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Honeybees can easily be trained to perform different types of discrimination tasks under controlled laboratory conditions. This review describes a range of experiments carried out with free-flying forager honeybees under such conditions. The research done over the past 30 or so years suggests that cognitive abilities (learning and perception) in insects are more intricate and flexible than was originally imagined. It has become apparent that honeybees are capable of a variety of visually guided tasks, involving decision making under challenging situations: this includes simultaneously making use of different sensory modalities, such as vision and olfaction, and learning to use abstract concepts such as “sameness” and “difference.” Many studies have shown that decision making in foraging honeybees is highly flexible. The trained animals learn how to solve a task, and do so with a high accuracy, but when they are presented with a new variation of the task, they apply the learnt rules from the earlier setup to the new situation, and solve the new task as well. Honeybees therefore not only feature a rich behavioral repertoire to choose from, but also make decisions most apt to the current situation. The experiments in this review give an insight into the environmental cues and cognitive resources that are probably highly significant for a forager bee that must continually make decisions regarding patches of resources to be exploited.
Cancer-related anemia is prevalent in cancer patients. Anemia negatively affects normal mental and physical function capacity with common symptoms s like fatigue, headache, or depression. Human erythropoietin (hEPO), a glycoprotein hormone regulating red blood cell formation, is approved for the treatment of cancer-related anemia. It has shown benefits in correcting anemia, and subsequently improving health-related quality of life and/or enhancing radio-, and chemotherapy. Several recent clinical trials have suggested that recombinant hEPO (rhEPO) may promote tumor growth that raises the questions concerning the safety of using rhEPO for cancer treatment. However in others, such effects were not indicated. As of today, the direct functional effect of rhEPO in tumor models remains controversial and needs to be further analyzed. Based on the GLV-1h68 backbone, the hEPO-expressing recombinant VACV strains (EPO-VACVs) GLV-1h210, GLV-1h211, GLV-1h212 and GLV-1h213 were generated by replacing the lacZ expression cassette at the J2R locus with hEPO under the control of different vaccinia promoters p7.5, pSE, pSEL, pSL, respectively. Also, GLV-1h209 was generated, which is similar to GLV-1h210 but expresses a mutated non-functinal EPO (R103A). The EPO-VACV strains were characterized for their oncolytic efficacy in lung (A549) cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed hEPO in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated. As expected, EPO-VACVs enhanced red blood cell (RBC) formation in xenograft model. The number of RBCs and hemoglobin (Hb) levels were significantly increased in EPO-VACVs-treated mice compared to GLV-1h68-treated or untreated control mice. However, the mean size of RBC or Hb content per RBC remained normal. Furthermore, over-expression of hEPO did not significantly affect numbers of lymphocytes, monocytes, leucocytes or platelets in the peripheral blood stream. The expression of hEPO in colonized tumors of mice treated with EPO-VACVs was demonstrated by immunohistological staining. Interestingly, there were 9 - 10 hEPO isoforms detected either in tumors, cells, or supernatant, while 3-4 basic isoforms were missing in blood serum, where only six hEPO isoforms were found. Tumor-bearing mice after treatment with EPO-VACVs showed enhanced tumor regression compared to GLV-1h68. The virus titers in tumors in EPO-VACVs-treated mice were 3-4 fold higher compared to GLV-1h68-treated mice. Nevertheless, no significant difference in virus titers among EPO-VACVs was found. The blood vessels in tumors were significantly enlarged while the blood vessel density remained unchanged compared to the GLV-1h68 treated mice, indicating that hEPO did not affect endothelial cell proliferation in this model. Meanwhile, rhEPO (Epoetin alfa) alone or in combination with GLV-1h68 did not show any signs of enhanced tumor growth when compared to untreated controls and GLV-1h68 groups, while doses used were clinical relevant (500 U/kg). These findings suggested that hEPO did not promote angiogenesis or tumor growth in the A549 tumor xenograft model. Human EPO has been reported to function as an immune modulator. In this study, however, we did not find any involvement of hEPO in immune cytokine and chemokine expression or innate immune cell infiltration (leucocytes, B cells, macrophages and dendritic cells) into infected tumors. The degree of immune infiltration and cytokine expression was directly correlated to the number of virus particles. Increased virus replication, led to more recruited immune cells and secreted cytokines/chemokines. It was proposed that tumor regression was at least partially mediated through activation of innate immune mechanisms. In conclusion, the novel EPO-VACVs were shown to significantly increase the number of RBCs, Hb levels, and virus replication in tumors as well as to enhance tumor regression in the A549 tumor xenograft model. Moreover, locally expressed hEPO did not promote tumor angiogenesis, tumor growth, and immune infiltration but was shown to causing enlarged tumoral microvessels which facilitated virus spreading. It is conceivable that in a possible clinical application, anemic cancer patients could benefit from the EPO-VACVs, where they could serve as “wellness pills” to decrease anemic symptoms, while simultaneously destroying tumors.
Trypanosome Motion Represents an Adaptation to the Crowded Environment ofthe Vertebrate Bloodstream
(2012)
Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy
Trypanosome Motion Represents an Adaptation to the Crowded Environment of the Vertebrate Bloodstream
(2012)
Blood is a remarkable habitat: it is highly viscous, contains a dense packaging of cells and perpetually flows at velocities varying over three orders of magnitude. Only few pathogens endure the harsh physical conditions within the vertebrate bloodstream and prosper despite being constantly attacked by host antibodies. African trypanosomes are strictly extracellular blood parasites, which evade the immune response through a system of antigenic variation and incessant motility. How the flagellates actually swim in blood remains to be elucidated. Here, we show that the mode and dynamics of trypanosome locomotion are a trait of life within a crowded environment. Using high-speed fluorescence microscopy and ordered micro-pillar arrays we show that the parasites mode of motility is adapted to the density of cells in blood. Trypanosomes are pulled forward by the planar beat of the single flagellum. Hydrodynamic flow across the asymmetrically shaped cell body translates into its rotational movement. Importantly, the presence of particles with the shape, size and spacing of blood cells is required and sufficient for trypanosomes to reach maximum forward velocity. If the density of obstacles, however, is further increased to resemble collagen networks or tissue spaces, the parasites reverse their flagellar beat and consequently swim backwards, in this way avoiding getting trapped. In the absence of obstacles, this flagellar beat reversal occurs randomly resulting in irregular waveforms and apparent cell tumbling. Thus, the swimming behavior of trypanosomes is a surprising example of micro-adaptation to life at low Reynolds numbers. For a precise physical interpretation, we compare our high-resolution microscopic data to results from a simulation technique that combines the method of multi-particle collision dynamics with a triangulated surface model. The simulation produces a rotating cell body and a helical swimming path, providing a functioning simulation method for a microorganism with a complex swimming strategy.
