Refine
Is part of the Bibliography
- yes (789)
Year of publication
Document Type
- Doctoral Thesis (780)
- Journal article (8)
- Book (1)
Language
- English (789) (remove)
Keywords
- Maus (32)
- Thrombozyt (30)
- Taufliege (20)
- Tissue Engineering (19)
- Genexpression (16)
- T-Lymphozyt (15)
- Dendritische Zelle (14)
- Biene (13)
- Entzündung (13)
- Angst (12)
Institute
- Graduate School of Life Sciences (789) (remove)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (7)
- Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg (2)
- Universitätsklinikum Münster (2)
- Zentrum für Infektionsforschung (ZINF) Würzburg (2)
- Bio-Imaging Center Würzburg (1)
- Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München (1)
- CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - the development agency of the Brazilian Federal Government (1)
- Carl-Ludwig-Institut für Physiologie, Universität Leipzig (1)
- Chair of Experimental Biomedicine I (1)
- DAAD - Deutscher Akademischer Austauschdienst (1)
Opioids have been, since centuries, the gold standard for pain treatment and relief. They exert their effects after binding to opioid receptors (OP) that are expressed and functional in the central (CNS) and peripheral nervous system (PNS). As their systemic application has many side effects, including sedation and respiratory depression, a peripheral application of opioids and selective targeting of µ-OP (MOP) in nociceptive axons would be extremely beneficial. MOP presence and function has been conclusively demonstrated at nerve terminals; however it is still controversial whether functional MOPs are available on the membrane of peripheral nociceptive axons to mediate opioid-induced antinociception. While under pathologic conditions (i.e. nerve injury) exogenous as well as endogenous MOP agonists applied at the damaged nerve can elicit potent antinociception or anti-allodynia, under physiological conditions no antinociception was seen in rats. This could be caused by either a lack of functional opioid receptors in the axonal membranes or by the inability of injected opioids to cross the intact perineurial barrier and to reach nociceptors. Previous behavioral test results showed an antinociceptive effect (up to 5h) following perisciatic application of the hydrophilic DAMGO (MOP agonist) if coinjected with hypertonic saline solution (HTS; 10% NaCl), a treatment suited to open the perineural barrier. The effect was inhibited by naloxone, a MOP antagonist, documenting its specific action via MOP. Fentanyl, a lipophilic opioid, elicited an effect, which was enhanced by HTS treatment, indicating that HTS may act not only on the barrier but also directly on axonal MOP presence and/or functionality. To provide a basis for testing this hypothesis, the present work was designed to study the axonal localization of MOP in experimental animals under different conditions using molecular and morphological methods.
Initially four different commercial antibodies were tested for MOP detection. Immunoreactions with these antibodies specifically detected MOP in the hippocampus and in amygdala, while in the peripheral nervous system the reactions showed varying labeling patterns pointing towards less specificity with low signal-to-noise ratio. Double labelling with calcitonin gene related peptide (CGRP), a neuropeptide expressed in sensory fibers, with the non-compacted myelin marker S100 or with the neuronal marker PGP9.5 documented significant immunoreaction signals outside sensory nerve fibers. Therefore, none of these antibodies appeared suitable. Taking advantage of a new commercial monoclonal rabbit antibody (RabMAb) and of genetically modified mice in which the fluorescent protein mcherry was inserted in the C-tail of MOP (MOP-mcherry knock-in mice), MOP fusion protein expression in rat and mouse CGRP+ sciatic nerve fibers and fiber bundles was confirmed by immunofluorescence labeling. Immunoelectron microscopic analysis indicated MOP/MOP-mcherry-localization in the cytoplasm and the membranes of unmyelinated axons organized in Remak bundles. Both antibodies detected bands of appropriate size in Western Blot in the CNS and additional larger bands in the PNS. Quantitative analyses 60 min after HTS-treatment revealed no change in MOP mRNA in the sciatic nerve and DRG as well as no change in MOP immunoreactivity in the sciatic nerve. Thus, the opioid-induced long lasting antinociception enhanced by perisciatic injection of HTS were not due to a sustained increased MOP expression or content in sensory, putative nociceptive axons.
In summary, the current study succeeded to unequivocally document the presence of MOP protein in intact sensory axons of rat and mouse sciatic nerve. Thus, axonal MOPs may indeed mediate antinociceptive opioid effects observed in behavioral studies in naive animals possibly via activation of potassium or calcium channels. As HTS treatment does not lead to a sustained increase in axonal MOP protein or MOP mRNA expression, other mechanisms might enhance MOP function, including inhibition of MOP recycling or changes in functional coupling. Future studies should further explore the axonal mechanisms of antinociception by opioids and enhancing treatments.
Eukaryotic cells are considered as evolutionary complex organisms because they possess organelles that enable them to regulate the spatio-temporal organization of cellular processes. Spatio-temporal organization of signal transduction cascades occurs in eukaryotic cells via organization of membrane-associated microdomains or lipid rafts. Lipid rafts are nanoscale-sized domains in the plasma membrane that are constituted by a specific set of lipids and proteins and harbor a number of proteins related to signal transduction and trafficking. The integrity of lipid rafts is important for the assembly and functional coordination of a plethora of signaling networks and associated processes. This integrity is partially mediated by a chaperone protein called flotillin. Disruption of lipid raft integrity, for example via depletion or overproduction of flotillin, alters raft-associated signal transduction cascades and causes severe diseases like Alzheimer’s, Parkinson’s disease or cardiovascular disease.
It was traditionally assumed that a sophisticated compartmentalization of cellular processes like the one exhibited in lipid rafts was exclusive to eukaryotic cells and therefore, lipid rafts have been considered as a hallmark in the evolution of cellular complexity, suggesting that prokaryotic cells were too simple organisms to organize such sophisticated membrane platforms. However, it was recently discovered that bacteria are also able to organize Functional Membrane Microdomains (FMMs) in their cellular membrane that are able to organize and catalyze the functionality of many diverse cellular processes. These FMMs of bacterial membranes contain flotillin-like proteins which play important roles in the organization of FMM-associated cellular processes.
In this dissertation I describe the structural and biological significance of the existence of two distinct flotillin proteins, FloA and FloT, in the FMMs of the bacterial model Bacillus subtilis. Localization studies, proteomic data and transcriptomic analyses show that FloA and FloT are individual scaffold proteins that activate different regulatory programs during bacterial growth. Using the tractable bacterial model system, I show that the functionality of important regulatory proteins, like the protease FtsH or the signaling kinases KinC, PhoR and ResE, is linked to the activity of FMMs and that this is a direct consequence of the scaffold activity of the bacterial flotillins. FloA and FloT distribute heterogeneously along the FMMs of B. subtilis thereby generating a heterogeneous population of FMMs that compartmentalize different signal transduction cascades. Interestingly, diversification of FMMs does not occur randomly, but rather in a controlled spatio-temporal program to ensure the activation of given signaling networks at the right place and time during cell growth.
Abstract
Background
HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases.
Results
As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde.
Conclusion
The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.
Abstract
Glioblastomas, primary brain tumors, represent a tumor entity with a dismal prognosis and a median survival of only about one year. Invasion into the healthy brain parenchyma contributes substantially to the malignancy of this type of brain tumor. Therefore, a better understanding of the mechanisms promoting the invasive behavior of these brain tumors is needed to identify new therapeutic targets.
Cofilin, an actin regulatory protein, has been shown to be an important regulator of the invasive behavior of tumor cells in other types of cancer and the actin cytoskeleton is involved in the formation of a variety of cellular structures important for cell migration and invasion. Cofilin is regulated by phosphorylation on a single residue, serine 3. The aim of this thesis was to examine the role of the cofilin regulatory phosphatase chronophin for glioma cell migration and invasion.
First, it was established that chronophin depletion in the cell line GBM6840 leads to an increase in the ratio of phosphorylated cofilin to total cofilin. Higher chronophin levels were correlated with a decrease in F-actin in the cell lines GBM6840 and U87 as measured in an actin spin down assay and in a flow cytometry based assay.
Furthermore, it was shown that knockdown of chronophin in two different cell lines, GBM6840 and DBTRG-05-MG, strongly increased their invasiveness in vitro. Expression of human chronophin in the cell line U87 decreased its invasiveness substantially. There was no difference in cell proliferation between GBM6840 and DBTRG-05-MG cells expressing a chronophin targeting shRNA or a control shRNA and U87 cells transfected with an empty vector or a human chronophin encoding plasmid. The increase in invasiveness after chronophin depletion could be correlated with an increase in directionality in cell migration under 2D culture conditions in the cell lines U87 and GBM6840. Moreover, treatment with the ROCK inhibitor Y-27632 decreased directionality in GBM6840 cells under 2D culture conditions and reduced the invasiveness of GBM6840 chronophin shRNA cells back to control levels.
