it might be logical to use an antioxidant therapy in obese individuals to reduce the side effects and oxidative stress dependent mutagenicity and cancer risk in these individuals. However, much more research will be needed to support this idea experimentally.
The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms’ behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly’s circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock’s interneuronal communication.
Drosophila melanogaster’s circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly’s locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature.
Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs).
The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs & l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day.
To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner.
However, the molecular link between clock neuron depolarization and shifts in the molecular clock’s cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock.
Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.
Effect of cytokine inhibition on peripheral memory B cells in patients with Rheumatoid arthtritis
(2015)
Objective: Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disease. Enhanced B cell activity has been proposed in the pathogenesis of RA along with different pro-inflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), critically involved in chronic inflammation. Biological agents targeting these cytokines IL-6 and TNF-α have considerably advanced treatment of autoimmunity. Enhanced B cell activity, particularly memory B cells gained particularly interest in evaluating response during therapies from biologics. Human peripheral memory B cells can be distinguished by the phenotypic expression of CD27 and IgD defining three major B cell subpopulations: CD27+IgD+ pre-switch, CD27+IgD- post-switch and CD27-IgD- double negative (DN) memory B cells. Therefore, we analyzed different memory populations during cytokine inhibition by using tocilizumab (anti-IL-6R, TCZ) and adalimumab (anti-TNF-α, ADA), with focus on DN B cells Suspended. DN B cells lacking the conventional memory marker CD27, but due to their mutational Ig repertoire (IgR) considered in the memory compartment. However, only scare data are available for this DN subpopulation in RA.
Methods: Phenotype analysis of activation markers (CD95 and ki-67) of B cell and their subsets were compared in RA patients (median age ~56 years) and in HD. DN memory B cells were phenotypically analyzed from RA patients during IL-6R or TNF-α inhibition at baseline week 12, week 24 and 1 year. Single B cell PCR approach was used to study Ig- receptors VH genes and isotype specific genes. Nonparametric Wilcoxon matched pair test and Mann-Whitney U test was used for statistical analysis by using GraphPadPrism 5. Univariate logistic regression was used to calculate odd ratios and correlation using Pearson r using SPSS statistics 22.
Results: Surface and intracellular staining of B cells showed a significantly higher percentage of CD95 and ki-67 expressions in RA, which was highest in post-switch memory B cells followed by pre-switch and DN memory B cells. During cytokines (IL-6R & TNF-α) inhibition, both CD95 and ki-67 expression were significantly reduced at week 12 and 24 along with reduction in their clinical parameters like DAS28, CRP, ESR. Furthermore, the phenotypic analysis in 107 RA patients and 49 healthy donors (HD) showed a significantly expanded population of DN B cells in RA which contain a heterogeneous mixture of IgA, IgG and IgM expressing cells with a clear dominance of IgG+ cells. Pre-therapy analysis of rearranged IgR sequences from patients (n=9) revealed that DN B cells carry rearranged heavy chain gene sequences with a diversified mutational pattern consistent with memory B cells. In contrast to tumor necrosis factor alpha (TNF-alpha) inhibition, a significant reduction in mutational frequency of BCR gene rearrangements at week 12, 24 and 1 year (p < 0.0001) was observed by in vivo IL-6R inhibition. These changes were observed for all BCR isotypes IgG, IgA and IgM at week 12, 24 and 1 year (p < 0.0001). IgA-RF, IgA serum level and IgA+ DN B cells decreased significantly (p < 0.05) at week 12 and week 24 during TCZ. Patients with a good European league against rheumatism (EULAR) response to TCZ had less DN B cells at baseline as compared to moderate responders (p = 0.006). Univariate logistic regression analysis revealed that the frequency of DN B cells at baseline is inversely correlated to a subsequent good EULAR response (p = 0.024) with an odds ratio of 1.48 (95% confidence interval as 1.05-2.06).
Conclusion: Both anti-TNF-α and anti-IL-6R could reduce higher B cell activity and improve disease activity tremendously in RA patients. The heterogeneous DN B cell compartment is expanded in RA and dominated by IgG isotype. TCZ can modulate the mutational status of DN Ig isotype receptors over 1 year. Interestingly, the frequency of DN B cells in RA may serve as a baseline predictor of subsequent EULAR response to TCZ.
Structural and biochemical characterization of gephyrin and various gephyrin-ligand complexes
(2014)
Efficient synaptic neurotransmission requires the exact apposition of presynaptic terminals and matching neurotransmitter receptor clusters on the postsynaptic side. The receptors are embedded in the postsynaptic density, which also contains scaffolding and regulatory proteins that ensure high local receptor concentrations. At inhibitory synapses the cytosolic scaffolding protein gephyrin assumes an essential organizing role within the postsynaptic density by the formation of self-oligomers which provide a high density of binding sites for certain -amino butyric acid type A (GABAA) and the large majority of glycine receptors (GlyR). Gephyrin contains two oligomerization domains: In isolation, the 20 kDa N-terminal G domain (GephG) and the 46 kDa E domain (GephE) trimerize and dimerize, respectively. In the full-length protein the domains are interconnected by a central ~150 amino acid linker, and only GephG trimerization is utilized, whereas GephE dimerization is prevented, thus suggesting the need for a trigger to release GephE autoinhibition, which would pave the way for the formation of higher oligomers and for efficient receptor clustering. The structural basis for this GephE autoinhibition has remained elusive so far, but the linker was reported to be sufficient for autoinhibition. This work dealt with the biochemical and structural characterization of apo-gephyrin and gephyrin in complexes with ligands which are known to promote the formation of synaptic gephyrin clusters (collybistin and neuroligin 2) and reorganize them (dynein light chain 1).
For full-length gephyrin no structural information has been available so far. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analyses described in this thesis disclosed that the gephyrin trimer forms a highly flexible assembly, which, due to the long linker, can switch between compact and extended conformational states in solution, with a preference for compact states. This partial compaction and potentially GephE autoinhibition are achieved by interactions of parts of the linker with the G and E domains, as suggested by circular dichroism spectroscopy. However, the linker on its own cannot account for GephE blockage, as size exclusion chromatography experiments coupled with multi angle light scattering detection (SEC-MALS) and SAXS analyses revealed that a gephyrin variant only encompassing the linker and GephE (GephLE) forms dimers and not monomers as suggested by an earlier study. The oligomeric state of GephLE and the observation that several gephyrin variants, in which linker segments of varying length were deleted, predominantly formed trimers, suggested the presence of a linker independent mechanism of GephE dimerization blockade. Taken together, the data indicated that linker-dependent and linker-independent mechanisms mediate gephyrin autoinhibition.
In the second project gephyrin’s interaction with DYNLL1 (Dynein LC8 Light Chain 1) was characterized. DYNLL1 is a 25 kDa dimer incorporated into the dynein motor and provides two binding sites, each of which can accommodate an octapeptide derived from gephyrin’s linker region (referred to as GephDB). Originally, DYNLL1 was regarded as a cargo adaptor, linking gephyrin-GlyR complexes to the dynein motor, thus driving their retrograde transport and leading to a decrease of synaptic gephyrin-GlyR complexes.