Background
Blood-born miRNA signatures have recently been reported for various tumor diseases. Here, we compared the miRNA signature in Wilms tumor patients prior and after preoperative chemotherapy according to SIOP protocol 2001.
Results
We did not find a significant difference between miRNA signature of both groups. However both, Wilms tumor patients prior and after chemotherapy showed a miRNA signature different from healthy controls. The signature of Wilms tumor patients prior to chemotherapy showed an accuracy of 97.5% and of patients after chemotherapy an accuracy of 97.0%, each as compared to healthy controls.
Conclusion
Our results provide evidence for a blood-born Wilms tumor miRNA signature largely independent of four weeks preoperative chemotherapy treatment.
Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade \(Milnesium\) \(tardigradum\) were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from \(Hypsibius\) \(dujardini\), revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for \(M.\) \(tardigradum\) are different from typical motifs known from higher animals. \(M.\) \(tardigradum\) and \(H.\) \(dujardini\) protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of \(M.\) \(tardigradum\). These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and \(M.\) \(tardigradum\) in particular so highly stress resistant.
Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy.
Animals acquire predictive values of sensory stimuli through reinforcement. In the brain of Drosophila melanogaster, activation of two types of dopamine neurons in the PAM and PPL1 clusters has been shown to induce aversive odor memory. Here, we identified the third cell type and characterized aversive memories induced by these dopamine neurons. These three dopamine pathways all project to the mushroom body but terminate in the spatially segregated subdomains. To understand the functional difference of these dopamine pathways in electric shock reinforcement, we blocked each one of them during memory acquisition. We found that all three pathways partially contribute to electric shock memory. Notably, the memories mediated by these neurons differed in temporal stability. Furthermore, combinatorial activation of two of these pathways revealed significant interaction of individual memory components rather than their simple summation. These results cast light on a cellular mechanism by which a noxious event induces different dopamine signals to a single brain structure to synthesize an aversive memory.
The Serotonergic Central Nervous System of the Drosophila Larva: Anatomy and Behavioral Function
(2012)
The Drosophila larva has turned into a particularly simple model system for studying the neuronal basis of innate behaviors and higher brain functions. Neuronal networks involved in olfaction, gustation, vision and learning and memory have been described during the last decade, often up to the single-cell level. Thus, most of these sensory networks are substantially defined, from the sensory level up to third-order neurons. This is especially true for the olfactory system of the larva. Given the wealth of genetic tools in Drosophila it is now possible to address the question how modulatory systems interfere with sensory systems and affect learning and memory. Here we focus on the serotonergic system that was shown to be involved in mammalian and insect sensory perception as well as learning and memory. Larval studies suggested that the serotonergic system is involved in the modulation of olfaction, feeding, vision and heart rate regulation. In a dual anatomical and behavioral approach we describe the basic anatomy of the larval serotonergic system, down to the single-cell level. In parallel, by expressing apoptosis-inducing genes during embryonic and larval development, we ablate most of the serotonergic neurons within the larval central nervous system. When testing these animals for naive odor, sugar, salt and light perception, no profound phenotype was detectable; even appetitive and aversive learning was normal. Our results provide the first comprehensive description of the neuronal network of the larval serotonergic system. Moreover, they suggest that serotonin per se is not necessary for any of the behaviors tested. However, our data do not exclude that this system may modulate or fine-tune a wide set of behaviors, similar to its reported function in other insect species or in mammals. Based on our observations and the availability of a wide variety of genetic tools, this issue can now be addressed.
Introduction: While it has been reported that the risk of contralateral breast cancer in patients from BRCA1 or BRCA2 positive families is elevated, little is known about contralateral breast cancer risk in patients from high risk families that tested negative for BRCA1/2 mutations.
Methods: A retrospective, multicenter cohort study was performed from 1996 to 2011 and comprised 6,235 women with unilateral breast cancer from 6,230 high risk families that had tested positive for BRCA1 (n = 1,154) or BRCA2 (n = 575) mutations or tested negative (n = 4,501). Cumulative contralateral breast cancer risks were calculated using the Kaplan-Meier product-limit method and were compared between groups using the log-rank test. Cox regression analysis was applied to assess the impact of the age at first breast cancer and the familial history stratified by mutation status.
Results: The cumulative risk of contralateral breast cancer 25 years after first breast cancer was 44.1% (95%CI, 37.6% to 50.6%) for patients from BRCA1 positive families, 33.5% (95%CI, 22.4% to 44.7%) for patients from BRCA2 positive families and 17.2% (95%CI, 14.5% to 19.9%) for patients from families that tested negative for BRCA1/2 mutations. Younger age at first breast cancer was associated with a higher risk of contralateral breast cancer. For women who had their first breast cancer before the age of 40 years, the cumulative risk of contralateral breast cancer after 25 years was 55.1% for BRCA1, 38.4% for BRCA2, and 28.4% for patients from BRCA1/2 negative families. If the first breast cancer was diagnosed at the age of 50 or later, 25-year cumulative risks were 21.6% for BRCA1, 15.5% for BRCA2, and 12.9% for BRCA1/2 negative families.