Expression of a non-phosphorylatable cofilin mutant, the S3A mutant, was able to reduce invasiveness and to reduce directionality under 2D culture conditions back to control levels in GBM6840 chronophin shRNA cells.
This provides important evidence for the involvement of cofilin phosphoregulation in the phenotypes described above.
In vivo, when injected into NOD-SCID mice, chronophin depleted cells showed a dramatic growth reduction as compared to control and rescue cells.
Transciptomic characterization of GBM6840 cells by microarray analysis and subsequent comparison of the data with microarray profiles of normal brain tissues and different glioma entities identified two specifically chronophin regulated transcripts potentially involved in tumor progression and invasion, MXI1 and EDIL3. Moreover, c-myc was identified as a significantly altered transcription factor after chronophin deregulation based on the number of c-myc target molecules in the microarray dataset.
MXI1 is a potential negative regulator of c-myc dependent transcription, and was strongly downregulated after chronophin knockdown in GBM6840. In line with this, the activity of a c-myc reporter plasmid was increased after chronophin depletion in GBM6840 and reduced after chronophin expression in U87 cells.
However, the protein level of the c-myc protein was reduced after chronophin depletion in GBM6840.
Finally, anaylsis of the expression of proteases known to be important for glioblastoma pathogenesis revealed no major changes in protease expression between chronophin depleted and control cells.
Therefore, a comprehensive analysis of chronophin in the context of glioma pathogenesis has been performed in this thesis. It has been shown that chronophin depletion strongly enhanced invasiveness of glioma cells and that it induced transcriptomic changes potentially involved in tumor progression. The proteins regulating cofilin phosphorylation are therefore valuable therapeutic targets for anti-invasive therapy in glioblastomas. Inhibitors for kinases upstream of cofilin, e.g. LIMKs and ROCKs, are available, and might be promising agents for anti-invasive therapy.
Adenosine receptors that belong to the rhodopsin-like G protein-coupled receptors (GPCRs) are involved in a lot of regulatory processes and are widely distributed throughout the body which makes them an attractive target for drugs. However, pharmacological knowledge of these receptors is still limited. A big advance regarding the structural knowledge of adenosine receptors was the development of the first crystal structure of the adenosine A2A receptor in 2008. The crystal structure revealed the amino acids that form the ligand binding pocket of the receptor and depicted the endpoint of receptor movement in the ligand binding process. Within the scope of this work two members of the adenosine receptor family were investigated, namely the adenosine A1 and the A2A receptor (A1R, A2AR). A1R was generated on base of the previously developed A2AR. Receptors were tagged with fluorophores, with the cyan fluorescent protein (CFP) at the C-terminal end of receptor and the Fluorescein Arsenical Hairpin binder (FlAsH) binding sequence within the third intracellular loop of receptors. Resulting fluorescent receptor sensors
A1 Fl3 CFP and A2A Fl3 CFP were investigated with help of Fluorescence Resonance Energy Transfer (FRET) measurements within living cells. FRET experiments enable the examination of alteration in the distance of two fluorophores and thus the observation of receptor dynamical movements.
For comparison of A1R and A2AR regarding receptor dynamical movement upon ligand binding, fluorescent receptor sensors A1 Fl3 CFP and A2A Fl3 CFP were superfused with various ligands and the outcomes of FRET experiments were compared regarding signal height of FRET ratio evoked by the distinct ligand that is correlated to the conformational change of receptor upon ligand binding. Beside the different direction of FRET ratio upon ligand binding at A1R and A2AR sensor, there were differences observable when signal height and association and dissociation kinetics of the various ligands investigated were compared to each other. Differences between the adenosine receptor subtypes were especially remarkable for the A1R subtype selective agonist CPA and the A2AR subtype selective agonist CGS 21680. Another part of the project was to investigate the influence of single amino acids in the ligand binding process within the fluorescent A1R sensor. Amino acid positions were derived from the crystal structure of the A2AR forming the ligand binding pocket and these amino acids were mutated in the A1R structure. Investigation of the A1R sensor and its mutants regarding confocal analysis showed involvement
of some amino acids in receptor localization. When these amino acids were mutated receptors were not expressed in the plasma membrane of cells. Some amino acids investigated were found to be involved in the ligand binding process in general whereas other amino acids were found to have an influence on the binding of distinct structural groups of the ligands investigated. In a further step, A1R and A2AR were N-terminally tagged with SNAP or CLIP which allowed to label receptor sensors with multiple fluorophores. With this technique receptor distribution in cells could be investigated with help of confocal analysis. Furthermore, ligand binding with fluorescent adenosine receptor ligands and their competition with help of a non-fluorescent antagonist was examined at the SNAP tagged A1R and A2AR. Finally the previously developed receptor sensors were combined to the triple labeled receptor sensors SNAP A1 Fl3 CFP and SNAP A2A Fl3 CFP which were functional regarding FRET experiments and plasma membrane expression was confirmed via confocal analysis. In the future, with the help of this technique, interaction between fluorescent ligand and SNAP tagged receptor can be monitored simultaneously with the receptor movement that is indicated by the distance alteration between FlAsH and CFP. This can
lead to a better understanding of receptor function and its dynamical movement upon ligand binding which may contribute to the development of new and more specific drugs for the A1R and A2AR in the future.
Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunologic research and for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However the large majority of human T-cells resides in tissues, especially in lymphatic organs, while only 1 % of the body’s T-cells circulate in the blood stream. Previous work in mice and humans had indicated that CD4 T-cells transiently lose antigen sensitivity when cellular contacts are lost, e.g. by leaving lymphoid organs such as lymph nodes (LNs) and entering the circulation. In this study, these findings were extended to CD8 T-cells. Thus, CD8 T-cell responses of the human tonsil show a significant drop in sensitivity to viral antigens if tissue-exit was simulated by keeping cells in dispersed culture at body temperature for two hours.
Conversely, tissue-like functionality in blood-derived CD8 T-cells was restored by applying the simple and robust RESTORE protocol. Indeed, application of the RESTORE protocol, i.e. pre-culturing PBMCs for two days at a high cell density before initiation of antigenic stimulation, demonstrated that CD8 T-cell responses to a broad range of viral and to tumor-associated antigens are greatly underestimated, and sometimes even remain undetected if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 (WT1)-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. My studies on the mechanism of the RESTORE protocol show that HD preculture of PBMCs does not involve antigen-or cytokine-driven clonal expansion of T-cells. Moreover, the gain in antigen sensitivity cannot be explained by a decreased activity of regulatory T-cells during the preculture step. The increased antigen sensitivity of CD8 T-cells from HD precultures of PBMCs is associated with tonic T-cell receptor signaling as indicated by enhanced tyrosine phosphorylation of the CD3 ζ chains and the tyrosine kinase Lck, thereby preparing T-cells for full responses. The upregulation of genes involved in aerobic glycolysis in “restored” CD8 memory T-cells relative to fresh cells might be an essential requirement for increased T-cell functionality including the regulation of IFN-γ production. Taken together, the RESTORE protocol, which was initially described for the CD4 T-cell response to the antibody TGN1412 permits a more meaningful monitoring of CD8 T-cell responses to viral infections and tumors. Furthermore, when generating T-cell lines for adoptive T-cell therapy, the RESTORE protocol allows the generation of CD8 T-cell lines with an improved representation of clones responding to low antigen concentrations.
In this work, a model-based acceleration of parameter mapping (MAP) for the determination of the tissue parameter T1 using magnetic resonance imaging (MRI) is introduced. The iterative reconstruction uses prior knowledge about the relaxation behavior of the longitudinal magnetization after a suitable magnetization preparation to generate a series of fully sampled k-spaces from a strongly undersampled acquisition. A Fourier transform results in a spatially resolved time course of the longitudinal relaxation process, or equivalently, a spatially resolved map of the longitudinal relaxation time T1.
In its fastest implementation, the MAP algorithm enables the reconstruction of a T1 map from a radial gradient echo dataset acquired within only a few seconds after magnetization preparation, while the acquisition time of conventional T1 mapping techniques typically lies in the range of a few minutes. After validation of the MAP algorithm for two different types of magnetization preparation (saturation recovery & inversion recovery), the developed algorithm was applied in different areas of preclinical and clinical MRI and possible advantages and disadvantages were evaluated.