Building on these studies, this thesis assessed the cargo hypothesis as well as the so far unclear stoichiometry of the gephyrin-DYNLL1 complex. The cargo scenario would require ternary complex formation between gephyrin, DYNLL1 and the dynein intermediate chain (DIC) of the dynein motor. However, such a complex could not be detected by analytical size exclusion chromatography (aSEC) experiments – presumably because gephyrin and DIC competed for a common binding site in DYNLL1. This finding was consistent with a single DYNLL1 dimer capturing two linker segments of a single gephyrin trimer as suggested by a 26 kDa mass increase of the gephyrin species in the presence of DYNLL1 in SEC-MALS experiments. aSEC experiments at even higher concentrations (~20 µM gephyrin and ~80 µM DYNLL1) indicated that the affinity of GephDB was significantly impaired in the context of full-length gephyrin but also in a variant that bears only GephG and the first 39 residues of the linker (GephGL220). Presumably due to avidity effects two linkers stably associated with a single DYNLL1 dimer, whereas the third DYNLL1 binding motif remained predominantly unoccupied unless high concentrations of GephGL220 (50 µM) and DYNLL1 (200 µM) were used. These findings indicate that an interplay between GephG and the N-terminal linker segment mediates the attenuation of GephDB affinity towards DYNLL1 and that preventing DYNLL1 from the induction of higher gephyrin oligomers is either advantageous for DYNLL1-mediated reorganization of gephyrin-GlyR clusters or that DYNLL1 exerts possibly two (concentration-dependent) actions on gephyrin.
The gephyrin-collybistin-neuroligin 2 complex was the subject of the third project. Previously, collybistin and gephyrin were observed to mutually trigger their translocation to the postsynaptic membrane, where the disordered cytoplasmic tail of the postsynaptic cell adhesion molecule NL2 (NL2cyt) causes the anchoring of collybistin 2 (CB2) by binding to its SH3 domain, thereby releasing SH3 domain mediated autoinhibiton of CB2 binding to the membrane phospholipid phosphatidylinositol-3-phosphate. Critical for this event is the binding of gephyrin to both CB2 and NL2, presumably via GephE.
Following up on these previous studies biochemical data presented in this thesis confirm the formation of the ternary complex. Unexpectedly, analyses by means of native polyacrylamide gel electrophoresis pointed to: (1) The existence of a complex containing NL2cyt and CB2 lacking the SH3 domain and consequently an additional NL2 binding site in CB2. (2) Attenuated gephyrin-collybistin complex formation in the presence of the SH3 domain. (3) A requirement for high NL2cyt concentrations (> 30 µM) during the formation of the ternary complex. This might allow for the regulation by other factors such as additional binding partners or posttranslational modifications. Although of preliminary character, these results provide a starting point for future studies, which will hopefully elucidate the interplay between gephyrin, collybistin, NL2 and certain GABAA receptors.
Structural and Biochemical Characterization of the GABA(A) Receptor Interacting Protein Muskelin
(2015)
In a study from 2011, the protein muskelin was described as a central coordinator of the retrograde transport of GABA(A) receptors in neurons. As muskelin governs the transport along actin filaments as well as microtubules, it might be the first representative of a novel class of regulators, which coordinate cargo transport across the borders of these two independent systems of transport paths and their associated motorproteins. To establish a basis for understanding the mode of operation of muskelin, the aim of this thesis was an in-depth biochemical and structural characterization of muskelin and its interaction with the GABA(A) receptor.
One focus of the work was the analysis of the oligomerization of muskelin. As could be demonstrated, the oligomerization is based on two independent interactions mediated by different domains of the protein: a known interaction of the N-terminal discoidin domain with the C-terminal portion, termed head-to-tail interaction, and a dimerization of the LisH motif in muskelin that was so far neglected in the literature. For the detailed studies of both binding events, the solution of a crystal structure of a fragment of muskelin, comprising the Discoidin domain and the LisH motif, was an important basis. The fragment crystallized as a dimer, with dimerization being mediated solely by the LisH motif. Biochemical analysis corroborated that the LisH motif in muskelin serves as a dimerization element, and, moreover, showed that the C-terminal domain of the protein substantially stabilizes this dimerization. In addition, the crystal structure revealed the molecular composition of the surface of the head in the head-to-tail interaction, namely the discoidin domain. This information enabled to map the amino acids contributing to binding, which showed that the binding site of the head-to-tail interaction coincides with the generic ligand binding site of the discoidin domain.
As part of the analyses, residues that are critical for LisH-dimerization and the head-to-tail binding, respectively, were identified, whose mutation specifically interfered with each of the interactions separately. These mutations allowed to investigate the interplay of these interactions during oligomerization. It could be shown that recombinant muskelin assembles into a tetramer to which both interactions, the LisH-dimerization and the head-to-tail binding, contribute independently. When one of the two interactions was disturbed, only a dimer mediated via the respective other interaction could be formed; when both interactions were disturbed, the protein was present as monomer. Furthermore, Frank Heisler in the group of Matthias Kneussel was able to show the drastic impact of an impaired LisH-dimerization on muskelin in cells using these mutations. Disturbing the LisH-dimerization led to a complete redistribution of the originally cytoplasmic muskelin to the nucleus which was accompanied by a severe impairment of its function during GABA(A) receptor transport. Following up on these results in an analysis of muskelin variants, for which alterations of the subcellular localization had been published earlier, the crucial influence of LisH-dimerization to the subcellular localization and thereby the role of muskelin in the cell was confirmed.
The biochemical studies of the interaction of muskelin and the alpha1 subunit of the GABA(A) receptor demonstrated a direct binding with an affinity in the low micromolar range, which is mediated primarily by the kelch repeat domain in muskelin. For the binding site on the GABA(A) receptor, it was confirmed that the thirteen most C-terminal residues of the intracellular domain are critical for the binding of muskelin. In accordance with the strong conservation of these residues among the alpha subunits of the GABA(A) receptor, it could be shown that an interaction with muskelin in vitro is also possible for the alpha2 and alpha5 subunits. Based on the comparison of the binding sites between the homologous subunits, tentative conclusions can be drawn about the details of the binding, which may serve as a starting point for follow-up studies.
This thesis thereby makes valuable contributions to the understanding of muskelin, in particular the significance of its oligomerization. It furthermore provides an experimental framework for future studies that address related topics, such as the characterization of other muskelin interaction partners, or the questions raised in this work.
Brain-computer interfaces (BCIs) could provide a muscle-independent communication channel to persons with severe paralysis by translating brain activity into device commands. As a means of communication, in particular BCIs based on event-related potentials (ERPs) as control signal have been researched. Most of these BCIs rely on visual stimulation and have been investigated with healthy participants in controlled laboratory environments. In proof-of-principle studies targeted end users gained control over BCI systems; however, these systems are not yet established as an assistive technology for persons who would most benefit from them. The main aim of this thesis is to advance the usability of ERP-BCIs for target users. To this end, five studies with BCIs have been conducted that enabled users to communicate by focusing their attention on external stimuli.
Two studies were conducted in order to demonstrate the advantages and to further improve the practical application of visual BCIs. In the first study, mental workload was experimentally manipulated during prolonged BCI operation. The study showed the robustness of the visual ERP-BCI since users maintained a satisfactory level of control despite constant distraction in the form of background noise. Moreover, neurophysiological markers that could potentially serve as indicators of high mental workload or fatigue were revealed. This is a first step towards future applications in which the BCI could adapt to the mental state of the user (e.g. pauses if high mental workload is detected to prevent false selections). In the second study, a head-mounted display (HMD), which assures that stimuli are presented in the field of view of the user, was evaluated. High accuracies and information transfer rates, similar to a conventional display, were achieved by healthy participants during a spelling task. Furthermore, a person in the locked-in state (LIS) gained control over the BCI using the HMD. The HMD might be particularly suited for initial communication attempts with persons in the LIS in situations, where mounting a conventional monitor is difficult or not feasible.
Visual ERP-BCIs could prove valuable for persons with residual control over eye muscles and sufficient vision. However, since a substantial number of target users have limited control over eye movements and/or visual impairments, BCIs based on non-visual modalities are required. Therefore, a main aspect of this thesis was to improve an auditory paradigm that should enable motor impaired users to spell by focusing attention on different tones. The two conducted studies revealed that healthy participants were able to achieve high spelling performance with the BCI already in the first session and stress the importance of the choice of the stimulus material. The employed natural tones resulted in an increase in performance compared to a previous study that used artificial tones as stimuli. Furthermore, three out of five users with a varying degree of motor impairments could gain control over the system within the five conducted sessions. Their performance increased significantly from the first to the fifth session - an effect not previously observed for visual ERP-BCIs. Hence, training is particularly important when testing auditory multiclass BCIs with potential users.