Conclusions: Contralateral breast cancer risk in patients from high risk families that tested negative for BRCA1/2 mutations is similar to the risk in patients with sporadic breast cancer. Thus, the mutation status should guide decision making for contralateral mastectomy.
In this thesis the Drosophila mutant loechrig (loe), that shows progressive degeneration of the nervous system, is further described. Loe is missing a neuronal isoform of the protein kinase AMPK γ subunit (AMP-activated protein kinase- also known as SNF4Aγ) The heterotrimeric AMPK controls the energy level of the cell, which requires constant monitoring of the ATP/AMP levels. It is activated by low energy levels and metabolic insults like oxygen starvation and regulates multiple important signal pathways that control cell metabolism. Still, its role in neuronal survival is unclear. One of AMPK’s downstream targets is HMGR (hydroxymethylglutaryl-CoA- reductase), a key enzyme in cholesterol and isoprenoid synthesis. It has been shown that manipulating the levels of HMGR affects the severity of the neurodegenerative phenotype in loe. Whereas the regulatory role of AMPK on HMGR is conserved in Drosophila, insects cannot synthesize cholesterol de novo. However, the synthesis of isoprenoids is a pathway that is evolutionarily conserved between vertebrates and insects. Isoprenylation of target proteins like small G-proteins provides a hydrophobic anchor that allows the association of these proteins with membranes and following activation. This thesis shows that the loe mutation interferes with the prenylation of Rho1 and the regulation of the LIM kinase pathway, which plays an important role in actin turnover and axonal outgrowth. The results suggest that the mutation in LOE, causes hyperactivity of the isoprenoid synthesis pathway, which leads to increased farnesylation of RHO1 and therefore higher levels of phospho-cofilin. A mutation in Rho1 improves the neurodegenerative phenotype and life span. The increased inactive cofilin amount in loe leads to an up regulation of filamentous actin. Actin is involved in neuronal outgrowth and experiments analyzing loe neurons gave valuable insights into a possible role of AMPK and accordingly actin on neurite growth and stability. It was demonstrated that neurons derived from loe mutants exhibit reduces axonal transport suggesting that changes in the cytoskeletal network caused by the effect of loe on the Rho1 pathway lead to disruptions in axonal transport and subsequent neuronal death. It also shows that actin is not only involved in neuronal outgrowth, its also important in maintenance of neurons, suggesting that interference with actin dynamics leads to progressive degeneration of neurons. Together, these results further support the importance of AMPK in neuronal function and survival and provide a novel functional mechanisms how alterations in AMPK can cause neuronal degeneration
The ITS2 Database
(2012)
The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution1 and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation.
The ITS2 Database presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank accurately reannotated. Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling. In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold.
The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE and ProfDistS for multiple sequence-structure alignment calculation and Neighbor Joining tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure.
In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.
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.
HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an Ebox motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression.
Recent development of proteomic approaches and generation of large-scale proteomic datasets calls for new methods for biological interpretation of the obtained results. Systems biological approaches such as integrated network analysis and functional module search have become an essential part of proteomic investigation. Proteomics is especially applied in anucleate cells such as platelets. The underlying molecular mechanisms of platelet activation and their pharmacological modulation are of immense importance for clinical research. Advances in platelet proteomics have provided a large amount of proteomic data, which has not yet been comprehensively investigated in a systems biological perspective. To this end, I assembled platelet specific data from proteomic and transcriptomic studies by detailed manual curation and worked on the generation of a comprehensive human platelet repository for systems biological analysis of platelets in the functional context of integrated networks (PlateletWeb) (http:/PlateletWeb.bioapps.biozentrum.uni-wuerzburg.de). I also added platelet-specific experimentally validated phosphorylation data and generated kinase predictions for 80% of the newly identified platelet phosphosites. The combination of drug, disease and pathway information with phosphorylation and interaction data makes this database the first integrative platelet platform available for platelet research. PlateletWeb contains more than 5000 platelet proteins, which can also be analyzed and visualized in a network context, allowing identification of all major signaling modules involved in platelet activation and inhibition. Using the wealth of integrated data I performed a series of platelet-specific analyses regarding the platelet proteome, pathways, drug targets and novel platelet phosphorylation events involved in crucial signaling events. I analyzed the statistical enrichment of known pathways for platelet proteins and identified endocytosis as a highly represented pathway in platelets. Further results revealed that highly connected platelet proteins are more often targeted by drugs. Using integrated network analysis offered by PlateletWeb, I analyzed the crucial activation signaling pathway of adenosine diphosphate (ADP), visualizing how the signal flow from receptors to effectors is maintained. My work on integrin inside-out signaling was also based on the integrated network approach and examined new platelet-specific phosphorylation sites and their regulation using kinase predictions. I generated hypothesis on integrin signaling, by investigating the regulation of Ser269 phosphorylation site on the docking protein 1 (DOK1). This phosphorylation site may influence the inhibiting effect of DOK1 on integrin a2bb3. Extending the integrated network approach to further cell lines, I used the assembled human interactome information for the analysis of functional modules in cellular networks. The investigation was performed with a previously developed module detection algorithm, which finds maximum-scoring subgraphs in transcriptomic datasets by using assigned values to the network nodes. We extended the algorithm to qualitative proteomic datasets and enhanced the module search by adding functional information to the network edges to concentrate the solution onto modules with high functional similarity. I performed a series of analyses to validate its performance in small-sized (virus-infected gastric cells) and medium-sized networks (human lymphocytes). In both cases the algorithm extracted characteristic modules of sample proteins with high functional similarity. The functional module search is especially useful in site-specific phosphoproteomic datasets, where kinase regulation of the detected sites is often sparse or lacking. Therefore, I used the module detection algorithm in quantitative phosphoproteomic datasets. In a platelet phosphorylation dataset, I presented a pipeline for network analysis of detected phosphorylation sites. In a second approach, the functional module detecting algorithm was used on a phosphoproteome network of human embryonic stem cells, in which nodes represented the maximally changing phosphorylation sites in the experiment. Additional kinases from the human phosphoproteome in PlateletWeb were included to the network to investigate the regulation of the signal flow. Results indicated important phosphorylation sites and their upstream kinases and explained changes observed in embryonic stem cells during differentiation. This work presents novel approaches for integrated network analysis in cells and introduces for the first time a systematic biological investigation of the human platelet proteome based on the platelet-specific knowledge base PlateletWeb. The extended methods for optimized functional module detection offer an invaluable tool for exploring proteomic datasets and covering gaps in complex large-scale data analysis. By combining exact module detection approaches with functional information data between interacting proteins, characteristic functional modules with high functional resemblance can be extracted from complex datasets, thereby focusing on important changes in the observed networks.