The oncogenic MYC protein is a transcriptional regulator of multiple cellular processes and is aberrantly activated in a wide range of human cancers. MYC is an unstable protein rapidly degraded by the ubiquitin-proteasome system. Ubiquitination can both positively and negatively affect MYC function, but its direct contribution to MYC-mediated transactivation remained unresolved.
To investigate how ubiquitination regulates MYC activity, a non-ubiquitinatable MYC mutant was characterized, in which all lysines are replaced by arginines (K-less MYC). The absence of ubiquitin-acceptor sites in K-less MYC resulted in a more stable protein, but did not affect cellular localization, chromatin-association or the ability to interact with known MYC interaction partners.
Unlike the wild type protein, K-less MYC was unable to promote proliferation in immortalized mammary epithelial cells. RNA- and ChIP-Sequencing analyses revealed that, although K-less MYC was present at MYC-regulated promoters, it was a weaker transcriptional regulator. The use of K-less MYC, a proteasomal inhibitor and reconstitution of individual lysine residues showed that proteasomal turnover of MYC is required for MYC target gene induction. ChIP-Sequencing of RNA polymerase II (RNAPII) revealed that MYC ubiquitination is dispensable for RNAPII recruitment and transcriptional initiation but is specifically required to promote transcriptional elongation. Turnover of MYC is required to stimulate histone acetylation at MYC-regulated promoters, which depends on a highly conserved region in MYC (MYC box II), thereby enabling the recruitment of BRD4 and P-TEFb and the release of elongating RNAPII from target promoters. Inhibition of MYC turnover enabled the identification of an intermediate in MYC-mediated transactivation, the association of MYC with the PAF complex, a positive elongation factor, suggesting that MYC acts as an assembly factor transferring elongation factors onto RNAPII. The interaction between MYC and the PAF complex occurs via a second highly conserved region in MYC’s amino terminus, MYC box I.
Collectively, the data of this work show that turnover of MYC coordinates histone acetylation with recruitment and transfer of elongation factors on RNAPII involving the cooperation of MYC box I and MYC box II.
SNAP25 (Synaptosomal-Associated Protein of 25 kDa; part of the SNARE complex) is involved in the docking and fusion of synaptic vesicles in presynaptic neurons necessary for the regulation of neurotransmitter release, as well as in axonal growth and synaptic plasticity. In humans, different single nucleotide polymorphisms of SNAP25 have repeatedly been associated with attention deficit/hyperactivity disorder (ADHD). Thus, in this study heterozygous Snap25 knockout mice were investigated as a model of ADHD.
Heterozygous (+/-) Snap25 knockout mice as well as their wild-type (+/+) littermates were reared under control conditions or underwent a Maternal Separation (MS) procedure. Starting at the age of 2 months, mice were tested for locomotor activity in a repeated long-term Open Field (OF) task, for attention deficits and impulsive behavior in the 5 Choice Serial Reaction Time Task (5CSRTT), for anxiety-like behavior in the Light-Dark Box (LDB) and for depression-like behavior in the Porsolt Forced Swim Test (FST). The brains of these mice were subsequently tested for the expression of several ADHD related genes in a quantitative Real-Time PCR (qRT-PCR) study. Another group of female mice (+/+; +/-) underwent a one hour OF test after oral administration of 45 mg/kg Methylphenidate (MPH) or placebo.
To find an optimized dosage for this MPH challenge, a pilot study was performed. Wild-type C57BL/6 mice were tested in a long-term OF with several dosages of MPH both intraperitoneally (i.p.) and orally. The brains of these animals were afterwards investigated for neurotransmitter concentrations. In this pilot study the dosages of MPH that were similarly behaviorally effective without causing symptoms of overdosing were 7.5-15 mg/kg intraperitoneally and 30-60 mg/kg orally. However, even though it was possible to find intraperitoneal and oral doses that correlate behaviorally, the neurochemistry was mostly different.
In the study on Snap25-deficient mice, unstressed controls showed a hyperactive phenotype in the second of two long-term OF sessions (60 min) spaced three weeks apart. Considering all groups, there was a significant interaction of stress and genotype in the second session, with animals subjected to MS being overall hyperactive with no genotype differences. In the training phase of the 5CSRTT only effects of stress were found, with MS animals finding and consuming fewer rewards. In the single test trial, several genotype effects became apparent, with tendencies for the number of correct nose pokes and the number of rewards eaten, and a significant effect for the number of rewards eaten directly after the correct response. In all of these variables +/- mice performed worse than their wild-type littermates. In the LDB +/- mice entered the lit compartment of the arena earlier than the controls, thus showing attenuated anxiety-like behavior. Regarding depressive-like behavior in the FST, male +/- mice spent significantly less time struggling than male +/+ mice. In the gene expression study, +/- mice had lower expression levels of Maoa and Comt, and higher expression levels of Nos1 than wild-types. Finally, the locomotor activity response to MPH was exaggerated in +/- mice as compared to controls.
Heterozygous Snap25 knockout mice show some of the behavioral characteristics of ADHD, as for example a mild hyperactivity in a familiar environment, difficulties in the correct execution of a given task and even some behavior that can be interpreted as delay aversion. Additionally, expression levels of three ADHD related genes were changed in these animals. Although the exaggerated locomotor activity response to MPH is not to be expected of an ADHD model, the difference in the response between +/+ and +/- mice nonetheless implicates a potential dysfunction of the brain dopaminergic system.
Burkitt lymphoma (BL) is a highly aggressive B cell malignancy. Rituximab, a humanized antibody against CD20, in a combination with chemotherapy is a current treatment of choice for B-cell lymphomas including BL. However, certain group of BL patients are resistant to Rituximab therapy. Therefore, alternative treatments targeting survival pathways of BL are needed.
In BL deregulation of MYC expression, together with additional mutations, inhibits differentiation of germinal centre (GC) B cells and drives proliferation of tumor cells. Pro-apoptotic properties of MYC are counteracted through the B-cell receptor (BCR) and phosphoinositide-3-kinase (PI3K) pathway to ensure survival of BL cells. In normal B-cells BCR triggering activates both NF-κB and NFAT-dependent survival signals. Since BL cells do not exhibit constitutive NF-κB activity, we hypothesized that anti-apoptotic NFATc1A isoform might provide a major survival signal for BL cells.
We show that NFATc1 is constitutively expressed in nuclei of BL, in BL cell lines and in Eµ-Myc–induced B cell lymphoma (BCL) cells. Nuclear residence of NFATc1 in these entities depends on intracellular Ca2+ levels but is largely insensitive to cyclosporine A (CsA) treatment and therefore independent from calcineurine (CN) activity. The protein/protein interaction between the regulatory domain of NFATc1 and DNA binding domain of BCL6 likely contributes to sustained nuclear residence of NFATc1 and to the regulation of proposed NFATc1-MYC-BCL6-PRDM1 network in B-cell lymphomas.
Our data revealed lack of strict correlation between the expression of six NFATc1 isoforms in different BL-related entities suggesting that both NFATc1/alphaA and -betaA isoforms provide survival functions and that NFATc1alpha/betaB and -alpha/betaC isoforms either do not possess pro-apoptotic properties in BL cells or these properties are counterbalanced. In addition, we show that in BL entities expression of NFATc1 protein is largely regulated at post-transcriptional level, including MYC dependent increase of protein stability.
Functionally we show that conditional inactivation of Nfatc1 gene in Eµ-Myc mice prevents development of BCL tumors with mature B cell immunophenotype (IgD+). Loss of NFATc1 expression in BCL cells ex vivo results in apoptosis of tumor cells.
Together our results identify NFATc1 as an important survival factor in BL cells and, hence, as a promising target for alternative therapeutic strategies for BL.
