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- Theodor-Boveri-Institut für Biowissenschaften (32) (remove)
Animals need to evaluate their experiences in order to cope with new situations they encounter. This requires the ability of learning and memory. Drosophila melanogaster lends itself as an animal model for such research because elaborate genetic techniques are available. Drosphila larva even saves cellular redundancy in parts of its nervous system. My Thesis has two parts dealing with associative olfactory learning in larval Drosophila. Firstly, I tackle the question of odour processing in respect to odour quality and intensity. Secondly, by focusing on the evolutionarily conserved presynaptic protein Synapsin, olfactory learning on the cellular and molecular level is investigated. Part I.1. provides a behaviour-based estimate of odour similarity in larval Drosophila by using four recognition-type experiments to result in a combined, task-independent estimate of perceived difference between odour-pairs. A further comparison of these combined perceived differences to published calculations of physico-chemical difference reveals a weak correlation between perceptual and physico-chemical similarity. Part I.2. focuses on how odour intensity is interpreted in the process of olfactory learning in larval Drosophila. First, the dose-effect curves of learnability across odour intensities are described in order to choose odour intensities such that larvae are trained at intermediate odour intensity, but tested for retention either with that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. A specificity of retention for the trained intensity is observed for all the odours used. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of associative olfactory memory trace formation. In part II.1. of the thesis, the cellular site and molecular mode of Synapsin function is investigated- an evolutionarily conserved, presynaptic vesicular phosphoprotein. On the cellular level, the study shows a Synapsin-dependent memory trace in the mushroom bodies, a third-order “cortical” brain region of the insects; on the molecular level, Synapsin engages as a downstream element of the AC-cAMP-PKA signalling cascade.
Is behaviour response or action? In this Thesis I study this question regarding a rather simple organism, the larva of the fruit fly Drosophila melanogaster. Despite its numerically simple brain and limited behavioural repertoire, it is nevertheless capable to accomplish surprisingly complex tasks. After association of an odour and a rewarding or punishing reinforcement signal, the learnt odour is able to retrieve the formed memory trace. However, the activated memory trace is not automatically turned into learned behaviour: Appetitive memory traces are behaviourally expressed only in absence of the rewarding tastant whereas aversive memory traces are behaviourally expressed in the presence of the punishing tastant. The ‘decision’ whether to behaviourally express a memory trace or not relies on a quantitive comparison between memory trace and current situation: only if the memory trace (after odour-sugar training) predicts a stronger sugar reward than currently present, animals show appetitive conditioned behaviour. Learned appetitive behaviour is best seen as active search for food – being pointless in the presence of (enough) food. Learned aversive behaviour, in turn, can be seen as escape from a punishment – being pointless in absence of punishment. Importantly, appetitive and aversive memory traces can be formed and retrieved independent from each other but also can, under appriate circumstances, summate to jointly organise conditioned behaviour. In contrast to learned behaviour, innate olfactory behaviour is not influenced by gustatory processing and vice versa. Thus, innate olfactory and gustatory behaviour is rather rigid and reflexive in nature, being executed almost regardless of other environmental cues. I suggest a behavioural circuit-model of chemosensory behaviour and the ‘decision’ process whether to behaviourally express a memory trace or not. This model reflects known components of the larval chemobehavioural circuit and provides clear hypotheses about the kinds of architecture to look for in the currently unknown parts of this circuit. The second chapter deals with gustatory perception and processing (especially of bitter substances). Quinine, the bitter tastant in tonic water and bitter lemon, is aversive for larvae, suppresses feeding behaviour and can act as aversive reinforcer in learning experiments. However, all three examined behaviours differ in their dose-effect dynamics, suggesting different molecular and cellular processing streams at some level. Innate choice behaviour, thought to be relatively reflexive and hard-wired, nevertheless can be influenced by the gustatory context. That is, attraction toward sweet tastants is decreased in presence of bitter tastants. The extent of this inhibitory effect depends on the concentration of both sweet and bitter tastant. Importantly, sweet tastants differ in their sensitivity to bitter interference, indicating a stimulus-specific mechanism. The molecular and cellular processes underlying the inhibitory effect of bitter tastants are unknown, but the behavioural results presented here provide a framework to further investigate interactions of gustatory processing streams.