Measuring and estimating biodiversity patterns is a fundamental task of the scientist working to support conservation and informmanagement decisions.Most biodiversity studies in temperate regions were often carried out over a very short period of time (e.g., a single season) and it is often—at least tacitly—assumed that these short-termfindings are representative of long-termgeneral patterns.However, should the studied biodiversity pattern in fact contain significant temporal dynamics, perhaps leading to contradictory conclusions. Here, we studied the seasonal diversity dynamics of arboreal spider communities dwelling in 216 European beeches (Fagus sylvatica L.) to assess the spider community composition in the following seasons: two cold seasons (I:November 2005–January 2006; II: February–April) and two warm seasons (III: May–July; IV: August–October). We show that the usually measured diversity of the warmseason community (IV: 58 estimated species) alone did not deliver a reliable image of the overall diversity present in these trees, and therefore, we recommend it should not be used for sampling protocols aimed at providing a full picture of a forest’s biodiversity in the temperate zones. In particular, when the additional samplings of other seasons (I, II, III) were included, the estimated species richness nearly doubled (108). Community I possessed the lowest diversity and evenness due to the harsh winter conditions: this community was comprised of one dominant species together with several species low in abundance. Similarity was lowest (38.6%) between seasonal communities I and III, indicating a significant species turnover due to recolonization, so that community III had the highest diversity. Finally, using nonparametric estimators, we found that further sampling in late winter (February–April) is most needed to complete our inventory. Our study clearly demonstrates that seasonal dynamics of communities should be taken into account when studying biodiversity patterns of spiders, and probably forest arthropods in general.
The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole (SCV) in host cells. We summarize latest results on metabolic requirements for Salmonella during infection. This includes intracellular phenotypes of mutant strains based on metabolic modeling and experimental tests, isotopolog profiling using (13)C-compounds in intracellular Salmonella, and complementation of metabolic defects for attenuated mutant strains towards a comprehensive understanding of the metabolic requirements of the intracellular lifestyle of Salmonella. Helpful for this are also genomic comparisons. We outline further recent studies and which analyses of intracellular phenotypes and improved metabolic simulations were done and comment on technical required steps as well as progress involved in the iterative refinement of metabolic flux models, analyses of mutant phenotypes, and isotopolog analyses. Salmonella lifestyle is well-adapted to the SCV and its specific metabolic requirements. Salmonella metabolism adapts rapidly to SCV conditions, the metabolic generalist Salmonella is quite successful in host infection.
ATP dependent chromatin remodeling complexes are multifactorial complexes that utilize the energy of ATP to rearrange the chromatin structure. The changes in chromatin structure lead to either increased or decreased DNA accessibility. SWI/SNF is one of such complex. The SWI/SNF complex is involved in both transcription activation and transcription repression. The ATPase subunit of SWI/SNF is called SWI2/SNF2 in yeast and Brahma, Brm, in Drosophila melanogaster. In mammals there are two paralogs of the ATPase subunit, Brm and Brg1. Recent studies have shown that the human Brm is involved in the regulation of alternative splicing. The aim of this study was to investigate the role of Brm in pre-mRNA processing. The model systems used were Chironomus tentans, well suited for in situ studies and D. melanogaster, known for its full genome information. Immunofluorescent staining of the polytene chromosome indicated that Brm protein of C. tentans, ctBrm, is associated with several gene loci including the Balbiani ring (BR) puffs. Mapping the distribution of ctBrm along the BR genes by both immuno-electron microscopy and chromatin immunoprecipitation showed that ctBrm is widely distributed along the BR genes. The results also show that a fraction of ctBrm is associated with the nascent BR pre-mRNP. Biochemical fractionation experiments confirmed the association of Brm with the RNP fractions, not only in C. tentans but also in D. melanogaster and in HeLa cells. Microarray hybridization experiments performed on S2 cells depleted of either dBrm or other SWI/SNF subunits show that Brm affects alternative splicing and 3´ end formation. These results indicated that BRM affects pre-mRNA processing as a component of SWI/SNF complexes. 1
Many organisms evolved an endogenous clock to adapt to the daily environmental changes caused by the earth’s rotation. Light is the primary time cue (“Zeitgeber”) for entrainment of circadian clocks to the external 24-h day. In Drosophila, several visual pigments are known to mediate synchronization to light: The blue-light photopigment Cryptochrome (CRY) and six well-described rhodopsins (Rh1-Rh6). CRY is present in the majority of clock neurons as well as in the compound eyes, whereas the location of rhodopsins is restricted to the photoreceptive organs – the compound eyes, the ocelli and the HB-eyelets. CRY is thought to represent the key photoreceptor of Drosophila’s circadian clock. Nevertheless, mutant flies lacking CRY (cry01) are able to synchronize their locomotor activity rhythms to light-dark (LD) cycles, but need significantly longer than wild-type flies. In this behavior, cry01 mutants strongly resemble mammalian species that do not possess any internal photoreceptors and perceive light information exclusively through their photoreceptive organs (eyes). Thus, a mammalian-like phase-shifting behavior would be expected in cry01 flies. We investigated this issue by monitoring a phase response curve (PRC) of cry01 and wild-type flies to 1-h light pulses of 1000 lux irradiance. Indeed, cry01 mutants produced a mammalian-similar so called type 1 PRC of comparatively