Sustained anxiety is considered as a chronic and future-oriented state of apprehension that does not belong to a specific object. It is discussed as an important characteristic of anxiety disorders including panic disorder, generalized anxiety disorder (GAD) and posttraumatic stress disorder (PTSD). Experimentally, sustained anxiety can be induced by contextual fear conditioning in which aversive events are unpredictably presented and therefore the whole context becomes associated with the threat. This thesis aimed at investigating important mechanisms in the development and maintenance of sustained anxiety: (1) facilitated acquisition and resistant extinction of contextual anxiety due to genetic risk factors (Study 1), and (2) the return of contextual anxiety after successful extinction using a new reinstatement paradigm (Study 2). To this end, two contextual fear conditioning studies were conducted in virtual reality (VR). During acquisition one virtual office was paired with unpredictable mildly painful electric stimuli (unconditioned stimulus, US), thus becoming the anxiety context (CXT+). Another virtual office was never paired with any US, thus becoming the safety context (CXT-). Extinction was conducted 24 h later, i.e. no US was presented, and extinction recall was tested another 24 h later on Day 3. In both studies context-evoked anxiety was measured on three different response levels: behavioral (anxiety-potentiated startle reflex), physiological (skin conductance level), and verbal (explicit ratings). In Study 1, participants were stratified for 5-HTTLPR (S+ risk allele vs. LL no risk allele) and NPSR1 rs324981 (T+ risk allele vs. AA no risk allele) polymorphisms, resulting in four combined genotype groups with 20 participants each: S+/T+, S+/LL, LL/T+, and LL/AA. Results showed that acquisition of anxiety-potentiated startle was influenced by a gene × gene interaction: only carriers of both risk alleles (S+ carriers of the 5-HTTLPR and T+ carriers of the NPSR1 polymorphism) exhibited significantly higher startle magnitudes in CXT+ compared to CXT-. However, extinction recall as measured with anxiety-potentiated startle was not affected by any genotype. Interestingly, the explicit anxiety level, i.e. valence and anxiety ratings, was only influenced by the NPSR1 genotype, in a way that no risk allele carriers (AA) reported higher anxiety and more negative valence in response to CXT+ compared to CXT-, whereas risk allele carriers (T+) did not. Study 2 adopted nearly the same paradigm with the modification that one group (reinstatement group) received one unsignaled US at the beginning of the experimental session on Day 3 before seeing CXT+ and CXT-. The second group served as a control group and received no US, but was immediately exposed to CXT+ and CXT-. Results showed a return of anxiety on the implicit and explicit level (higher startle responses and anxiety ratings in response to CXT+ compared to CXT-) in the reinstatement group only. Most important, the return of contextual anxiety in the reinstatement group was associated with a change of state anxiety and mood from extinction to test, that is the more anxiety and negative mood participants experienced before the reinstatement procedure, the higher their return of anxiety was. In sum, results of Study 1 showed that facilitated contextual fear conditioning on an implicit behavioral level (startle response) could be regarded as an endophenotype for anxiety disorders, which can contribute to our understanding of the etiology of anxiety disorders. Results of Study 2 imply that anxiety and negative mood after extinction could be an important facilitator for the return of anxiety. Furthermore, the present VR-based contextual fear conditioning paradigm seems to be an ideal tool to experimentally study mechanisms underlying the acquisition and the return of anxiety. Future studies could investigate clinical samples and extend the VR paradigm to evolutionary-relevant contexts (e.g., heights, darkness, open spaces).
Spreading drug resistances among Gram-negative pathogens and the paucity of new agents on the antibacterial drug market against these tenacious bacteria create a pressing need for the development of new antibiotics. The bacterial fatty acid biosynthesis pathway FAS-II, especially the enoyl-ACP reductase catalyzing the last step of the elongation cycle, is an established drug target against tuberculosis but has not been extensively exploited for drug design against other bacterial pathogens. In this thesis the enoyl-ACP reductases of the Gram-negative biothreat organisms Burkholderia pseudomallei and Yersinia pestis were targeted in a structure-based drug design approach. The structure of the most recently identified enoyl-ACP isoenzyme FabV was characterized by X-ray crystallography and could be determined in three different states. FabV from B. pseudomallei was obtained in the apo-form of the enzyme, whereas FabV from Y. pestis was characterized in a binary complex with the cofactor NADH as well as in a ternary complex with NADH and the triclosan-based 2-pyridone inhibitors PT172 and PT173. Analysis of the FabV structure revealed the typical fold of the short chain dehydrogenase/reductase superfamily with the NADH-binding Rossmann fold and a substrate-binding pocket with a conserved active site geometry compared to the related isoenzyme FabI. Additional structural elements of FabV are located around the active site. The monomeric form of the enzyme is thereby stabilized and the substrate-binding loop is kept in a closed, helical conformation. The ternary complexes of FabV exhibited a similar inhibitor-binding mode as observed for triclosan inhibition in FabI and point to a potential substrate-binding mechanism. B. pseudomallei possesses FabI as an additional enoyl-ACP reductase isoenzyme, which was structurally characterized in the apo form and in ternary complexes with NAD+ and the diphenyl ether inhibitors triclosan, PT02, PT12 or PT404 as well as the 4-pyridone inhibitor PT155. The structural data of the ternary enoyl-ACP reductases complexes of B. pseudomallei and Y. pestis hold the promise for the possibility to develop antibacterials targeting FabV or even both isoenzymes, FabI and FabV, based on the triclosan scaffold.
Molecular and developmental characterization of the Echinococcus multilocularis stem cell system
(2014)
The metacestode larva of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most dangerous zoonotic diseases in the Northern Hemisphere. Unlike “typical” metacestode larvae from other tapeworms, it grows as a mass of interconnected vesicles which infiltrates the liver of the intermediate host, continuously forming new vesicles in the periphery. From these vesicles, protoscoleces (the infective form for the definitive host) are generated by asexual budding. It is thought that in E. multilocularis, as in other flatworms, undifferentiated stem cells (so-called germinative cells in cestodes and neoblasts in free-living flatworms) are the sole source of new cells for growth and development. Therefore, this cell population should be of central importance for the progression of AE.
In this work, I characterized the germinative cells of E. multilocularis, and demonstrate that they are indeed the only proliferating cells in metacestode vesicles. The germinative cells are a population of undifferentiated cells with similar morphology, and express high levels of transcripts of a novel non-autonomous retrotransposon family (ta-TRIMs). Experiments of recovery after hydroxyurea treatment suggest that individual germinative cells have extensive self-renewal capabilities. However, germinative cells also display heterogeneity at the molecular level, since only some of them express conserved homologs of fgfr, nanos and argonaute genes, suggesting the existence of several distinct sub-populations. Unlike free-living flatworms, cestode germinative cells lack chromatoid bodies. Furthermore, piwi and vasa orthologs are absent from the genomes of cestodes, and there is widespread expression of some conserved neoblast markers in E. multilocularis metacestode vesicles. All of these results suggest important differences between the stem cell systems of free-living flatworms and cestodes.
Furthermore, I describe molecular markers for differentiated cell types, including the nervous system, which allow for the tracing of germinative cell differentiation. Using these molecular markers, a previously undescribed nerve net was discovered in metacestode vesicles. Because the metacestode vesicles are non-motile, and the nerve net of the vesicle is independent of the nervous system of the protoscolex, we propose that it could serve as a neuroendocrine system. By means of bioinformatic analyses, 22 neuropeptide genes were discovered in the E. multilocularis genome. Many of these genes are expressed in metacestode vesicles, as well as in primary cell preparations undergoing complete metacestode regeneration. This suggests a possible role for these genes in metacestode development. In line with this hypothesis, one putative neuropeptide (RGFI-amide) was able to stimulate the proliferation of primary cells at a concentration of 10-7 M, and the corresponding gene was upregulated during metacestode regeneration.
Atherosclerosis is accepted to be a chronic inflammatory disease of the arterial vessel wall. Several cellular subsets of the immune system are involved in its initiation and progression, such as monocytes, macrophages, T and B cells. Recent research has demonstrated that dendritic cells (DCs) contribute to atherosclerosis, too. DCs are defined by their ability to sense and phagocyte antigens, to migrate and to prime other immune cells, such as T cells. Although all DCs share these functional characteristics, they are heterogeneous with respect to phenotype and origin. Several markers have been used to describe DCs in different lymphoid and non-lymphoid organs; however, none of them has proven to be unambiguous. The expression of surface molecules is highly variable depending on the state of activation and the surrounding tissue. Furthermore, DCs in the aorta or the atherosclerotic plaque can be derived from designated precursor cells or from monocytes. In addition, DCs share both their marker expression and their functional characteristics with other myeloid cells like monocytes and macrophages. The repertoire of aortic DCs in healthy and atherosclerotic mice has just recently started to be explored, but yet there is no systemic study available, which describes the aortic DC compartment. Because it is conceivable that distinct aortic DC subsets exert dedicated functions, a detailed description of vascular DCs is required. The first part of this thesis characterizes DC subsets in healthy and atherosclerotic mice. It describes a previously unrecognized DC subset and also sheds light on the origin of vascular DCs. In recent years, microRNAs (miRNAs) have been demonstrated to regulate several cellular functions, such as apoptosis, differentiation, development or proliferation. Although several cell types have been characterized extensively with regard to the miRNAs involved in their regulation, only few studies are available that focus on the role of miRNAs in DCs. Because an improved understanding of the regulation of DC functions would allow for new therapeutic options, research on miRNAs in DCs is required. The second part of this thesis focuses on the role of the miRNA cluster miR- 17~92 in DCs by exploring its functions in healthy and atherosclerotic mice. This thesis clearly demonstrates for the first time an anti-inflammatory and atheroprotective role for the miR17-92 cluster. A model for its mechanism is suggested.