Protection of healthy tissues from infection with systemically administered vaccinia virus strains
(2012)
Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host’s own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell’s own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3’end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.
Non–Small-Cell Lung Cancer (NSCLC) is the most frequent human lung cancer and a major cause of death due to its high rate of metastasis1. These facts emphasize the urgent need for the investigation of new targets for anti-metastatic therapy. Up to now a number of genes and gene products have been identified that positively or negatively affect the probability of established human tumor cell lines to metastasize2. Previously, together with the group of Professor Ulf Rapp, we have described the first conditional mouse model for metastasis of NSCLC and identified a gene, c-MYC, that is able to orchestrate all steps of this process. We could identify potential markers for detection of metastasis and highlighted GATA4, which is exclusively expressed during lung development, as a target for future therapeutic intervention2. However, the mechanism underlying this metastatic conversion remained to be identified, and was therefore the focus of the present work. Here, GATA4 is identified as a MYC target in the development of metastasis and epigenetic alterations at the GATA4 promoter level are shown after MYC expression in NSCLC in vivo and in vitro. Such alterations include site-specific demethylation that accompanies the displacement of the MYC-associated zinc finger protein (MAZ) from the GATA4 promoter, which leads to GATA4 expression. Histone modification analysis of the GATA4 promoter revealed a switch from repressive histone marks to active histone marks after MYC binding, which corresponds to active GATA4 expression. This work identifies a novel epigenetic mechanism by which MYC activates GATA4 leading to metastasis in NSCLC, suggesting novel potential targets for the development of anti-metastatic therapy.
A metacommunity approach will be a useful framework to assess and predict changes in biodiversity in spatially structured landscapes and changing environments. However, the relationship between two core elements of metacommunity dynamics, dispersal and species interaction are not well understood. Most theoretical studies on dispersal evolution assume that target species are in isolation and do not interact with other species although the species interactions and community structure should have strong interdependence with dispersal. On the one hand, a species interaction can change the cost and benefit structure of dispersing in relation to non-dispersing individuals. On the other hand, with dispersal, an individual can follow respectively avoid species partners. Moreover, it is also important to explore the interdependence between dispersal and species interaction with spatial and temporal heterogeneity of environment because it would allow us to gain more understanding about responses of community to disturbances such as habitat destruction or global climate change, and this aspect is up to now not well-studied. In this thesis, I focus on the interactive and evolutionary feedback effects between dispersal and various types of interspecific interactions in different environmental settings. More specifically, I contrast dispersal evolution in scenarios with different types of interactions (chapter 2), explore the concurrent evolution of dispersal and habitat niche width (specialization) in spatial heterogeneous landscape (chapter 3) and consider (potential) multidimensional evolutionary responses under climate change (chapter 4). Moreover, I investigate consequences of different dispersal probability and group tolerance on group formation respectively group composition and the coexistence of ‘marker types’ (chapter 5). For all studies, I utilize individual-based models of single or multiple species within spatially explicit (grid-based) landscapes. In chapter 5, I also use an analytical model in addition to an individual-based model to predict phenomenon in group recognition and group formation. ...
Computer Science approaches (software, database, management systems) are powerful tools to boost research. Here they are applied to metabolic modelling in infections as well as health care management. Starting from a comparative analysis this thesis shows own steps and examples towards improvement in metabolic modelling software and health data management. In section 2, new experimental data on metabolites and enzymes induce high interest in metabolic modelling including metabolic flux calculations. Data analysis of metabolites, calculation of metabolic fluxes, pathways and their condition-specific strengths is now possible by an advantageous combination of specific software. How can available software for metabolic modelling be improved from a computational point of view? A number of available and well established software solutions are first discussed individually. This includes information on software origin, capabilities, development and used methodology. Performance information is obtained for the compared software using provided example data sets. A feature based comparison shows limitations and advantages of the compared software for specific tasks in metabolic modeling. Often found limitations include third party software dependence, no comprehensive database management and no standard format for data input and output. Graphical visualization can be improved for complex data visualization and at the web based graphical interface. Other areas for development are platform independency, product line architecture, data standardization, open source movement and new methodologies. The comparison shows clearly space for further software application development including steps towards an optimal user friendly graphical user interface, platform independence, database management system and third party independence especially in the case of desktop applications. The found limitations are not limited to the software compared and are of course also actively tackled in some of the most recent developments. Other improvements should aim at generality and standard data input formats, improved visualization of not only the input data set but also analyzed results. We hope, with the implementation of these suggestions, metabolic software applications will become more professional, cheap, reliable and attractive for the user. Nevertheless, keeping these inherent limitations in mind, we are confident that the tools compared can be recommended for metabolic modeling for instance to model metabolic fluxes in bacteria or metabolic data analysis and studies in infection biology. ...