low amplitude (< 25% of wild-type) with phase delays to light pulses during the early subjective night and phase advances to light pulses during the late subjective night (~1 h each). Despite the predominant role of CRY, the visual system contributes to the light sensitivity of the fly’s circadian clock, mainly around dawn and dusk. Furthermore, this phase shifting allows for the slow re-entrainment which we observed in cry01 mutants to 8-h phase delays of the LD 12 h:12 h cycle. However, cry01 also showed surprising differences in their shifting ability: First of all, their PRC was characterized by a second dead zone in the middle of the subjective night (ZT17-ZT19) in addition to the usual unresponsiveness during the subjective day. Second, in contrast to wild-type flies, cry01 mutants did not increase their shift of activity rhythms neither in response to longer stimuli nor to light pulses of higher irradiance. In contrast, both 6-h light pulses of 1000 lux and 1-h light pulses of 10,000 lux light intensity during the early subjective night even resulted in phase advances instead of the expected delays. Thus, CRY seems to be not only responsible for the high light sensitivity of the wild-type circadian clock, but is apparently also involved in integrating and processing light information. Rhodopsin 7 (Rh7) is a yet uncharacterized protein, but became a good photoreceptor candidate due to sequence similarities to the six known Drosophila Rhs. The second part of this thesis investigated the expression pattern of Rh7 and its possible functions, especially in circadian photoreception. Furthermore, we were interested in a potential interaction with CRY and thus, tested cry01 and rh70 cry01 mutants as well. Rh1 is the main visual pigment of the Drosophila compound eye and expressed in six out of eight photoreceptors cells (R1-R6) in each of the ~800 ommatidia. Motion vision depends exclusively on Rh1 function but, moreover, Rh1 plays an important structural role and assures proper photoreceptor cell development and maintenance. In order to investigate its possible photoreceptive function, we expressed Rh7 in place of Rh1. Rh7 was indeed able to overtake the role of Rh1 in both aspects: It prevented retinal degeneration and mediated the optomotor response (OR), a motion vision-dependent behavior. At the transcriptional level, rh7 is expressed at approximately equal amounts in adult fly brains and retinas. Due to a reduced specificity of anti-Rh7 antibodies, we could not verify this result at the protein level. However, analysis of rh7 null mutants (rh70) suggested different Rh7 functions in vivo. Previous experiments strongly indicated an increased sensitivity of the compound eyes in the absence of Rh7 and suggested impaired light adaptation. We aimed to test this hypothesis at the levels of circadian photoreception. Locomotor activity rhythms are a reliable output of the circadian clock. Rh70 mutant flies generally displayed a wild-type similar bimodal activity pattern comprising morning (M) and evening (E) activity bouts. Activity monitoring supported the proposed “shielding” function, since rh70 mutants behaved like wild-type flies experiencing high irradiances. Under all investigated conditions, their activity peaks lay further apart resulting in a prolonged midday break. The behavior of cry01 mutants was mainly characterized by an unexpectedly high flexibility in the timing of M and E activity bouts which allowed tracking of lights-on and lights-off even under extreme photoperiods. Activity profiles of the corresponding rh70 cry01 double mutants reflected neither synergistic nor antagonistic effects of Rh7 and CRY and were dominated by a broad E activity peak. In the future, the different circadian phenotypes will be further investigated on the molecular level by analysis of clock protein cycling in the underlying pacemaker neurons. The work of this thesis confirmed that Rh7 is indeed able to work as a photoreceptor and to initiate the classical phototransduction cascade. On the other hand, it provided further evidence at the levels of circadian photoreception that Rh7 might serve as a shielding pigment for Rh1 in vivo, thereby mediating proper light adaptation.
The development of insecticides requires valid risk assessment procedures to avoid causing harm to beneficial insects and especially to pollinators such as the honeybee Apis mellifera. In addition to testing according to current guidelines designed to detect bee mortality, tests are needed to determine possible sublethal effects interfering with the animal's vitality and behavioral performance. Several methods have been used to detect sublethal effects of different insecticides under laboratory conditions using olfactory conditioning. Furthermore, studies have been conducted on the influence insecticides have on foraging activity and homing ability which require time-consuming visual observation. We tested an experimental design using the radiofrequency identification (RFID) method to monitor the influence of sublethal doses of insecticides on individual honeybee foragers on an automated basis. With electronic readers positioned at the hive entrance and at an artificial food source, we obtained quantifiable data on honeybee foraging behavior. This enabled us to efficiently retrieve detailed information on flight parameters. We compared several groups of bees, fed simultaneously with different dosages of a tested substance. With this experimental approach we monitored the acute effects of sublethal doses of the neonicotinoids imidacloprid (0.15-6 ng/bee) and clothianidin (0.05-2 ng/bee) under field-like circumstances. At field-relevant doses for nectar and pollen no adverse effects were observed for either substance. Both substances led to a significant reduction of foraging activity and to longer foraging flights at doses of >= 0.5 ng/bee (clothianidin) and >= 1.5 ng/bee (imidacloprid) during the first three hours after treatment. This study demonstrates that the RFID-method is an effective way to record short-term alterations in foraging activity after insecticides have been administered once, orally, to individual bees. We contribute further information on the understanding of how honeybees are affected by sublethal doses of insecticides.