Platelet activation and aggregation are essential processes for the sealing of injured vessel walls and preventing blood loss. Under pathological conditions, however, platelet aggregation can lead to uncontrolled thrombus formation, resulting in irreversible vessel occlusion. Therefore, precise regulation of platelet activation is required to ensure efficient platelet plug formation and wound sealing but also to prevent uncontrolled thrombus formation. Rapid elevations in the intracellular levels of cations are a core signaling event during platelet activation. In this thesis, the roles of Ca2+ and Mg2+ channels in the regulation of platelet function were investigated.
Orai1, the major store-operated calcium (SOC) channel in platelets, is not only vital for diverse signaling pathways, but may also regulate receptor-operated calcium entry (ROCE). The coupling between the Orai1 signalosome and canonical transient receptor potential channel (TRPC) isoforms has been suggested as an essential step in the activation of store-operated calcium entry (SOCE) and ROCE in human platelets. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms still remains to be answered. In the first part of this thesis, the functional crosstalk between Orai1 and TRPC6 was addressed. Orai1-mediated SOCE was found to enhance the activity of phospholipases (PL) C and D, to increase diacylglycerol (DAG) production and finally to regulate TRPC6-mediated ROCE via DAG, indicating that the regulation of TRPC6 channel activity seems to be independent of the physical interaction with Orai1. Furthermore, Orai1 and TRPC6 double deficiency led to a reduced Ca2+ store content and basal cytoplasmic Ca2+ concentrations, but surprisingly also enhanced ATP secretion, which may enhance Ca2+ influx via P2X1 and compensate for the severe Ca2+ deficits seen in double mutant platelets. In addition, Orai1 and TRPC6 were not essential for G protein-coupled receptor (GPCR)-mediated platelet activation, aggregation and thrombus formation.
Transient receptor potential melastatin-like 7 (TRPM7) contains a cytosolic serine/threonine protein kinase. To date, a few in vitro substrates of the TRPM7 kinase have been identified, however, the physiological role of the kinase remains unknown. In the second part of this thesis, mice with a point mutation which blocks the catalytic activity of the TRPM7 kinase (Trpm7KI) were used to study the role of the TRPM7 kinase in platelet function. In Trpm7KI platelets phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism and Ca2+ mobilization were severely impaired upon glycoprotein (GP) VI activation, indicating that the TRPM7 kinase regulates PLC function. This signaling defect in Trpm7KI platelets resulted in impaired aggregate formation under flow and protected animals from arterial thrombosis and ischemic brain infarction. Altogether, these results highlight the kinase domain of TRPM7 as a pivotal signaling moiety implicated in the pathogenesis of thrombosis and cerebrovascular events.
Background: Nicotine addiction is the most prevalent type of drug addiction that has been described as a cycle of spiraling dysregulation of the brain reward systems. Imaging studies have shown that nicotine addiction is associated with abnormal function in prefrontal brain regions that are important for cognitive emotion regulation. It was assumed that addicts may perform less well than healthy nonsmokers in cognitive emotion regulation tasks. The primary aims of this thesis were to investigate emotional responses to natural rewards among smokers and nonsmokers and to determine whether smokers differ from nonsmokers in cognitive regulation of positive and negative emotions. To address these aims, two forms of appraisal paradigms (i.e., appraisal frame and reappraisal) were applied to compare changes in emotional responses of smokers with that of nonsmokers as a function of appraisal strategies. Experiment 1: The aim of the first experiment was to evaluate whether and how appraisal frames preceding positive and negative picture stimuli affect emotional experience and facial expression of individuals. Twenty participants were exposed to 125 pairs of auditory appraisal frames (either neutral or emotional) followed by picture stimuli reflecting five conditions: unpleasant-negative, unpleasant-neutral, pleasant-positive, pleasant-neutral and neutral-neutral. Ratings of valence and arousal as well as facial EMG activity over the corrugator supercilii and the zygomaticus major were measured simultaneously. The results indicated that appraisal frames could alter both subjective emotional experience and facial expressions, irrespective of the valence of the pictorial stimuli. These results suggest and support that appraisal frame is an efficient paradigm in regulation of multi-level emotional responses. 8 Experiment 2: The second experiment applied the appraisal frame paradigm to investigate how smokers differ from nonsmokers on cognitive emotion regulation. Sixty participants (22 nonsmokers, 19 nondeprived smokers and 19 12-h deprived smokers) completed emotion regulation tasks as described in Experiment 1 while emotional responses were concurrently recorded as reflected by self-ratings and psychophysiological measures (i.e., facial EMG and EEG). The results indicated that there was no group difference on emotional responses to natural rewards. Moreover, nondeprived smokers and deprived smokers performed as well as nonsmokers on the emotion regulation task. The lack of group differences in multiple emotional responses (i.e., self-reports, facial EMG activity and brain EEG activity) suggests that nicotine addicts have no deficit in cognitive emotion regulation of natural rewards via appraisal frames. Experiment 3: The third experiment aimed to further evaluate smokers’ emotion regulation ability by comparing performances of smokers and nonsmokers in a more challenging cognitive task (i.e., reappraisal task). Sixty-five participants (23 nonsmokers, 22 nondeprived smokers and 20 12-h deprived smokers) were instructed to regulate emotions by imagining that the depicted negative or positive scenario would become less negative or less positive over time, respectively. The results showed that nondeprived smokers and deprived smokers responded similarly to emotional pictures and performed as well as nonsmokers in down-regulating positive and negative emotions via the reappraisal strategy. These results indicated that nicotine addicts do not have deficit in emotion regulation using cognitive appraisal strategies. In sum, the three studies consistently revealed that addicted smokers were capable to regulate emotions via appraisal strategies. This thesis establishes the groundwork for therapeutic use of appraisal instructions to cope with potential self-regulation failures in nicotine addicts.
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease of the brain, which is characterized by a progressive loss of memory and spatial orientation. Only less than 5-10% of AD sufferers are familial cases due to genetic mutations in the amyloid precursor protein (APP) gene or presenilin (PS) 1 and 2 genes. The cause of sporadic AD (sAD) which covers > 95% of AD patients is still unknown. Current research found interactions between aging, diabetes and cognitive decline including dementia in general and in AD in particular. Disturbances of brain glucose uptake, glucose tolerance and utilization and impairment of the insulin/insulin receptor (IR) signaling cascade are thought to be key targets for the development of sAD.
In the brain of AD patients, neural plasticity is impaired indicated by synaptic and neuronal loss. Adult neurogenesis (AN), the generation of functional neurons in the adult brain, may be able to restore neurological function deficits through the integration of newborn neurons into existing neural networks. The dentate gyrus of the hippocampus is one out of few brain regions where life-long AN exists. However, there is a big controversy in literature regarding the involvement of AN in AD pathology. Most animal studies used transgenic mice based on the Amyloid ß (Aß) hypothesis which primarily act as models for the familial form of AD. Findings from human post mortem AN studies were also inconstistent. In this thesis, we focused on the possible involvement of AN in the pathogenesis of the sporadic form of AD. Streptozotocin intracerebroventricularily (STZ icv) treated rats, which develop an insulin-resistant brain state and learning and memory deficits preceding Aß pathology act as an appropriate animal model for sAD. We used STZ treatment for both parts of my work, for the in vivo and in vitro study.
In the first part of my thesis, my coworkers and I investigated STZ icv treatment effects on different stages of AN in an in vivo approach. Even if STZ icv treatment does not seem to considerably influence stem cell proliferation over a short-term (1 month after STZ icv treatment) as well as in a long-term (3 months after STZ icv treatment) period, it results in significantly less immature and newborn mature neurons 3 months after STZ icv treatment. This reduction detected after 3 months was specific for the septal hippocampus, discussed to be important for spatial learning. Subsequently we performed co-localization studies with antibodies detecting BrdU (applied appr. 27 days before sacrifice) and cell-type specific markers such as NeuN, and GFAP, we found that STZ treatment does not affect the differentiation fate of newly generated cells. Phenotype analysis of BrdU-positive cells in the hilus and molecular layer revealed that some of the BrdU-positive cells are newborn oligodendrocytes but not newborn microglia.