A prerequisite for user satisfaction is that the BCI technology matches user requirements. In this context, it is important to compare BCIs with already established assistive technology. Thus, the fifth study of this dissertation evaluated gaze dependent methods (EOG, eye tracking) as possible control signals for assistive technology and a binary auditory BCI with a person in the locked-in state. The study participant gained control over all tested systems and rated the ease of use of the BCI as the highest among the tested alternatives, but also rated it as the most tiring due to the high amount of attention that was needed for a simple selection. Further efforts are necessary to simplify operation of the BCI.
The involvement of end users in all steps of the design and development process of BCIs will increase the likelihood that they can eventually be used as assistive technology in daily life. The work presented in this thesis is a substantial contribution towards the goal of re-enabling communication to users who cannot rely on motor activity to convey their thoughts.
The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age – related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation – undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse – forager transition.
In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging.
Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring.
To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.
The change of day and night is one of the challenges all organisms are exposed to, as they have to adjust their physiology and behavior in an appropriate way. Therefore so called circadian clocks have evolved, which allow the organism to predict these cyclic changes of day and night. The underlying molecular mechanism is oscillating with its endogenous period of approximately 24 hours in constant conditions, but as soon as external stimuli, so called Zeitgebers, are present, the clocks adjust their period to exactly 24h, which is called entrainment. Studies in several species, including humans, animals and plants, showed that light is the most important Zeitgeber synchronizing physiology and behavior to the changes of day and night. Nevertheless also other stimuli, like changes in temperature, humidity or social interactions, are powerful Zeitgebers for entraining the clock. This thesis will focus on the question, how light influences the locomotor behavior of the fly in general, including a particular interest on the entrainment of the circadian clock. As a model organism Drosophila melanogaster was used.
During the last years several research groups investigated the effect of light on the circadian clock and their results showed that several light input pathways to the clock contribute to wild-type behavior. Most of the studies focused on the photopigment Cryptochrome (CRY) which is expressed in about half of the 150 clock neurons in the fly. CRY is activated by light, degrades the clock protein Timeless (TIM) and hence entrains the clock to the light-dark (LD)-cycle resulting from changes of day and night. However, also flies lacking CRY are still able to entrain their clock mechanism as well as their activity-rest-rhythm to LD-cycles, clearly showing that the visual system of the fly also contributes to clock synchronization. The mechanism how light information from the visual system is transferred to the clock is so far still unknown. This is also true for so-called masking-effects which are changes in the behavior of the animal that are directly initiated by external stimuli and therefore independent of the circadian clock. These effects complement the behavior of the animals as they enable the fly to react quickly to changes in the environment even during the clock-controlled rest state.
Both of these behavioral features were analyzed in more detail in this study. On the one hand, we investigated the influence of the compound eyes on the entrainment of the clock neurons and on the other hand, we tried to separate clock-controlled behavior from masking. To do so "nature-like" light conditions were simulated allowing the investigation of masking and entrainment within one experiment. The simulation of moonlight and twilight conditions caused significant changes in the locomotor behavior. Moonlit nights increased nocturnal activity levels and shifted the morning (M) and evening (E) activity bouts into the night. The opposite was true for the investigation of twilight, as the activity bouts were shifted into the day. The simulation of twilight and moonlight within the same experiment further showed that twilight appears to dominate over moonlight, which is in accordance to the assumption that twilight in nature is one of the key signals to synchronize the clock as the light intensity during early dawn rises similarly in every season. By investigating different mutants with impaired visual system we showed that the compound eyes are essential for the observed behavioral adaptations. The inner receptor cells (R7 and R8) are important for synchronizing the endogenous clock mechanism to the changes of day and night. In terms of masking, a complex interaction of all receptor cells seems to adjust the behavioral pattern, as only flies lacking photopigments in inner and outer receptor cells lacked all masking effects. However, not only the compound eyes seem to contribute to rhythmic activity in moonlit nights. CRY-mutant flies shift their E activity bout even more into the night than wild-type flies do. By applying Drosophila genetics we were able to narrow down this effect to only four CRY expressing clock neurons per hemisphere. This implies that the compound eyes and CRY in the clock neurons have antagonistic effects on the timing of the E activity bout. CRY advances activity into the day, whereas the compound eyes delay it. Therefore, wild-type behavior combines both effects and the two light inputs might enable the fly to time its activity to the appropriate time of day.
But CRY expression is not restricted to the clock neurons as a previous study showed a rather broad distribution within the compound eyes. In order to investigate its function in the eyes we collaborated with Prof. Rodolfo Costa (University of Padova). In our first study we were able to show that CRY interacts with the phototransduction cascade and thereby influences visual behavior like phototaxis and optomotor response. Our second study showed that CRY in the eyes affects locomotor activity rhythms. It appears to contribute to light sensation without being a photopigment per se. Our results rather indicate that CRY keeps the components of the phototransduction cascade close to the cytoskeleton, as we identified a CRY-Actin interaction in vitro. It might therefore facilitate the transformation of light energy into electric signals.
In a further collaboration with Prof. Orie Shafer (University of Michigan) we were able to shed light on the significance of the extraretinal Hofbauer-Buchner eyelet for clock synchronization. Excitation of the eyelet leads to Ca2+ and cAMP increases in specific clock neurons, consequently resulting in a shift of the flies´ rhythmic activity.
Taken together, the experiments conducted in this thesis revealed new functions of different eye structures and CRY for fly behavior. We were furthermore able to show that masking complements the rhythmic behavior of the fly, which might help to adapt to natural conditions.
The protozoan parasite Trypanosoma brucei is the causal agent of sleeping sickness and besides its epidemiological importance it has been used as model organism for the study of many aspects of cellular and molecular biology especially the post-transcriptional control of gene expression.
Several studies in the last 30 years have shown the importance of mRNA processing and stability for gene regulation. In T. brucei genes are unusually arranged in polycistronic transcription units (PTUs) and a coupled process of trans-splicing and polyadenylation produces the mature mRNAs. Both processes, mRNA processing and stability, cannot completely explain the control of gene expression in the different life cycle stages analyzed in T. brucei so far.
In recent years, the relevance of expression regulation at the level of translation has become evident in other eukaryotes. Therefore, in the first part of my thesis I studied the impact of translational regulation by means of a genome-wide ribosome profiling approach. My data suggest that translational efficiencies vary between life cycle stages of the parasite as well as between genes within one life cycle stage. Furthermore, using ribosome profiling I was able to identify many new putative un-annotated coding sequences and to evaluate the coding potential of upstream open reading frames (uORF). Comparing my results with previously published proteomic and RNA interference (RNAi) target sequencing (RIT-seq) datasets allowed me to validate some of the new coding sequences and to evaluate their relevance for the fitness of the parasite.
In the second part of my thesis I used the transcriptomic and translatomic profiles obtained from the ribosome profiling analysis for the identification of putative non-coding RNAs (ncRNAs). These results led to the analysis of the coding potential in the regions upstream and downstream of the expressed variant surface glycoprotein (VSG), which is outlined in the third part of the results section. The region upstream of the VSG, the co-transposed region (CTR), has been implicated in an increase of the in situ switching rate upon its deletion. The ribosome profiling results indicated moderate transcription but not translation in this region. These results raised the possibility that the CTR may be transcribed into ncRNA. Therefore, in the third part of my thesis, I performed a primary characterization of the CTR-derived transcripts based on northern blotting and RACE. The results suggested the presence of a unique transcript species of about 1,200 nucleotides (nt) and polyadenylated at the 3’-end of the sequence.