The mechanisms that enable cells to regulate their gene expression and thus their metabolism, proliferation or cellular behaviour are not only important to understand the basic biology of a living cell, but are also of crucial interest in cancerogenesis. Highly interwoven and tightly regulated pathways are the basis of a robust but also flexible regulatory network. Interference with these pathways can be either causative for tumorigenesis or can modify its outcome. The receptor tyrosine kinase (RTK) and RAS dependent pathways leading to AKT or ERK1/2 activation are of particular interest in melanoma. These signaling modules are commonly activated by different mutations that can be found in various pathway components like NRAS, BRAF or PTEN. The first part of this work deals with the diverse and versatile functions of the ERK1/2 pathway feedbackregulator MKP2 in different cellular, melanoma relevant settings. In addition, a functional role of the AP1-complex member FOSL1, an ERK1/2 transcriptional target being implicated in the regulation of proliferation, is demonstrated. Secondly, aspects of direct pharmacological inhibition of the ERK1/2 pathway with regard to the induction of apoptosis have been analysed. Due to the high frequency of melanoma related mutations occurring in the RAS/RAF/MEK/ERK pathway (e.g. NRASQ61K, BRAFV600E), inhibition of this signaling cascade is deemed to be a promising therapeutic strategy for the treatment of malignant melanoma. However, although in clinical trials mono-therapeutic treatment with MEK- or RAF inhibitors was successful in the short run, it failed to show satisfactory long-lasting effects. Hence, combination therapies using a MAPK pathway inhibitor and an additional therapy are currently under investigation. I was able to demonstrate that inhibition of MEK using the highly specific inhibitor PD184352 can have a protective effect on melanoma cells with regard to their susceptibility towards the apoptosis inducing agent cisplatin. Single application of cisplatin led to strong DNA damage and the induction of caspase-dependent apoptosis. Additional administration of the MEK inhibitor, however, strongly reduced the apoptosis inducing effect of cisplatin in several melanoma cell lines, These cells displayed an increased activation of the serine/threonine kinase AKT after MEK inhibition. This AKT activation concomitantly led to the phosphorylation of FOXO transcription factors, attenuating the cisplatin induced expression of the BH3-only protein PUMA. PUMA in turn was important to mediate the apoptosis machinery after cisplatin treatment. My results also indicate a participation of RTKs, in particular EGFR, in mediating MEK inhibitor induced activation of AKT. These results demonstrate that inhibition of the RAS/RAF/MEK/ERK signaling pathway in melanoma cell lines does not necessilary have favourable effects in a cytotoxic co-treatment situation. Instead, it can even enhance melanoma survival under pro-apoptotic conditions.
Understanding the emergence of species' ranges is one of the most fundamental challenges in ecology. Early on, geographical barriers were identified as obvious natural constraints to the spread of species. However, many range borders occur along gradually changing landscapes, where no sharp barriers are obvious. Mechanistic explanations for this seeming contradiction incorporate environmental gradients that either affect the spatio-temporal variability of conditions or the increasing fragmentation of habitat. Additionally, biological mechanisms like Allee effects (i.e. decreased growth rates at low population sizes or densities), condition-dependent dispersal, and biological interactions with other species have been shown to severely affect the location of range margins. The role of dispersal has been in the focus of many studies dealing with range border formation. Dispersal is known to be highly plastic and evolvable, even over short ecological time-scales. However, only few studies concentrated on the impact of evolving dispersal on range dynamics. This thesis aims at filling this gap. I study the influence of evolving dispersal rates on the persistence of spatially structured populations in environmental gradients and its consequences for the establishment of range borders. More specially I investigate scenarios of range formation in equilibrium, periods of range expansion, and range shifts under global climate change ...
We identified eukaryotic translation elongation factor 1A (eEF1A) Raf-mediated phosphorylation sites and defined their role in the regulation of eEF1A half-life and of apoptosis of human cancer cells. Mass spectrometry identified in vitro S21 and T88 as phosphorylation sites mediated by B-Raf but not C-Raf on eEF1A1 whereas S21 was phosphorylated on eEF1A2 by both B-and C-Raf. Interestingly, S21 belongs to the first eEF1A GTP/GDP-binding consensus sequence. Phosphorylation of S21 was strongly enhanced when both eEF1A isoforms were preincubated prior the assay with C-Raf, suggesting that the eEF1A isoforms can heterodimerize thus increasing the accessibility of S21 to the phosphate. Overexpression of eEF1A1 in COS 7 cells confirmed the phosphorylation of T88 also in vivo. Compared with wt, in COS 7 cells overexpressed phosphodeficient (A) and phospho-mimicking (D) mutants of eEF1A1 (S21A/D and T88A/D) and of eEF1A2 (S21A/D), resulted less stable and more rapidly proteasome degraded. Transfection of S21 A/D eEF1A mutants in H1355 cells increased apoptosis in comparison with the wt isoforms. It indicates that the blockage of S21 interferes with or even supports C-Raf induced apoptosis rather than cell survival. Raf-mediated regulation of this site could be a crucial mechanism involved in the functional switching of eEF1A between its role in protein biosynthesis and its participation in other cellular processes.
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
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.
Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1
(2012)
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.
Plant communities in the European Alps are assumed to be highly affected by climate change since temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010 an experiment simulating advanced and delayed snowmelt as well as drought event was conducted along an altitudinal transect ca. every 250m (600-2000 m a.s.l.) in the Berchtesgaden National Park, Germany. The study showed that flower phenology is strongly affected by altitude; however there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes might be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes since changes may be more pronounced at higher altitudes. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.
Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An ‘ideal’ virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.
The bloodstream developmental forms of pathogenic African trypanosomes are uniquely susceptible to killing by small hydrophobic peptides. Trypanocidal activity is conferred by peptide hydrophobicity and charge distribution and results from increased rigidity of the plasma membrane. Structural analysis of lipid-associated peptide suggests a mechanism of phospholipid clamping in which an internal hydrophobic bulge anchors the peptide in the membrane and positively charged moieties at the termini coordinate phosphates of the polar lipid headgroups. This mechanism reveals a necessary phenotype in bloodstream form African trypanosomes, high membrane fluidity, and we suggest that targeting the plasma membrane lipid bilayer as a whole may be a novel strategy for the development of new pharmaceutical agents. Additionally, the peptides we have described may be valuable tools for probing the biosynthetic machinery responsible for the unique composition and characteristics of African trypanosome plasma membranes.