In the second part of my thesis I worked with cultured neural stem cells (NSCs) isolated from the adult rat hippocampus to reveal STZ effects on the proliferation of of NSCs, and on the survival and differentiation of their progeny. Furthermore, this in vitro approach enabled me to study cellular mechanisms underlying the observed impaired neurogenesis in the hippocampus of STZ-treated rats. In contrast to our findings of the STZ icv in vivo study we revealed that STZ supplied with the cell culture medium inhibits the proliferation of NSCs in a dose-dependent and time-dependent manner. Moreover, performing immunofluorescence studies with antibodies detecting cell-type specific markers after triggering NSCs to differentiate, we could show that STZ treatment affects the number of newly generated neurons but not of astrocytes. Analyzing newborn cells starting to differentiate and migrate I was able to demonstrate that STZ has no effect on the migration of newborn cells. Trying to reveal cellular mechanisms underlying the negative influence of STZ on hippocampal AN, we performed qRT-PCR and immunofluorescence staining and thus could show that in NSCs the expression of glucose transporter (GLUT)3 mRNA as well as IR and GLUT3 protein levels are reduced after STZ treatment. Therefore, the inhibition of the proliferation of NSCs may be (at least partially) caused by these two molecules. Interestingly, the effect of STZ on differentiating cells was shown to be different, as IR protein expression was not significantly changed but GLUT3 protein levels were decreased in consequence of STZ treatment.
In summary, this project delivered further insights into the interrelation between AN the sporadic form of sAD and thus provides a basis of new therapeutic approaches in sAD treatment through intervening AN. Discrepancies between the results of the two parts of my thesis, the in vivo and in vitro part, were certainly caused to a certain extent by the missing microenvironment in the in vitro approach with cultured NSCs. Future studies e.g. using co-culture systems could at least minimize the effect of a missing natural microenvironment of cultured NSCs, so that the use of an in vitro approach for the investigation of STZ treatment underlying cellular mechanisms can be improved.
Accurate information transfer between neurons governs proper brain function. At chemical synapses, communication is mediated via neurotransmitter release from specialized presynaptic intercellular contact sites, so called active zones. Their molecular composition constitutes a precisely arranged framework that sets the stage for synaptic communication.
Active zones contain a variety of proteins that deliver the speed, accuracy and plasticity inherent to neurotransmission. Though, how the molecular arrangement of these proteins influences active zone output is still ambiguous. Elucidating the nanoscopic organization of AZs has been hindered by the diffraction-limited resolution of conventional light microscopy, which is insufficient to resolve the active zone architecture on the nanometer scale. Recently, super-resolution techniques entered the field of neuroscience, which yield the capacity to bridge the gap in resolution between light and electron microscopy without losing molecular specificity. Here, localization microscopy methods are of special interest, as they can potentially deliver quantitative information about molecular distributions, even giving absolute numbers of proteins present within cellular nanodomains.
This thesis puts forward an approach based on conventional immunohistochemistry to quantify endogenous protein organizations in situ by employing direct stochastic optical reconstruction microscopy (dSTORM). Focussing on Bruchpilot (Brp) as a major component of Drosophila active zones, the results show that the cytomatrix at the active zone is composed of units, which comprise on average ~137 Brp molecules, most of which are arranged in approximately 15 heptameric clusters. To test for a quantitative relationship between active zone ultrastructure and synaptic output, Drosophila mutants and electrophysiology were employed. The findings indicate that the precise spatial arrangement of Brp reflects properties of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression. Moreover, functional diversification could be connected to a heretofore unrecognized ultrastructural gradient along a Drosophila motor neuron.
In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule–1 (MAdCAM-1) and T cell responses in Peyer’s patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD.
The Notch signaling pathway is crucial for mammalian heart development. It controls cell-fate decisions, coordinates patterning processes and regulates proliferation and differentiation. Critical Notch effectors are Hey bHLH transcription factors (TF) that are expressed in atrial (Hey1) and ventricular (Hey2) cardiomyocytes (CM) and in the developing endocardium (Hey1/2/L). The importance of Hey proteins for cardiac development is demonstrated by knockout (KO) mice, which suffer from lethal cardiac defects, such as ventricular septum defects (VSD), valve defects and cardiomyopathy. Despite this clear functional relevance, little is known about Hey downstream targets in the heart and the molecular mechanism by which they are regulated.
Here, I use a cell culture system with inducible Hey1, Hey2 or HeyL expression to study Hey target gene regulation in HEK293 cells, in murine embryonic stem cells (ESC) and in ESC derived CM. In HEK293 cells, I could show that genome wide binding sites largely overlap between all three Hey proteins, but HeyL has many additional binding sites that are not bound by Hey1 or Hey2. Shared binding sites are located close to transcription start sites (TSS) where Hey proteins preferentially bind to canonical E boxes, although more loosely defined modes of binding exist. Additional sites only bound by HeyL are more scattered across the genome. The ability of HeyL to bind these sites depends on the C-terminal part of the protein. Although there are genes which are differently regulated by HeyL, it is unclear whether this regulation results from binding of additional sites by HeyL.
Additionally, Hey target gene regulation was studied in ESC and differentiated CM, which are more relevant for the observed cardiac phenotypes. ESC derived CM contract in culture and are positive for typical cardiac markers by qRT PCR and staining. According to these markers differentiation is unaffected by prolonged Hey1 or Hey2 overexpression. Regulated genes are largely redundant between Hey1 and Hey2. These are mainly other TF involved in e.g. developmental processes, apoptosis, cell migration and cell cycle. Many target genes are cell type specifically regulated causing a shift in Hey repression of genes involved in cell migration in ESC to repression of genes involved in cell cycle in CM.
The number of Hey binding sites is reduced in CM and HEK293 cells compared to ESC, most likely due to more regions of dense chromatin in differentiated cells. Binding sites are enriched at the proximal promoters of down-regulated genes, compared to up-or non-regulated genes. This indicates that up-regulation primarily results from indirect effects, while down-regulation is the direct results of Hey binding to target promoters. The extent of repression generally correlates with the amount of Hey binding and subsequent recruitment of histone deacetylases (Hdac) to target promoters resulting in histone H3 deacetylation.
However, in CM the repressive effect of Hey binding on a subset of genes can be annulled, likely due to binding of cardiac specific activators like Srf, Nkx2-5 and Gata4. These factors seem not to interfere with Hey binding in CM, but they recruit histone acetylases such as p300 that may counteract Hey mediated histone H3 deacetylation. Such a scenario explains differential regulation of Hey target genes between ESC and CM resulting in gene and cell-type specific regulation.
LIM and SH3 protein 1 (LASP1) is a nucleocytoplasmic scaffolding protein. LASP1 interacts with various cytoskeletal proteins via its domain structure and is known to participate in physiological processes of cells. In the present study, a detailed investigation of the expression pattern of LASP1 protein in normal skin, melanocytic nevi and melanoma was carried out and the melanocyte–specific function of LASP1 was analyzed. LASP1 protein was identified in stratum basale of skin epidermis and a very high level was detected in nevi, the benign tumor of melanocyte. In the highly proliferative basal cells, an additional distinct nuclear localization of the protein was noted. In different tumor entities, an elevated LASP1 expression and nuclear localization, correlated positively with malignancy and tumor grade. However, LASP1 level was determined to be very low in melanoma and even reduced in metastases. Melanoma is distinguished as the first tumor tested to date – that displayed an absence of elevated LASP1 expression. In addition no significant relation was observed between LASP1 protein expression and clinicopathological parameters in melanoma.
The epidermal melanin unit of skin comprises of melanocytes and keratinocytes. Melanocytes are specialized cells that synthesize the photo protective coloring pigment, melanin inside unique organelles called melanosomes. The presence of LASP1 in melanocytes is reported for the first time through this study and the existence was confirmed by immunoblotting analysis in cultured normal human epidermal melanocyte (NHEM) and in melanoma cell lines, along with the immunohistostaining imaging in normal skin and in melanocytic nevi. LASP1 depletion in MaMel2 cells revealed a moderate increase in the intracellular melanin level independently of de novo melanogenesis, pointing to a partial hindrance in melanin release. Immunofluorescence images of NHEM and MaMel2 cells visualized co-localization of LASP1 with dynamin and tyrosinase concomitant with melanosomes at the dendrite tips of the cells. Melanosome isolation experiments by sucrose density gradient centrifugation clearly demonstrated the presence of LASP1 and the melanosome specific markers tyrosinase and TRP1 in late stage melanosomes.