The deletion of the CTR sequence promoting and increase of the in situ switching rates was performed around 20 years ago by means of inserting reporter genes. With the recent development of endonuclease-based tools for genome editing, it is now possible to delete sequences in a marker-free way. In the fourth part of my thesis, I show the results on the implementation of the highly efficient genome-editing CRISPR-Cas9 system in T. brucei using episomes. As a proof of principle, I inserted the sequence coding for the enhanced green fluorescent protein (eGFP) at the end of the SCD6 coding sequence (CDS). Fluorescent cells were observed as early as two days after transfection. Therefore, after the successful set up of the CRISPR-Cas9 system it will be possible to modify genomic regions with more relevance for the biology of the parasite, such as the substitution of codons present in gene tandem arrays.
The implementation of ribosome profiling in T. brucei opens the opportunity for the study of translational regulation in a genome-wide scale, the re-annotation of the currently available genome, the search for new putative coding sequences, the detection of putative ncRNAs, the evaluation of the coding potential in uORFs and the role of unstranslated regions (UTRs) in the regulation of translation. In turn, the implementation of the CRISPR-Cas9 system offers the possibility to manipulate the genome of the parasite at a nucleotide resolution and without the need of including resistant makers. The CRISPR-Cas9 system is a powerful tool for editing ncRNAs, UTRs, multicopy gene families and CDSs keeping their endogenous UTRs. Moreover, the system can be used for the modification of both alleles after just one round of transfection and of codons coding for amino acids carrying post-translational modifications (PTMs) among other possibilities.
Cuticles cover all above-ground primary plant organs and are lipoid in nature consisting of a cutin matrix with cuticular waxes embedded within or deposited on its surface. The foremost function of the plant cuticle is the limitation of transpirational water loss into the surrounding atmosphere. Transpiration of water vapour from plants differs between stomatal and cuticular transpiration. Stomatal closure minimises the stomatal water loss and the remaining, much lower water transpiration occurs through the plant cuticle.
Temperature influence on the transpiration barrier properties of intact leaves is not yet known, despite the importance of the cuticular transpiration especially under drought and heat conditions. The present study focuses on the temperature-dependent minimum water permeability of whole leaves, in comparison to the temperature effect on the cuticular permeance of isolated, astomatous cuticles (Chapter I - III).
The minimum water permeability was determined gravimetrically from leaf drying curves and represents the cuticular water permeability of intact, stomatous leaves under conditions of complete stomatal closure. The temperature effect on the transpiration barrier of the desert plant Rhazya stricta and the Mediterranean sclerophyll Nerium oleander exposed a continuous increase of minimum water permeabilities with an increase in temperature. In contrast to other published studies, no abrupt and steep increase of the water permeability at high temperatures was detected. This steep increase indicates structural changes of the barrier properties of isolated cuticular membranes with a drastic decrease of efficiency. A stabilising impact of the cell wall on the plant cuticle of intact leaves was proposed. This steadying effect was confirmed with different experimental approaches measuring the cuticular water permeability of Prunus laurocerasus intact leaves.
Physiological analysis of water transport on isolated, astomatous leaf cuticles indicated a drastic decline of the barrier properties at elevated temperatures for Prunus laurocerasus but not for Nerium oleander. Cuticular components were quantitatively and qualitatively analysed by gas chromatography with a flame ionisation detector and a mass spectrometric detector, respectively. A high accumulation of pentacyclic triterpenoids as cuticular wax components in relation to the cutin monomer coverage was detected for Nerium oleander and for Rhazya stricta leaves, too. Accordingly, reinforcing of the cutin matrix by triterpenoids was proposed to improve the mechanical strength and to reduce the extensibility of plant cuticles. Thus, structural changes of the cuticular barrier properties were potentially suppressed at elevated temperatures.
The function of the cuticular wax amount and/or wax composition and its relation with the cuticular water permeability remains to be elucidated. In the second part of this work the cuticular wax quantity and quality as well as its impact on the transpiration barrier properties was analysed in order to deduce a potential relation between chemistry and function of plant cuticles (Chapter IV - V).
Chemical analyses of the cuticular wax components of a wide range of plant species, including one tropical (Vanilla planifolia), temperate (Juglans regia, Plantago lanceolata), Mediterranean (Nerium oleander, Olea europaea) and one desert (Rhazya stricta) plant species, were conducted. The cuticular wax compositions of nine characteristic plant species from xeric limestone sites naturally located in Franconia (Southern Germany) were determined for the first time. The corresponding minimum or cuticular water permeabilities of both stomatous and astomatous leaf surfaces were measured to detect a potential relationship between the cuticular wax amount, wax composition and the cuticular barrier properties.
It was demonstrated that abundant cuticular wax amounts did not constitute more efficient transpiration barriers. However, 55% of the cuticular barrier function can be attributed to the very-long-chain aliphatic wax coverages. These new findings provide evidence that the acyclic wax constituents play a pivotal role establishing efficient transpiration barriers. Additionally, these findings strengthen the hypothesis that cyclic components, such as pentacyclic triterpenoids, do not hinder the water diffusion through plant cuticles as effectively as acyclic constituents. For the first time a relationship between the cuticular wax composition and the transpiration barrier properties of a wide range of plant species proved insights into the potential relation between chemistry and function of plant cuticles.
The Fanconi anemia (FA) pathway is a replication-dependent DNA repair mechanism which is essential for the removal of interstrand crosslink (ICL) DNA damages in higher eukaryotes (Moldovan and D’Andrea, 2009). Malfunctions in this highly regulated repair network lead to genome instability (Deans and West, 2011). Pathological phenotypes of the disease FA which is caused by mutations in the eponymous pathway are very heterogeneous, involving congenital abnormalities, bone-marrow failure, cancer predisposition and infertility (Auerbach, 2009). The FA pathway comprises a complex interaction network and to date 16 FA complementation groups and associated factors have been identified (Kottemann and Smogorzewska, 2013). Additionally, components of nucleotide excision repair (NER), homologous recombination repair (HRR), and translesion synthesis (TLS) are involved and coordinated by the FA proteins (Niedzwiedz et al., 2004; Knipscheer et al., 2009). One of the FA proteins is the DEAH helicase FANCM. In complex with its binding partners FAAP24 and MHF1/2 it binds the stalled replication fork and activates the FA damage response (Wang et al., 2013). However, the exact steps towards removal of the ICL damage still remain elusive.
To decipher the underlying process of FA initiation by FANCM, this thesis mainly focuses on the archaeal FANCM homolog helicase-associated endonuclease for fork-structured DNA (Hef). Hef from the archaeal organism Thermoplasma acidophilum (taHef) differs from other archaeal Hef proteins and exclusively comprises an N-terminal helicase entity with two RecA and a thumb-like domain while others additionally contain a nuclease portion at the C-terminus. I solved the crystal structure of full-length taHef at a resolution of 2.43 Å. In contrast to the crystal structure of the helicase domain of Hef from Pyrococcus furiosus (pfHef), taHef exhibits an extremely open conformation (Nishino et al., 2005b) which implies that a domain movement of the RecA-like helicase motor domains of 61° is possible thus highlighting the flexibility of helicases which is required to translocate along the DNA. However, small-angle x-ray scattering (SAXS) measurements confirm an intermediate conformation of taHef in solution indicating that both crystal structures represent rather edge states. Most
importantly, proliferating cell nuclear antigen (PCNA) was identified as an interaction partner of Hef. This interaction is mediated by a highly conserved canonical PCNA interacting peptide (PIP) motif. Intriguingly, the presence of PCNA does not alter the ATPase nor the helicase activity of taHef, thus suggesting that the interaction is entirely dedicated to recruit taHef to the replication fork to fulfill its function. Due to a high level of flexibility the taHef-taPCNA complex could not be crystallized and therefore SAXS was utilized to determine a low-resolution model of this quaternary structure.