High background noise is an impediment to signal detection and perception. We report the use of multiple solutions to improve signal perception in the acoustic and visual modality by the Bornean rock frog, Staurois parvus. We discovered that vocal communication was not impaired by continuous abiotic background noise characterised by fast-flowing water. Males modified amplitude, pitch, repetition rate and duration of notes within their advertisement call. The difference in sound pressure between advertisement calls and background noise at the call dominant frequency of 5578 Hz was 8 dB, a difference sufficient for receiver detection. In addition, males used several visual signals to communicate with conspecifics with foot flagging and foot flashing being the most common and conspicuous visual displays, followed by arm waving, upright posture, crouching, and an open-mouth display. We used acoustic playback experiments to test the efficacy-based alerting signal hypothesis of multimodal communication. In support of the alerting hypothesis, we found that acoustic signals and foot flagging are functionally linked with advertisement calling preceding foot flagging. We conclude that S. parvus has solved the problem of continuous broadband low-frequency noise by both modifying its advertisement call in multiple ways and by using numerous visual signals. This is the first example of a frog using multiple acoustic and visual solutions to communicate in an environment characterised by continuous noise.
The actin cytoskeleton is essential for many cellular functions, such as the regulation of cell morphology, cell migration and vesicle transport processes. The functional diversity of actin structures is reflected in a variety of distinct molecular mechanisms regulating the polymerization of actin filaments. The spontaneous polymerization of actin however is inhibited, by both the instability of small actin oligomers and by actin monomer binding proteins, which prevent the formation of such oligomers. Actin nucleation factors help to overcome this kinetic barrier of filament initiation and are essential for the generation of novel actin filaments at specified subcellular compartments. Spir proteins are the founding members of the novel class of WH2 domain containing actin nucleation factors. They initiate actin polymerization by binding of actin monomers to four WH2 domains in the central part of the protein. Despite their ability to nucleate actin polymerization in vitro by themselves, Spir proteins form a regulatory complex with the distinct actin nucleators of the formin subgroup of formins. Spir functions in the regulation of vesicular originated filamentous actin structures, vesicle transport processes and the assembly of the cleavage furrow during asymmetric meiotic cell divisions. The mammalian genome encodes two spir genes, spir-1 and spir-2. The corresponding proteins have an identical structural array and share a high degree of homology. In order to elucidate the Spir function in developing and adult mouse tissues, the yet unknown expression of the mouse spir-2 gene was addressed. Real-time PCR analysis revealed highest expression of spir-2 in oocytes, the brain, throughout the gastrointestinal tract, testis and kidney of adult mice. In situ hybridizations were performed to substantiate the cellular nature of spir gene expression. During embryogenesis in situ hybridizations show spir-2 to be expressed in the developing nervous system and intestine. In adult mouse tissues highest expression of spir-2 was detected in the epithelial cells of the digestive tract, in neuronal cells of the nervous system and in spermatocytes. In contrast to the more restricted expression of the mouse spir-1 gene, which is mainly found in the nervous system, oocytes and testis, the data presented here show a distinct and broader expression pattern of the spir-2 gene and by this support a more general cell biological function of the novel actin nucleators. In order to address the function of Spir proteins in the developing and adult nervous system, Spir-1 deficient mice were generated by a gene trap method. Spir-1 deficient mice are viable and provide a perfect tool to address the neurobiological function of the Spir-1 protein. Analyses of primary cortical neurons from Spir-1 deficient mice revealed a specific reduction of dendritic branchpoints and are the first description of a neuronal Spir-1 function. Further, a transgenic mouse line (thy1-GFP-M) was employed that expresses the green fluorescent protein (GFP) under the control of neuron specific elements from the thy1 promoter. GFP is thereby expressed in only a subset of neurons and labels the neurons in their entirety. Spir-1 deficient mice carrying the GFP transgene were generated and analyzed. It was found that Spir-1 deficient mice exhibit a reduced number of dendritic spines in the entorhinal cortex compared to wildtype littermates. All together this study gives novel information about the cell biological function of Spir and provides insights how cytoskeletal functions structure the mammalian neuronal network.
Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.
Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus
(2012)
The production of antimicrobial peptides (AMPs) is a major defense mechanism against pathogen infestation and of particular importance for insects relying exclusively on an innate immune system. Here, we report on the characterization of three AMPs from the carpenter ant Camponotus floridanus. Due to sequence similarities and amino acid composition these peptides can be classified into the cysteine-rich (e.g. defensin) and glycine-rich (e.g. hymenoptaecin) AMP groups, respectively. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. We characterized two different defensin genes. The defensin-2 gene has a single intron, whereas the defensin-1 gene has two introns. The deduced amino acid sequence of the C. floridanus defensins is very similar to other known ant defensins with the exception of a short C-terminal extension of defensin-1. The hymenoptaecin gene has a single intron and a very peculiar domain structure. The corresponding precursor protein consists of a signal- and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains. Each of the hymenoptaecin domains is flanked by an EAEP-spacer sequence and a RR-site known to be a proteolytic processing site. Thus, proteolytic processing of the multipeptide precursor may generate several mature AMPs leading to an amplification of the immune response. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity.
Sensory limitation plays an important role in the evolution of animal behaviour. Animals have to find objects of interest (e.g. food, shelters, predators). When sensory abilities are strongly limited, animals adjust their behaviour to maximize chances for success. Bats are nocturnal, live in complex environments, are capable of flight and must confront numerous perceptual challenges (e.g. limited sensory range, interfering clutter echoes). This makes them an excellent model for studying the role of compensating behaviours to decrease costs of finding resources. Cavity roosting bats are especially interesting because the availability of tree cavities is often limited, and their quality is vital for bats during the breeding season. From a bat's sensory point of view, cavities are difficult to detect and finding them requires time and energy. However, tree cavities are also long lasting, allowing information transfer among conspecifics. Here, we use a simple simulation model to explore the benefits of tree selection, memory and eavesdropping (compensation behaviours) to searches for tree cavities by bats with short and long perception range. Our model suggests that memory and correct discrimination of tree suitability are the basic strategies decreasing the cost of roost finding, whereas perceptual range plays a minor role in this process. Additionally, eavesdropping constitutes a buffer that reduces the costs of finding new resources (such as roosts), especially when they occur in low density. We conclude that natural selection may promote different strategies of roost finding in relation to habitat conditions and cognitive skills of animals.