The study identified LASP1 and dynamin as novel binding partners in melanocytes and provides first evidence for the existence of LASP1 and dynamin (a protein well–known for its involvement in vesicle formation and budding) in melanosomes. Co-localization of LASP1 and dynamin along the dendrites and at the tips of the melanocytes indicates a potential participation of the two proteins in the membrane vesicle fission at the plasma membrane.
In summary, a possible involvement of LASP1 in the actin–dynamin mediated membrane fission and exocytosis of melanin laden melanosome vesicles into the extracellular matrix is suggested.
Myocardial infarction (MI) is a leading cause of death worldwide. Timely restoration of coronary blood flow to ischemic myocardium significantly reduces acute infarct mortality and attenuates ventricular remodeling. However, surviving MI patients frequently develop heart failure, which is associated with reduced quality of life, high mortality rate (10% annually), as well as high healthcare expenditures. The main processes involved in the evolution of heart failure post-MI are the great loss of contractile cardiomyocytes during ischemia-reperfusion and the subsequent complex structural and functional alterations, which are rooted in modifications at molecular and cellular levels in both the infarcted and non-infarcted myocardium. However, we still lack efficient treatments to prevent the development and progression of left ventricular remodeling. The improved survival rate of acute MI patients combined with the lack of effective therapy for post-MI remodeling contributes to the high prevalence of heart failure. Cardiac Magnetic Resonance Imaging (MRI) is an important tool for diagnosis and assessment of MI. With the advancement of this technology, the frontier of MRI has been extended to probing molecular and cellular events in vivo and non-invasively. In combination with assessment of morphology and function, the visualization of essential molecular and cellular markers in vivo could provide comprehensive, multifaceted views of the healing process in infarcted hearts, which might give new insight for the treatment of acute MI. In this thesis, molecular and cellular cardiac MRI methods were established to visualize and investigate inflammation and calcium flux in the healing process of acute MI in vivo, in a clinically relevant rat model.
The role of host dendritic cells during the effector phase of intestinal graft-versus-host disease
(2014)
Monocytes can be functionally divided in two subsets, both capable to differentiate into dendritic cells (DCs): CX3CR1loCCR2+ classical monocytes, actively recruited to the sites of inflammation and direct precursors of inflammatory DCs; and CX3CR1hiCCR2− non-classical monocytes, characterized by CX3CR1-dependent recruitment to non-inflamed tissues. Yet, the function of non-classical monocyte-derived DCs (nc-mo-DCs), and the factors, which trigger their recruitment and DC differentiation, have not been clearly defined to date. Here we show that in situ differentiated nc-moDCs mediate immunosuppression in the context of intestinal graft-versus-host disease (GVHD).
Employing multi-color confocal microscopy we observed a dramatic loss of steady state host-type CD103+ DC subset immediately after transplantation, followed by an enrichment of immune-regulatory CD11b+ nc-moDCs. Parabiosis experiments revealed that tissue-resident non-classical CX3CR1+ monocytes differentiated in situ into intestinal CD11b+ nc-moDCs after allogeneic hematopoietic cell transplantation (allo-HCT). Differentiation of this intestinal DC subset depended on CSF-1 but not on Flt3L, thus defining the precursors as monocytes and not pre-DCs. Importantly, CX3CR1 but not CCR2 was required for this DC subset differentiation, hence defining the precursors as non-classical monocytes. In addition, we identify PD-L1 expression by CX3CR1+ nc-moDCs as the major mechanism they employ to suppress alloreactive T cells during acute intestinal GVHD. All together, we demonstrate that host nc-moDCs surprisingly mediate immunosuppression in the context of murine intestinal GVHD – as opposed to classical “inflammatory” monocyte-derived dendritic cells (mo-DCs) – via coinhibitory signaling. This thorough study unravels for the first time a biological function of a - so far only in vitro and phenotypically described - DC subset. Our identification of this beneficial immunoregulatory DC subset points towards alternate future strategies in underpinning molecular pathways to foster their function. We describe an unexpected mechanism of nc-moDCs in allo-HCT and intestinal GVHD, which might also be important for autoimmune disorders or infections of the gastrointestinal tract.
Stress has been shown to influence neuroplasticity and is suspected to increase the risk for psychiatric disorders such as major depression and anxiety disorders. Additionally, the short variant of the human serotonin transporter (5-HTT) length polymorphism (5-HTTLPR) is suggested to increase the risk for the development of such disorders. While stress as well as serotonergic signaling are not only discussed to be involved in the development of psychiatric disorders, they are also known to influence hippocampal adult neurogenesis (aN). Therefore, it has long been suspected that aN is involved in the etiology of these illnesses. The exact role of aN in this context however, still remains to be clarified.
In the present doctoral thesis, I am introducing two different studies, which had been carried out to assess possible changes in neuroplasticity and behavior as a result of 5-HTT genotype by stress interactions. In both studies, animals of the 5-HTT knock-out (5-HTT-/-) mouse line were used, which have been found to exhibit increased anxiety- and depression-related behavior, an altered stress response and decreased aggressive behavior. The aim of the first study, the so-called Spatial Learning study, had been to evaluate whether mice with altered levels of brain 5-HT as a consequence of lifelong 5-HTT deficiency perform differently in two spatial memory tests, the Morris Water Maze (WM) and the Barnes Maze (BM) test prospectively differing in aversiveness. Mice of the Spatial Learning study were of male sex and six months of age, and where subjected to a total of 10 (BM) or 15 (WM) trials. My particular interest was to elucidate if there are genotype by treatment interactions regarding blood plasma corticosterone levels and, if neurobiological equivalents in the brain to the found behavioral differences exist. For this purpose I carried out a quantitative immunohistochemistry study, investigating stem cell proliferation (via the marker Ki67) and aN (via the immature neuron marker NeuroD), as well as expression of the two immediate early genes (IEGs) Arc and cFos as a markers for neuronal activity in the hippocampus. The aim of the second study, the chronic mild stress (CMS) study had been to evaluate whether the innate divergent depression-like and anxiety-like behavior of mice with altered levels of brain 5-HT as a consequence of 5-HTT-deficiency is altered any further after being subjected to a CMS paradigm. Two cohorts of one-year-old female mice had been subjected to a variety of unpredictable stressors. In order to exclude possible interfering influences of behavioral testing on corticosterone levels and the outcome of the quantitative immunohistochemistry study the first cohort had been behaviorally tested after CMS while the second one had remained behaviorally untested. The objective of my part of the study was to find out about possible genotype by treatment interactions regarding blood plasma corticosterone as well as regarding aN in the hippocampus of the mice that had been subjected to CMS. For this purpose I performed a quantitative immunohistochemistry study in order to investigate the phenomenon of adult neurogenesis (via Ki67, NeuroD and the immature neuron marker DCX).
Both studies led to interesting results. In the CMS study, we could not replicate the increased innate anxiety- and depression-like behavior in 5-HTT-/- mice known from the literature. However, with regard to the also well documented reduced locomotor activity, as well as the increased body weight of 5-HTT-/- mice compared to their 5-HTT+/- and 5-HTT+/+ littermates, we could demonstrate that CMS leads to increased explorative behavior in the Open Field Test and the Light/Dark Box primarily in 5-HTT+/- und 5-HTT+/+ mice. The Spatial learning study revealed that increased stress sensitivity of 5-HTT-/- mice leads to a poorer performance in the WM test in relation to their 5-HTT+/+ and 5-HTT+/- littermates. As the performance of 5-HTT-/- mice in the less aversive BM was undistinguishable from both other genotypes, we concluded that the spatial learning ability of 5-HTT-/- mice is comparable to that of both other genotypes. As far as stress reactivity is concerned, the experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels, irrespective of the 5-HTT genotype. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Blood plasma corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. In the CMS study, the corticosterone assessment of mice of cohort 1, which had undergone behavioral testing before sacrifice, resulted in significantly elevated corticosterone levels in 5-HTT-/- mice in relation to their 5-HTT+/+ controls. Contrary, corticosterone levels in mice of cohort 1, which had remained behaviorally untested, were shown to be elevated / increased after CMS experience regardless of the 5-HTT genotype. Regarding neuroplasticity, the Spatial Learning study revealed higher baseline levels of cFos- and Arc-ir cells as well as more proliferation (Ki67-ir cells) and higher numbers of neuronal progenitor cells (NeuroD-ir cells) in 5-HTT-/- compared to 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we could demonstrate that learning performance in the WM correlates with the extent of aN. The CMS study, in which aN (DCX-ir cells), has also been found to be increased in 5-HTT-/- mice compared to their 5-HTT+/+ littermates, yet only in control animals, did show hampered proliferation (Ki67-ir cells) in the hippocampus of all 5-HTT genotypes following CMS experience. Interestingly, the number of immature neurons (DCX-ir cells) was diminished exclusively in 5-HTT-/- mice in response to CMS.