This newly discovered PCNA interaction could also be validated for the eukaryotic FANCM homolog Mph1 from the thermophilic fungus Chaetomium thermophilum (ctMph1). As the first step towards the characterization of this interaction I solved the crystal structure of PCNA from Chaetomium thermophilum (ctPCNA).
Furthermore, it was possible to achieve preliminary results on the putative interaction between the human proteins FANCM and PCNA (hsFANCM, hsPCNA). In collaboration with Detlev Schindler (Human Genetics, Würzburg) and Weidong Wang (National Institute on Aging, Baltimore, USA) co-immunoprecipitation (CoIP) experiments were performed using hsFANCM and hsPCNA expressed in HEK293 cells. Although an interaction was reproducibly observed in hydroxyurea stimulated cells
further experiments and optimization procedures are required and ongoing.
Use of polyhexanide and nanomedicine approach for effective treatments of cutaneous leishmaniasis
(2015)
Despite huge suffering caused by cutaneous leishmaniasis (CL), there is no effective and affordable treatment strategy against CL and no licensed vaccines. The current treatments show limited efficacy and high toxicity. Improved therapies through discovery of novel drugs and/or an alternative treatment approaches are/is urgently needed. We aimed at identifying a novel antileishmanial agent and developing an innovative nanoparticle (NP) based platform for safe and effective treatments against CL. We discovered that polyhexanide (PHMB), a widely used antimicrobial polymer and wound antisepsis, shows an inherent antileishmanial activity at submicromolar concentrations. PHMB appears to kill L. major parasites via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation. However, host chromosomes binding appear to be limited by exclusion from mammalian cell nuclei. Moreover, we attempted to establish effective drug delivery systems that overcome the various shortcomings in the present treatment of CL. In this scenario, we initially studied the cellular interactions of NPs and their uptake mechanisms into mammalian cells before applying them in drug delivery system. We obtained clear evidence for the involvement of multiple endocytic routes to internalize NPs. Physicochemical properties of NPs, cell type, temperature and pathogenesis of the target diseases were shown to be determinant factors. Thereafter, a mechanism based host- and pathogen-directed combination therapy comprising PHMB and CpG ODN immunomodulator was established for overall synergistic effect against CL. It simultaneously targets the pathogen and the host immunity with effective delivery system. The results show that PHMB binds to CpG ODN and form stable nanopolyplexes for efficient cell entry and therapy. The nanopolyplexes displayed enhanced cellular uptake and antileishmanial potency while drastically reducing the toxicity against mammalian cells. In conclusion, our findings clearly indicate that PHMB can be used as effective candidate drug against CL and as non-viral delivery of immunomodulatorynucleic acids. Moreover, our proof-of concept study showed nanomedicine approaches are effective strategy to challenge CL and other human diseases.
Studies on receptor signaling and regulation in platelets and T cells from genetically modified mice
(2014)
Receptors with tyrosine-based signaling motifs control essential functions of hematopoietic cells, including lymphocytes and platelets. Downstream of the platelet receptor glycoprotein (GP) VI and the T cell receptor (TCR) the immunoreceptor tyrosine-based activation motif (ITAM) initiates a signaling cascade that involves kinases, adapter and effector proteins and finally leads to cellular activation. This thesis summarizes the results of three studies investigating different aspects of receptor signaling and regulation in platelets and T cells.
In the first part, the impact of constitutive Ca2+ influx on TCR signaling and T cell physiology was investigated using a transgenic mouse line with a mutation in the Ca2+ sensor stromal interaction molecule 1 (STIM1). The elevated cytoplasmic Ca2+ level resulted in an altered phosphorylation pattern of the key enzyme phospholipase (PL) Cγ1 in response to TCR stimulation, but without affecting its enzymatic activity. Withdrawal of extracellular Ca2+ or inhibition of the phosphatase calcineurin restored the normal phosphorylation pattern. In addition, there was a decrease in the release of Th2-type cytokines interleukin 4, 5 and 13 upon stimulation in vitro.
The second part of the thesis deals with the role of the adapter protein growth factor receptor-bound protein 2 (Grb2) in platelets using a megakaryocyte/platelet-specific knockout mouse line. Loss of Grb2 severely impaired signaling of GPVI and C-type lectin-like receptor 2 (CLEC-2), a related hemITAM receptor. This was attributed to defective stabilization of the linker for activation of T cells (LAT) signalosome and resulted in reduced adhesion, aggregation, Ca2+ mobilization and procoagulant activity downstream of (hem)ITAM-coupled receptors in vitro. In contrast, the signaling pathways of G protein-coupled receptors (GPCRs) and the integrin αIIbβ3, which do not utilize the LAT signalosome, were unaffected. In vivo, the defective (hem)ITAM signaling caused prolonged bleeding times, however, thrombus formation was only affected under conditions where GPCR signaling was impaired (upon acetylsalicylic acid treatment). These results establish Grb2 as an important adapter protein in the propagation of GPVI- and CLEC-2-induced signals.
Finally, the proteolytic regulation of the immunoreceptor tyrosine-based switch motif (ITSM)-bearing receptor CD84 in platelets was investigated. This study demonstrated that in mice CD84 is cleaved by two distinct and independent proteolytic mechanisms upon platelet activation: shedding of the extracellular part, which is exclusively mediated by a disintegrin and metalloproteinase (ADAM) 10 and cleavage of the intracellular C-terminus by the protease calpain. Finally, the analysis of soluble CD84 levels in the plasma of transgenic mice revealed that shedding of CD84 by ADAM10 occurs constitutively in vivo.
Development Of Three-Dimensional Liver Models For Drug Development And Therapeutical Applications
(2015)
Primary human liver cells such as hepatocytes when isolated and cultured in 2D monolayers, de-differentiate and lose their phenotypic characteristics. In order to maintain the typical polygonal shape of the hepatocytes and their polarization with respect to the neighbouring cells and extra cellular matrix (ECM), it is essential to culture the cells in a three-dimensional (3D) environment. There are numerous culturing techniques available to retain the 3D organization including culturing hepatocytes between two layers of collagen and/or MatrigelTM (Moghe et al. 1997) or in 3D scaffolds (Burkard et al. 2012).
In this thesis, three different 3D hepatic models were investigated.
1. To reflect the in vivo situation, the hepatocytes were cultured in 3D synthetic scaffolds called Mimetix®. These were generated using an electrospinning technique using biodegradable polymers. The scaffolds were modified to increase the pore size to achieve an optimal cell function and penetration into the scaffolds, which is needed for good cell-cell contact and to retain long-term phenotypic functions. Different fibre diameters, and scaffold thicknesses were analyzed using upcyte® hepatocytes. The performance of upcyte® hepatocytes in 3D scaffolds was determined by measuring metabolic functions such as cytochrome P450 3A4 (CYP3A4) and MTS metabolism.
2. Apart from maintaining the hepatocytes in 3D orientation, co-culturing the hepatocytes with other non-parenchymal cell types, such as liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs), better reflects the complexity of the liver. Three different upcyte® cell types namely, hepatocytes, LSECs and MSCs, were used to generated 3D liver organoids. The liver organoids were generated and cultured in static and dynamic conditions. Dynamic conditions using Quasi-vivo® chambers were used to reflect the in vivo blood flow. After culturing the cells for 10 days, the structural orientation of cells within the organoids was analyzed. Functional integrity was investigated by measuring CYP3A4 activities. The organoids were further characterized using in situ hybridization for the expression of functional genes, albumin and enzymes regulating glutamine and glucose levels.