The neurodegenerative disorder Alzheimer's disease (AD) is the cause of approximately 60% of the world's 35 million patients suffering from dementia. Current research focuses here are on association with other diseases such as diabetes type 2 (T2DM), possible genetic markers, specific signal transduction pathways within the brain and potential protein modification, because the pathogenesis and etiology of AD are still not fully understood. Specifically association of T2DM with AD came to the focus with the so-called "Rotterdam study" in 1999, indicating that T2DM doubles the risk of developing AD. In the meantime, it is known that the prevalence rate in patients with T2DM is 30%. Drugs commonly used in the treatment of T2DM such as peroxisome proliferator-activated receptors gamma (PPARγ) agonists show improvement of the cognitive abilities in patients with early stage of dementia, with potential therapeutically relevance. Therefore it is important not only to investigate a link between these diseases, but also to investigate the insulin signaling pathway in the brain of AD patients. In order to investigate this complex issue in more details and demonstrate additional links between T2DM and AD, the present study used several basic biological methods to clarify the question: "Is impaired insulin signaling pathway within the brain crucial for the development of AD?" from several points of view. The methods used in this work have been i) an analysis of single nucleotide (SNP) polymorphism of the insulin-degrading enzyme gene (IDE) in relation to risk of AD and / or of T2DM, ii) post-mortem histochemical studies of brain tissue of patients with only AD, with AD combined with T2DM and with only T2DM compared with an age-matched control group, and iii.) investigations of neurochemical pathways and gene/protein expression changes of a human cell culture as a consequences of amyloid β (Aβ) treatment. After analysis of the IDE SNP polymorphism in the selected VITA (Vienna Trans Danube Aging) cohort disease-specific effects were discovered. The upstream polymorphism (IDE2) was found to influence AD risk in a protective manner, while the downstream polymorphism (IDE7) modified the T2DM risk. Based on the SNP results, the presented study delineate the model that IDE promoter and 3‟ untranslated region/downstream variation can have different effects on IDE expression, maybe a relevant endophenotype with disorder-specific effects on AD and T2DM susceptibility. Furthermore, the human post-mortem studies could show that both AD as well as T2DM patients had a significantly lower density of the insulin receptor (IR) in the hippocampus, whereas a significantly increased density of inactive phosphorylated PPARγ has been found and this persisted even in patients with both diseases. Summarizing the histological study, it was possible to reveal common histological features of AD and T2DM, but no direct connection between the two diseases. Although AD is nowadays not only characterized by amyloid-containing plaque deposits and by the hyperphosphorylation of tau protein, the excessive Aβ42 presence in the brains of AD patients is still playing a key role. Up to date it is still not entirely clear which physical form of Aβ42 is responsible for the development of AD. The present work investigated, what impact has the state of aggregation of Aβ42 on genes and proteins of the insulin signaling pathway and the amyloid cascade. It could be shown that the oligomeric variant enhanced specifically the gene and protein expression of glycogen synthase kinase (GSK) 3β and also the enzyme activity was significantly increased, but has in turn strongly inhibited the IR gene and protein expression. Additionally, the effect of Aβ42 on monoamine oxidase B (MAO-B) was examined. An effect of both aggregated forms of Aβ42 had on enzyme activity was discovered. However, the fibrillar variants led to significantly increased activity of MAO-B while the oligomeric variants inhibited the enzyme activity. Several previous studies have demonstrated the involvement of increased MAO-B activity in AD, but the present work provides for the first time a direct link between the states of aggregation of Aβ42 to enzyme activity. Finally the results of the presented thesis can be summarized to following conclusion: Although AD and T2DM sharing some degrees of common features, still there is a lack of direct association, and therefore the diseases must be considered more independent rather than linked. But the impaired cerebral insulin signaling pathway seems to be another manifested hallmark of AD.