The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15\(^{Ink4}\)), cdkn1a (p21\(^{Cip1}\)), and cdkn1c (p57\(^{Kip2}\)). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.
MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.
Chlamydiales are obligate intracellular gram-negative bacteria that have gained high medical relevance. These important human pathogens cause diverse diseases including trachoma and wide spread sexually transmitted diseases. Chlamydia establishes membrane bound inclusions in the host cell and loots the host for nutritional requirements. Infections are usually recognized by the host immune system and eliminated systematically, by triggering apoptosis. However, the pathogen Chlamydia has evolved various strategies to prevent the detection as well as protect the invaded cell against apoptosis or any other form of cell death. The evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. The present study was aimed at investigating the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Caspases were differentially regulated upon Sn infection. Caspase-3 and -9 were not activated upon Sn infection and apoptosis induction; whereas caspases-8 was activated in Sn infected cells even without apoptosis induction. This indicates that, Sn utilizes death receptor association independent caspase activation for thriving in the host environment. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Proteins (IAP-1/2 and XIAP) and the Akt/PI3K pathway. Sn infection also 20 activated the pro-survival transcription factor NF-кB. Blocking any of these survival pathways sensitized the infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. The NF-кB mutant cells also showed reduced infectivity of Sn, which indicated an essential role of NF-кB in Sn infection. It was interesting to observe that, Acanthamoeba castellanii, a natural host of Sn, survived maintaining its trophozoite forms after infection with Sn upon starvation. The metacaspases, responsible for encystment could be regulated by Sn upon infection. This suggests an early level of gene regulation indicating how the pathogen evolved its ability to inhibit apoptosis in higher organisms. The resistance to apoptosis pathways subverted in Sn-infected cells was similar but not identical to those modulated by Chlamydia. Together, the data supports the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.
Myc is a global transcriptional regulator and one of the most frequently overexpressed oncoproteins in human tumors. It is well established that activation of Myc leads to enhanced cell proliferation but can also lead to increased apoptosis. The use of animal models expressing deregulated levels of Myc has helped to both elucidate its function in normal cells and give insight into how Myc initiates and maintains tumorigenesis. Analyses of the medaka (Oryzias latipes) genome uncovered the unexpected presence of two Myc gene copies in this teleost species. Comparison of these Myc versions to other vertebrate species revealed that one gene, myc17, differs by the loss of some conserved regulatory protein motifs present in all other known Myc genes. To investigate how such differences might affect the basic biological functions of Myc, we generated a tamoxifeninducible in vivo model utilizing a natural, fish-specific Myc gene. Using this model we show that, when activated, Myc17 leads to increased proliferation and to apoptosis in a dose-dependent manner, similar to human Myc. We have also shown that long-term Myc17 activation triggers liver hyperplasia in adult fish, allowing this newly established transgenic medaka model to be used to study the transition from hyperplasia to liver cancer and to identify Myc-induced tumorigenesis modifiers.
Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.
Staphylococcus aureus uses a plethora of virulence factors to accommodate a diversity of niches in its human host. Aside from the classical manifestations of S. aureus-induced diseases, the pathogen also invades and survives within mammalian host cells. The survival strategies of the pathogen are as diverse as strains or host cell types used. S. aureus is able to replicate in the phagosome or freely in the cytoplasm of its host cells. It escapes the phagosome of professional and non-professional phagocytes, subverts autophagy, induces cell death mechanisms such as apoptosis and pyronecrosis, and even can induce anti-apoptotic programs in phagocytes. The focus of this review is to present a guide to recent research outlining the variety of intracellular fates of S. aureus.
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.
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.
Background: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress. Results: In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurablemetabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration. Conclusions: The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.
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.
We have studied the responses of honey bees at different life stages (Apis mellifera) to controlled infection with acute bee paralysis virus and have identified the haemolymph of infected larvae and adult worker bees as the compartment where massive propagation of ABPV occurs. Insects respond with a broad spectrum of induced innate immune reactions to bacterial infections, whereas defence mechanisms based on RNA interference play a major role in antiviral immunity. In this study, we have determined that honey bee larvae and adult workers do not produce a humoral immune reaction upon artificial infection with ABPV, in contrast to control individuals challenged with Escherichia coli. ABPV-infected bees produced neither elevated levels of specific antimicrobial peptides (AMPs), such as hymenoptaecin and defensin, nor any general antimicrobial activity, as revealed by inhibition-zone assays. Additionally, adult bees did not generate melanised nodules upon ABPV infection, an important cellular immune function activated by bacteria and viruses in some insects. Challenge of bees with both ABPV and E. coli showed that innate humoral and cellular immune reactions are induced in mixed infections, albeit at a reduced level.
Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
To highlight human impact on biodiversity in the Lamto region, termites were studied with regard to their use as bio-indicators of habitat change in the tropics. Using a standardized method, termites were sampled in the three most common habitat types, i.e., in semi-deciduous forest, savanna woodland, and annually burned savanna, all inside Lamto Reserve and its surrounding rural domain. Termite species richness fell from 25 species in the Lamto forest to 13 species in the rural area, involving strong modification in the species composition (species turnover = 59 %). In contrast, no significant change in diversity was found between the Lamto savannas and the rural ones. In addition, the relative abundance of termites showed a significantly greater decline in the rural domain, even in the species Ancistrotermes cavithorax (Sjostedt) (Isoptera: Termitidae), which is known to be ecologically especially versatile. Overall, the findings of this study suggest further investigation around Lamto Reserve on the impact of human activities on biodiversity, focusing on forest conversion to land uses (e.g. agricultural and silvicultural systems).
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.