3. An ex vivo bioreactor employing a decellularized organic scaffold called a “Biological Vascularized Scaffold” (BioVaSc) was established. Jejunum of the small intestine from pigs was chemically decellularized by retaining the vascular system. The vascular tree of the
BioVaSc was repopulated with upcyte® microvascular endothelial cells (mvECs). The lumen of the BioVaSc was then used to culture the liver organoids generated using upcyte® hepatocytes, LSECs and MSCs. The structural organisation of the cells within the organoids was visualized using cell-specific immunohistochemical stainings. The performance of liver organoids in the BioVaSc was determined according to metabolic functions (CYP3A4 activities).
This thesis also addresses how in vitro models can be optimized and then applied to drug development and therapy.
A comprehensive evaluation was conducted to investigate the application of second-generation upcyte® hepatocytes from 4 donors for inhibition and induction assays, using a selection of reference inhibitors and inducers, under optimized culture conditions. CYP1A2, CYP2B6, CYP2C9 and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC50 values for each compound correctly classified them as potent inhibitors. Upcyte® hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9 and CYP3A4 inducers, confirming that they have functional AhR, CAR and PXR mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or non-inducers of CYP3A4 and CYP2B6 were tested. Three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUCu/F2 and Cmax,u/Ind50 were analyzed. In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were also demonstrated.
Haemophilia A occurs due to lack of functional Factor VIII (FVIII) protein in the blood. Different types of cells from hepatic and extrahepatic origin produce FVIII. Supernatants harvested from primary LSECs were evaluated for the presence of secreted functional FVIII. In order to increase the FVIII production, different upcyte® endothelial cells such as blood outgrowth endothelial cells (BOECs), LSECs and mvECs were transduced with lentiviral particles carrying a FVIII transgene. Also, to reflect a more native situation, primary mvECs were selected and modified by transducing them with FVIII lentivirus and investigated as a potential method for generating this coagulation factor.
Deregulated MYC expression contributes to cellular transformation as well as progression and
maintenance of human tumours. Interestingly, in the absence of additional genetic alterations,
potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced
apoptosis has long been recognised as a major barrier against cancer development.
However, it is largely unknown how cells discriminate physiological from supraphysiological levels
of MYC in order to execute an appropriate biological response.
The experiments described in this thesis demonstrate that induction of apoptosis in mammary
epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene
expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required
to invade low-affinity binding sites and repress target genes of the serum response factor SRF.
These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely
needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1-
dependent gene repression and increases AKT phosphorylation and downstream function.
Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC’s
transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive
role for the MYC/MIZ1 complex in epithelial cells.
Wasps of the genus Polistes comprise over 200 species and are nearly cosmopolitan. They show a lack of physiological caste differentiation and are therefore considered as primitively eusocial. Furthermore, paper wasps are placed between the solitary living Eumenidae and the highly social organized Vespinae. Hence, they are often called a “key genus” for understanding the evolution of sociality. Particularly, Polistes dominula, with its small easy manageable nests and its frequent occurrence and wide distribution range is often the subject of studies.
In Europe, the invasion of this species into northern regions is on the rise. Since little was known about the nesting behaviour of P. dominula in Central Europe, the basic principles about nesting were investigated in Würzburg, Germany (latitude 49°) by conducting a comprehensive field-study spanning three consecutive years. Furthermore, the thermoregulation of individual wasps in their natural habitat had not yet been investigated in detail. Therefore, their ability to respond to external hazards with elevated thorax temperatures was tested. In addition, different types of nest thermoregulation were investigated using modern methods such as infrared thermography and temperature data logger.
In the present work, the investigation of basic nesting principles revealed that foundress groups (1-4 foundresses) and nests are smaller and that the nesting season is shorter in the Würzburg area than in other regions. The mean size of newly founded nests was 83 cells and the average nesting season was around 4.6 months. The queens neither preferred single (54%) nor multiple founding (46%) in this study. The major benefit of multiple founding is an increased rate of survival. During the three years of observation, only 47% of single-foundress colonies survived, whereas 100% of colonies that were built by more than two queens, survived. However, an influence of the number of foundresses on the productivity of colonies in terms of number of cells and pupae per nest has not shown up. However, the length of the nesting season as well as the nest sizes varied strongly depending on the climatic conditions of the preceding winter during the three consecutive years.
In order to investigate the thermoregulatory mechanisms of individual adult P. dominula wasps, I presented artificial threats by applying smoke or carbon dioxide simulating fire and predator attacks, respectively, and monitored the thorax temperature of wasps on the nest using infrared thermography. The results clearly revealed that P. dominula workers recognized smoke and CO2 and reacted almost instantaneously and simultaneously with an increase of their thorax temperature. The maximal thorax temperature was reached about 65 s after the application of both stressors, but subsequently the wasps showed a different behaviour pattern. They responded to a longer application of smoke with moving to the exit and fled, whereas in case of CO2 the wasps started flying and circling the nest without trying to escape. No rise of the thorax temperature was detectable after an air blast was applied or in wasps resting on the nest. Additionally, the thorax temperatures of queens were investigated during dominance battles. I found that the thorax temperature of the dominant queens rose up to 5°C compared to that of subordinate queens that attacked the former.
The study of active mechanisms for nest thermoregulation revealed no brood incubation or clustering behaviour of P. dominula. Furthermore, I found out that wing fanning for cooling the nest was almost undetectable (4 documented cases). However, I could convincingly record that water evaporation is most effective for nest cooling. By the direct comparison of active (with brood and adults) and non-active (without brood and adults) nests, the start of cooling by water evaporation was detected above maximum outside temperatures of 25°C or at nest temperatures above 35°C. The powerful role of water in nest cooling was manifested by an average decrease of temperature of a single cell of about 8°C and a mean duration of 7 min until the cell reached again its initial temperature. The investigation of passive thermoregulatory mechanisms revealed that the nest site choice as well as nest orientation appears to be essential for P. dominula wasps. Furthermore, I was able to show that the architecture of the nests plays an important role. Based on the presented results, it can be assumed that the vertical orientation of cells helps maintaining the warmth of nests during the night, whereas the pedicel assists in cooling the nest during the day.
Feedback efficiency and training effects during alpha band modulation over the sensorimotor cortex
(2015)
Neural oscillations can be measured by electroencephalography (EEG) and these oscillations can be characterized by their frequency, amplitude and phase. The mechanistic properties of neural oscillations and their synchronization are able to explain various aspects of many cognitive functions such as motor control, memory, attention, information transfer across brain regions, segmentation of the sensory input and perception (Arnal and Giraud, 2012). The alpha band frequency is the dominant oscillation in the human brain. This oscillatory activity is found in the scalp EEG at frequencies around 8-13 Hz in all healthy adults (Makeig et al., 2002) and considerable interest has been generated in exploring EEG alpha oscillations with regard to their role in cognitive (Klimesch et al., 1993; Hanselmayr et al., 2005), sensorimotor (Birbaumer, 2006; Sauseng et al., 2009) and physiological (Lehmann, 1971; Niedermeyer, 1997; Kiyatkin, 2010) aspects of human life. The ability to voluntarily regulate the alpha amplitude can be learned with neurofeedback training and offers the possibility to control a brain-computer interface (BCI), a muscle independent interaction channel. BCI research is predominantly focused on the signal processing, the classification and the algorithms necessary to translate brain signals into control commands than on the person interacting with the technical system. The end-user must be properly trained to be able to successfully use the BCI and factors such as task instructions, training, and especially feedback can therefore play an important role in learning to control a BCI (Neumann and Kübler, 2003; Pfurtscheller et al., 2006, 2007; Allison and Neuper, 2010; Friedrich et al., 2012; Kaufmann et al., 2013; Lotte et al., 2013).