Primary contact with human polyomaviruses is followed by lifelong asymptomatic persistence of viral DNA. Under severe immunosuppression JCV activation may lead to unrestricted virus growth in the CNS followed by development of progressive multifocal leukoencephalopathy (PML). Besides the kidney and the brain, target cells of persistent infection were also found in the hematopoietic system. This included the presence of JCV genomes in peripheral blood cells (PBCs). In the attempt to understand the role of PBCs for the JCV infection in humans, we asked for the type of cells affected as well as for virus interaction with PBCs. Analysis of separated subpopulations by highly sensitive and specific polymerase chain reaction and Southern blot hybridization revealed the presence of JCV DNA mostly in circulating granulocytes. These cells have important functions in innate immunity and are professional phagocytes. This suggested that PCR amplified DNA might be the result of an extranuclear association of the virus due to membrane attachment or phagocytosis rather than JCV infection with presence of viral DNA in the nucleus. In the attempt to answer this question JCV DNA was subcellularly localized in the blood of 22 healthy donors by JCV specific fluorescence in situ hybridization (FISH). Granulocytes and peripheral blood mononuclear cells (PBMCs) were separated by Percoll gradient centrifugation. Intracellular JCV DNA was hybridized with Digoxigenin-labeled JCV specific DNA probes covering half of the viral genome. As the sensitivity of the anti-digoxigenin antibody system was lower than the PCR detection level, a chemical amplification step was included consisting of peroxidase labeled secondary antibody precipitating biotinylated tyramide followed by detection with streptavidin-Texas-Red and fluorescence microscopy. Comparison of the number of cells affected in healthy individuals with 15 HIV-1 infected patients with and without PML revealed that the rate of affected PBMCs was comparable in both groups (2.5±0.4 and 14.5±0.9 per 1000). In contrast, the rate of JCV positive granulocytes in the immunosuppressed group was 92.6±1.7% compared to 4±1.4% in healthy donors thus confirming that granulocytes are the major group of circulating cells affected by JCV and that HIV-1 associated immune impairment has an important effect on the virus-cell association. Localization revealed that JCV DNA was predominantly located within the cytoplasm, although hybridizing signals occasionally covered the nuclear compartment. The fluorescent glow of chemical amplification combined with classical fluorescence microscopy did not allow an unequivocal localization of viral DNA. However, confocal microscopy of 24 sections through single cells combined with FISH without chemical amplification confirmed cytoplasmic localization of JCV DNA in a large number of cells. Additionally, it clearly demonstrated that JCV DNA was also located in the nucleus and nuclear localization directly correlated with the number of cells affected. Calculation of the virus load in subcellular compartments revealed that up to 50% of the JCV genomes were located in the nucleus thus pointing to viral infection at least in the granulocytes of HIV-1 infected patients. This may contribute to the distribution of the virus from sites of peripheral infection to the CNS and may promote the development of active PML in the severely immune impaired patients.