The main purpose of this thesis was to investigate how end-users can efficiently be trained to perform alpha band modulation recorded over their sensorimotor cortex. The herein presented work comprises three studies with healthy participants and participants with schizophrenia focusing on the effects of feedback and training time on cortical activation patterns and performance. In the first study, the application of a realistic visual feedback to support end-users in developing a concrete feeling of kinesthetic motor imagery was tested in 2D and 3D visualization modality during a single training session. Participants were able to elicit the typical event-related desynchronisation responses over sensorimotor cortex in both conditions but the most significant decrease in the alpha band power was obtained following the three-dimensional realistic visualization. The second study strengthen the hypothesis that an enriched visual feedback with information about the quality of the input signal supports an easier approach for motor imagery based BCI control and can help to enhance performance. Significantly better performance levels were measurable during five online training sessions in the groups with enriched feedback as compared to a conventional simple visual feedback group, without significant differences in performance between the unimodal (visual) and multimodal (auditory–visual) feedback modality. Furthermore, the last study, in which people with schizophrenia participated in multiple sessions with simple feedback, demonstrated that these patients can learn to voluntarily regulate their alpha band. Compared to the healthy group they required longer training times and could not achieve performance levels as high as the control group. Nonetheless, alpha neurofeedback training lead to a constant increase of the alpha resting power across all 20 training session.
To date only little is known about the effects of feedback and training time on BCI performance and cortical activation patterns. The presented work contributes to the evidence that healthy individuals can benefit from enriched feedback: A realistic presentation can support participants in getting a concrete feeling of motor imagery and enriched feedback, which instructs participants about the quality of their input signal can give support while learning to control the BCI. This thesis demonstrates that people with schizophrenia can learn to gain control of their alpha oscillations recorded over the sensorimotor cortex when participating in sufficient training sessions. In conclusion, this thesis improved current motor imagery BCI feedback protocols and enhanced our understanding of the interplay between feedback and BCI performance.
Background
GDF-15 is a divergent member of the TGF-superfamily, which was first described as macrophage inhibitory cytokine-1 (MIC-1), revealing an immune modulatory function. GDF-15 is a soluble protein which is, under physiological conditions, highly expressed in the placenta and found in elevated levels in blood sera of pregnant women. Apart from the placenta, GDF-15 is expressed in healthy tissue, albeit to a lower extent and overexpressed in many solid tumors. A variety of different functions are attributed to GDF-15 in healthy as well as diseased humans. On the one hand, GDF-15 is required for successful pregnancy and low GDF-15 serum levels during pregnancy correlate with fetal abortion. On the other hand, overexpression of GDF-15, which can be observed in several malignancies is correlated with a poor prognosis. Furthermore, tumor derived GDF-15 leads to cancer associated anorexia-cachexia syndrome in mice. The aim of my PhD thesis was to further investigate the role of GDF-15 as an immune modulatory factor in cancer, in particular, by inhibiting the target molecule in vitro and in vivo. Therefore, the main focus was placed on the generation and characterization of monoclonal GDF-15 specific blocking antibodies, which were tested in vitro and in vivo, which represents a substantial part of my work.
Results
Here, GDF-15 was shown to be highly expressed in human gynecological cancer and brain tumors. We could then demonstrate that GDF-15 modulates effector immune cells in vitro. GDF-15 mediated a slight downregulation of the activating NKG2D receptor on NK and CD8+ T cells, which is crucial for proper anti-tumoral immune responses. Furthermore, we could demonstrate that GDF-15 reduces the adhesion of CD4+ and CD8+ T cells on endothelial cells in vitro. A negatively affected trans-endothelial migration of leukocytes into inflamed tissue could explain the low T cell infiltration in GDF-15 expressing tumors, which were observed in vivo, where mice bearing (shRNA mediated) GDF-15 deficient glioma cells revealed enhanced immune cell infiltrates in the tumor microenvironment, compared with the GDF-15 expressing control group. Those animals further exhibited a decreased tumor growth and prolonged survival. GDF-15 is a soluble protein, secreted by more than 50 % of solid tumors and associated with grade of malignancy. Therefore a neutralizing monoclonal antibody to GDF-15 was assumed to be an auspicious therapeutically anti-cancer tool. Such an antibody was thus generated in GDF-15 knock out mice against human GFD-15. Amongst many clones, the GDF-15 antibody clone B1-23 was found to be applicable in Western Blot as well as in ELISA techniques, detecting a three-dimensional epitope of the mature GDF-15 dimer with high affinity and specificity. To enable the humanization for a later administration in humans, the variable regions of antibody B1-23 were identified by a special PCR method using degenerate primers and cloned into a sequencing vector. The sequence obtained thereby enabled the generation of chimeric and humanized B1-23 variants. After further comprehensive characterization, the original mouse antibody B1-23 as well as the chimeric antibody (ChimB1-23) and the humanized B1-23 antibody (H1L5) were applied in a melanoma xenograft study in vivo. None of the antibodies could significantly inhibit tumor growth. .However of utmost importance, body weight loss mediated by tumor derived GDF-15 could be significantly prevented upon administration of all three GDF-15 specific antibodies, which confirmed the antagonizing functionality of the immunoglobulin.
Conclusion
GDF-15 is a promising cancer target, involved in tumor progression and cancer related cachexia. A monoclonal GDF-15 antibody was generated, which served on one hand as a tool for molecular biological applications (Western Blot, ELISA, etc.) and on the other hand was applied as an antagonizing antibody in vitro and in vivo. Even though tumor growth inhibition by GDF-15 depletion in T cell deficient athymic mice failed using B1-23, the same antibody and derivates thereof (chimeric and humanized) impressively prevented tumor associated cachexia in UACC-257 melanoma bearing nude mice. The missing anti-tumor effect in our own melanoma model in nude mice can only partially be explained by the missing secondary immunity, in particular cytotoxic T cells, in the athymic animals, since in a similar melanoma model, performed by an external company, a tumor reduction in immunocompromised animals was observed, when B1-23 was administered. These findings support the idea that T cells are substantial for an effective tumor immunity and are in line with the results of the syngeneic, T cell comprising, mouse glioma model, where silencing of tumor expressed GDF-15 led to an enhanced intratumoral T cell infiltration and a prolonged survival.
Taken together our data allow for the conclusion that tumor associated cachexia can be combatted with the GDF-15 antibody B1-23. Further, B1-23 might elicit direct anti-tumor effects in immune competent models, which contain T cells, rather than in an athymic, T cell deficient nude mouse model.
Whereas most currently used antibiotics act by interfering with essential bacterial processes, a smaller group of antibacterials disturbs the integrity of the cell membrane. Since fatty acids are a vital component of membrane phospholipids, the type-II fatty acid biosynthesis pathway (FAS-II) of bacteria constitutes a promising drug target. The front-line anti-tuberculosis prodrug isoniazid blocks the FAS-II pathway in M. tuberculosis thereby leading to morphological changes and finally to cell lysis. When it became evident that the enoyl-ACP reductase in the FAS-II pathway is the target of the activated isoniazid, several programs were initiated to develop novel inhibitors directed against this protein in different pathogens. The S. aureus enoyl-ACP reductase (saFabI) is of particular interest since three promising drug candidates inhibiting this homologue have reached clinical trials. However, despite these prospects, no crystal structures of saFabI were publicly available at the time the present work was initiated. Thus, one major goal of this thesis was the generation of high-resolution atomic models by means of X-ray crystallography. The development of a highly reproducible approach to co-crystallize saFabI in complex with NADP+ and diphenyl ether-based inhibitors led to crystal structures of 17 different ternary complexes. Additional crystallographic experiments permitted the view into two apo-structures and two atomic models of saFabI in complex with NADPH and 2-pyridone inhibitors. Based on the established saFabI structure, molecular dynamics (MD) simulations were performed to improve our understanding of the conformational mobility of this protein. Taken together, these investigations of the saFabI structure and its flexibility served as an ideal platform to address important questions surrounding substrate and inhibitor recognition by this enzyme. Intriguingly, our saFabI structures provide several vastly different snapshots along the reaction coordinate of ligand binding and hydride transfer, including the closure of the flexible substrate binding loop (SBL). The extraordinary mobility of saFabI was confirmed by MD simulations suggesting that conformational motions indeed play a pivotal role during substrate delivery and turnover. A water chain linking the active site with a water-basin inside the homo-tetrameric enzyme was found likely to be crucial for the closure and opening of the SBL and, thus, for the catalyzed reaction. Notably, the induced-fit ligand binding process involves a dimer-tetramer transition, which could be related to the observed positive cooperativity of cofactor and substrate binding. Overall, saFabI displays several unique characteristics compared to FabI proteins from other organisms that might be necessary for the synthesis of branched-chain fatty acids, which in turn are required for S. aureus fitness in vivo. This finding may explain why S. aureus is sensitive to FAS-II inhibitors even in the presence of exogenous fatty acids. Accordingly, saFabI remains a valid drug target and our structures can be used as a molecular basis for rational drug design efforts. In fact, binding affinity trends of diphenyl ether inhibitors and, more importantly, the correlated residence times could be rationalized at the molecular level. Furthermore, the structure of saFabI in complex with the 2-pyridone inhibitor CG400549 revealed unique interactions in the wider binding crevice of saFabI compared to other FabI homologues explaining the narrow activity spectrum of this clinical candidate with proven human efficacy. In summary, these studies provide an ideal platform for the development of new, effective saFabI inhibitors as exemplified by the promising 4-pyridone PT166. In the context of this dissertation, crystal structures of the condensing enzyme KasA in complex with several analogs of the naturally occurring inhibitor thiolactomycin have been solved.