Ovarian cancer currently causes ~6,000 deaths per year in Germany alone. Since only palliative treatment is available for ovarian carcinomas that have developed resistance against platinum-based chemotherapy and paclitaxel, there is a pressing medical need for the development of new therapeutic approaches. As survival is strongly influenced by immunological parameters, immunotherapeutic strategies appear promising. The research of our group thus aims at overcoming tumour immune escape by counteracting immunosuppressive mechanisms in the tumour microenvironment. In this context, we found that tumour-infiltrating myeloid-derived suppressor cells (MDSC) or tumour associated macrophages (TAM) which are abundant in ovarian cancer express high levels of the enzyme 11β-hydroxysteroid dehydrogenase1 (11-HSD1). This oxido-reductase enzyme is essential for the conversion of biologically inactive cortisone into active cortisol. In line with this observation, high endogenous cortisol levels could be detected in serum, ascitic fluid and tumour exudates from ovarian cancer patients. Considering that cortisol exerts strong anti-inflammatory and immunosuppressive effects on immune cells, it appears likely that high endogenous cortisol levels contribute to immune escape in ovarian cancer. We thus hypothesised that local activation of endogenous glucocorticoids could suppress beneficial immune responses in the tumour microenvironment and thereby prevent a successful immunotherapy. To investigate the in vivo relevance of this postulated immune escape mechanism, irradiated PTENloxP/loxP loxP-Stop-loxP-krasG12D mice were reconstituted with hematopoietic stem cells from either glucocorticoid receptor (GR) expressing mice (GRloxP/loxP) or from mice with a T cell-specific glucocorticoid receptor knock-out (lck-Cre GRloxP/loxP) mice. In the host mice, the combination of a conditional PTEN knock-out with a latent oncogenic kras leads to tumour development when a Cre-encoding adenovirus is injected into the ovarian bursa. Using this model, mice that had been reconstituted with GC-insensitive T cells showed better intratumoural T cell infiltration than control mice that had received functionally unaltered GRloxP/loxP cells via adoptive transfer. However, tumour-infiltrating T cells mostly assumed a Foxp3+ (regulatory) phenotype and survival was even shortened in mice with cortisol-insensitive T cells. Thus, endogenous cortisol seems to inhibit immune cell infiltration in ovarian cancer, but productive anti-tumour immune responses might still be prevented by further factors from the tumour microenvironment. Thus, our data did not provide a sufficiently strong rationale to further pursue the antagonisation of glucocorticoid signalling in ovarian cancer patients, Moreover, glucocorticoids are frequently administered to cancer patients to reduce inflammation and swelling and to prevent chemotherapy-related toxic side effects like nausea or hypersensitivity reactions associated with paclitaxel therapy. Thus, we decided to address the question whether specific signalling pathways in innate immune cells, preferentially in NK cells, could still be activated even in the presence of GC. A careful investigation of the various activating NK cell receptors (i.e. NKp30, NKp44, NKp46), DNAM-1 and NKG2D) was thus performed which revealed that NKp30, NKp44 and NKG2D are all down-regulated by cortisol whereas NKp46 is actually induced by cortisol. Interestingly, NKp46 is the only known receptor that is strictly confined to NK cells. Its activation via crosslinking leads to cytokine release and activation of cytotoxic activity. Stimulation of NK cells via NKp46 may contribute to immune-mediated tumour destruction by triggering the lysis of tumour cells and by altering the cytokine pattern in the tumour microenvironment, thereby generating more favourable conditions for the recruitment of antigen-specific immune cells. Accordingly, our observation that even cortisol-treated NK cells can still be activated via NKp46 and CD2 might become valuable for the design of immunotherapies that can still be applied in the presence of endogenous or therapeutically administered glucocorticoids.
Nowadays, agriculturally used areas form a major part of the German landscape. The conversion from natural habitats to agriculturally used grasslands fundamentally influences the diversity of plants and animals. Intensive use of these areas increases indeed the productivity of crop or biomass on meadows as food source for cattle. How these influences affect biodiversity, ecosystems and trophic interactions over years is still not understood completely. To understand biodiversity functions in an agriculturally used area my study focused on the influence of land use (fertilization, grazing and mowing) on a herbivore-parasitoid system of Plantago lanceolata. The ribwort plantain is a generalist herb of cosmopolitan distribution. It can grow in a very broad range of ground conditions (both in wet and dry habitats), which makes P. lanceolata an ideal model system for investigating tritrophic interactions in a gradient of land use intensity. The weevils Mecinus labilis and M. pascuorum feed and oviposit on P. lanceolata. Mesopolobus incultus is a generalist parasitoid that parasitizes different insect orders. However its only hosts on P. lanceolata are the two weevil species mentioned before. The intention of my study was to investigate the influence of land use on a tritrophic system and its surrounding vegetation (structure, density and species richness) at different spatial scales like subplot, plot and landscape level in three different regions (north, middle and south of Germany). I studied the influence of land use intensity not only correlative but also experimentally. Additionally I aimed to reveal how vegetation composition changes host plant metabolites and whether these changes impact higher trophic levels in the field.