The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell’s physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.
The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed “Dual RNA-seq”. Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host’s immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.
Bariatric surgery represents the first-line treatment for morbid obesity, resulting in weight loss and improved diabetes control. The positive effect of bariatric surgery on type-2 diabetes is unclear. Increased secretion of insulin regulating enterohormone glucagon-like-peptide 1 (GLP-1) has been observed in rats with experimental type 2-like diabetes following duodenal-jejunal bypass (DJB) and ileal transposition (IT). Sodium dependent glucose co-transporter (SGLT1) is involved in the secretion of GLP-1 that in turn regulates insulin secretion. In the present study, an attempt was made to elucidate the impact of DJB and IT on SGLT1 mediated glucose transport. Transport measurements using phlorizin inhibited uptake of SGLT1-specific glucose analogue [14C] α-Methyl-D-glucopyranoside (AMG) were performed to determine the changes in SGLT1 transport upon these surgical procedures. The data indicated that DJB decreased SGLT1-mediated glucose absorption in the small intestine which contributes to the body-weight independent improvement of type 2 diabetes. However, IT did not change the SGLT1-mediated glucose transport. Immunohistochemical analysis revealed that in IT, the transposed ileum showed increased diameter, increased villi length and increased number of GLP-1 secreting L-cells. The weight-independent improvement in glycemic control after IT is not related to SGLT1-mediated glucose absorption but may be linked to increased GLP-1 secretion.
Along with this, the study also focused on the regulation of SGLT1 by several RS1 derived tripeptides in mouse and human intestinal tissues (ex vivo). Phlorizin inhibited uptake of AMG was measured without and with tripeptides. QEP and thiophosphorylated QSP down-regulated SGLT1 activity in small intestine in a concentration-dependent manner. Among the tested tripeptides, QEP showed higher activity and further analysis in various species demonstrated its universal role in SGLT1 regulation. The data thus indicates that RS1 derived tripeptides QEP and thiophosphorylated QSP may be employed for the treatment of type 2 diabetes.
Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis.
Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock.
In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats.
We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD.
It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity.
Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication.
Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.
The integrity of our genome is continuously endangered by DNA damaging factors. Several cellular mechanisms have evolved to recognize and remove different types of DNA lesions. Despite the wealth of information on the three-dimensional structure and the catalytic mechanism of DNA repair enzymes, the essential process of target site search and identification remains more elusive. How can a small number of repair proteins find and detect the rare sites of damage rapidly and efficiently over an excess of millions of undamaged bases?
To address this pivotal question in DNA repair, I focused on the central players from the two DNA damage excision repair pathways in my studies: nucleotide excision repair (NER) and base excision repair (BER). As examples for completely different approaches of damage search, recognition and verification, I compared the NER protein Xeroderma pigmentosum group D (XPD) with the BER proteins human thymine DNA glycosylase (hTDG) and human 8-oxoguanine glycosylase (hOgg1).
In particular, the single molecule approach of atomic force microscopy (AFM) imaging and complementary biochemical and biophysical techniques were applied. I established a simple, optimized preparation approach, which yields homogeneous and pure samples of long (several hundreds to thousands of base pairs) DNA substrates suitable for the AFM studies with DNA repair proteins. Via this sample preparation, a single target site of interest can be introduced into DNA at a known position, which allows separate analysis of specific protein-DNA complexes bound to the lesion site and nonspecific complexes bound to non-damaged DNA.
The first part of the thesis investigates the XPD protein involved in eukaryotic NER. In general, the NER mechanism removes helix-distorting lesions – carcinogenic UV light induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs) as well as bulky DNA adducts. The 5’-3’ helicase XPD has been proposed to be one of the key players in DNA damage verification in eukaryotic NER, which is still a matter of hot debate. In the studies, I focused on XPD from the archaeal species Thermoplasma acidophilum (taXPD), which shares a relatively high sequence homology with the sequence of the human protein and may serve as a good model for its eukaryotic counterpart. Based on AFM experiments and accompanying DNA binding affinity measurements with the biosensor technology Biolayer Interferometry (BLI), a clear role of XPD in damage verification was deciphered. Specifically, the data suggested that the ATP-dependent 5’-3’ helicase activity of XPD was blocked by the presence of damage leading to stalled XPD-DNA damage verification complexes at the lesion sites.
Successful damage verification led to ATP-dependent conformational changes visible by a significant transition in DNA bend angles from ~ 50° to ~ 65° at the site of the bound protein. Remarkably, this DNA bend angle shift was observed both in the presence of ATP and ATPγs (non-hydrolyzable ATP analog) indicating that ATP-binding instead of ATP hydrolysis was sufficient to induce repair competent conformational changes of XPD. Most importantly, detailed protein binding position and DNA bend angle analyses revealed for the first time that XPD preferably recognizes a bulky fluorescein lesion on the translocated strand, whereas a CPD lesion is preferentially detected on the opposite, non-translocated strand. Despite the different recognition strategies for both types of damages, they share a common verification complex conformation, which may serve as a signal for the recruitment of further NER factors.
In the second part of the thesis, AFM imaging and a 2-Aminopurine fluorescence-based base-flipping assay were combined to investigate damage search and recognition by DNA glycosylases in BER. Exemplarily, I chose to study hTDG as a representative of the vast glycosylase family. hTDG excises thymine and uracil from mutagenic G:T and G:U mispairs contributing to cancer and genetic disease. The AFM data suggested that hTDG uses the intrinsic flexibility of G:T and G:U wobble pairs for initial damage sensing, while scanning DNA as a search complex (SC, slightly bent DNA). Remarkably, hTDG has been indicated to continuously switch between the search and interrogation conformation (IC, stronger bent DNA) during damage search. In the IC, target bases are interrogated by extrahelical base flipping, which is facilitated by protein-induced DNA bending and enhanced DNA flexibility at mismatches. AFM and fluorescence analyses revealed that the flipped base is stabilized via hTDG’s arginine finger. Correct target bases are perfectly stabilized within the enzyme’s catalytic pocket resulting in prolonged residence time and enhanced excision probability. To test for the generalizability of the proposed hTDG damage search model to BER glycosylases, identical studies were performed with a second glycosylase, hOgg1. The data on hOgg1, which removes structurally more stable 8-oxoguanine lesions, supported the hypothesis developed for lesion recognition by hTDG as a common strategy employed by BER glycosylases