From the Spatial Learning study we concluded, that increased IEG expression and aN levels observed in the hippocampus of 5-HTT deficient mice can be the neurobiological correlate of emotion circuit dysfunction and heightened anxiety of these mice and that 5-HTT-/- animals per se display a “stressed” phenotype as a consequence of long-life 5-HTT deficiency. Due to the different age and sex of the mice in the two studies, they cannot be compared easily. However, although the results of the CMS study seem to contradict the results of the Spatial Learning study at the first glance, they do support the conclusion of the Spatial Learning study by demonstrating that although CMS does have an impact on 5-HTT-/- mice on the neurobiological level (e.g. manifesting in a decrease of DXC-ir cells following CMS) CMS experience cannot add onto their heightened inborn stress-level and is almost ineffective regarding further changes of the behavior of 5-HTT-deficient mice. I thus propose, that 5-HTT-/- mice as a result of lifelong altered 5-HT signaling display a stressed phenotype which resembles a state of lethargy and is paralleled by baseline heightened IEG expression and aN. It cannot be altered or increased by CMS, but it becomes most visible in stressful situations such as repeated spatial learning tests like the WM in which locomotor activity is required.
Platelet activation and aggregation at sites of vascular injury are essential processes to limit blood loss but they also contribute to arterial thrombosis, which can lead to myocardial infarction and stroke. Stable thrombus formation requires a series of events involving platelet receptors which contribute to adhesion, activation and aggregation of platelets. Regulation of receptor expression by (metallo-)proteinases has been described for several platelet receptors, but the molecular mechanisms are ill-defined. The signaling lymphocyte activation molecule (SLAM) family member CD84 is expressed in immune cells and platelets, however its role in platelet physiology was unclear. In this thesis, CD84 deficient mice were generated and analyzed. In well established in vitro and in vivo assays testing platelet function and thrombus formation, CD84 deficient mice displayed phenotypes indistinguishable from wild-type controls. It was concluded that CD84 in platelets does not function as modulator of thrombus formation, but rather has other functions. In line with this, in the second part of this thesis, a novel regulation mechanism for platelet CD84 was discovered and elucidated. Upon platelet activation, the N-terminus of CD84 was found to be cleaved exclusively by the a disintegrin and metalloproteinase 10 (ADAM10), whereas the intracellular part was cleaved by calpain. In addition, regulation of the platelet activating collagen receptor glycoprotein VI (GPVI) was studied and it was shown that GPVI is in contrast to CD84 differentially regulated by ADAM10 and ADAM17. A novel role of CD84 under pathophysiological conditions was revealed as CD84 deficient mice were protected from ischemic stroke in the model of transient middle cerebral artery occlusion and this protection was based on the lack of CD84 in T cells. Ca2+ is an essential second messenger that facilitates activation of platelets and diverse functions in different eukaryotic cell types. Store-operated Ca2+ entry (SOCE) represents the major mechanism leading to rise in intracellular Ca2+ concentration in non-excitable cells. The Ca2+ sensor STIM1 (stromal interaction molecule 1) and the SOC channel subunit protein Orai1 are established mediators of SOCE in platelets. STIM2 is the major STIM isoform in neurons, but the role of the SOC channel subunit protein Orai2 in platelets and neurons has remained elusive. In the third part of this thesis, Orai2 deficient mice were generated and analyzed. Orai2 was dispensable for platelet function, however, Orai2 deficient mice were protected from ischemic neurodegeneration and this phenotype was attributed to defective SOCE in neurons.
Oncolytic viral therapies have shown great promise pre-clinically and in human clinical trials for the treatment of various cancers. Oncolytic viruses selectively infect and replicate in cancer cells, destroying tumor tissue via cell lysis, while leaving noncancerous tissues unharmed. Vaccinia virus (VACV) is arguably one of the safest viruses, which has been intensively studied in molecular biology and pathogenesis as a vaccine for the eradication of smallpox in more than 200 million people. It has fast and efficient replication, and cytoplasmic replication of the virus lessens the chance of recombination or integration of viral DNA into the genome of host cells. Anti-tumor therapeutic efficacy of VACV has been demonstrated for human cancers in xenograft models with a variety of tumor types. In addition recombinant oncolytic VACVs carrying imaging genes represent an advance in treatment strategy that combines tumor-specific therapeutics as well as diagnostics.
As for other targeted therapies, a number of challenges remain for the clinical translation of oncolytic virotherapy. These challenges include the potential safety risk of replication of oncolytic virus in non-tumor tissue, the relatively poor virus spread throughout solid tumor tissue and the disadvantageous ratio between anti-viral and anti-tumoral immunity. However, manipulation of components of the tumor microenvironment may help oncolytic virus infection in killing the tumor tissue and thereby increasing the anti-tumor efficacy. Furthermore, dogs with natural cancer are considered as one of the best animal models to develop new drugs for cancer therapy. Traditionally, rodent cancer models have been used for development of cancer therapeutics. However, they do not adequately represent several features that define cancer in humans, including biology of initiation of tumor, the complexity of cancer recurrence and metastasis and outcomes to novel therapies. However, the tumor microenvironment, histopathology, molecular and genomics data from dog tumors has significant similarities with corresponding human tumors. These advantages of pet dog cancers provide a unique opportunity to integrate canine cancer patients in the studies designed for the development of new cancer drugs targeted against both human and canine cancers. This dissertation centers on the use of VACV strains in canine cancer xenografts with the aim of understanding the effects of modulation of tumor microenvironment on VACV-mediated tumor therapy.
In the first studies, wild-type VACV strain LIVP6.1.1 was tested for its oncolytic efficiency in canine soft tissue sarcoma (STSA-1) and canine prostate carcinoma (DT08/40) cells in culture and xenografts models. LIVP6.1.1 infected, replicated within, and killed both STSA-1 and DT08/40 cells in cell culture. The replication of virus was more efficient in STSA-1 cells compared to DT08/40 cells. In xenograft mouse models, LIVP6.1.1 was safe and effective in regressing both STSA-1 and DT08/40 xenografts. However, tumor regression was faster in STSA-1 xenografts compared to DT08/40 xenografts presumably due to more efficient replication of virus in STSA-1 cells. Biodistribution profiles revealed persistence of virus in tumors 5 and 7 weeks post virus injection in STSA-1 and DT08/40 xenografts, respectively, with the virus mainly cleared from all other major organs. Immunofluorescence staining detected successful colonization of VACV in the tumor. Consequently, LIVP6.1.1 colonization in the tumor showed infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. These findings suggest that virotherapy-mediated anti-tumor mechanism in xenografts could be a combination of direct viral oncolysis of tumor cells and virus-dependent infiltration of tumor-associated host immune cells.
In further studies, the effects of modulation of tumor angiogenesis of VACV therapy were analyzed in canine cancer xenografts. GLV-1h109 VACV strain (derived from prototype virus GLV-1h68) encoding the anti-VEGF single chain antibody GLAF-1 was characterized for its oncolytic efficacy in STSA-1 and DT08/40 cancer cells in culture and tumor xenografts. Concomitantly, the effects of locally expressed GLAF- 1 in tumors on virus replication, host immune infiltration, tumor vascularization and tumor growth were also evaluated.
GLV-1h109 was shown to be similar to the parental virus GLV-1h68 in expression of the two marker genes that both virus strains have in common (Ruc-GFP and gusA) in cell cultures. Additionally, the anti-VEGF single-chain antibody GLAF-1 was expressed by GLV-1h109 in both cell cultures and tumor xenografts. The insertion of GLAF-1 did not significantly affect the replication and cytotoxicity of GLV-1h109 in the STSA-1 and DT08/40 cell lines, although at early time points (24-48 hpi), the replication of GLV-1h109 was higher in STSA-1 cells compared to DT08/40 cells. In addition, STSA-1 cells were more susceptible to lysis with GLV-1h109 than DT08/40 cells. GLV-1h109 achieved a significant inhibition of tumor growth in both STSA-1 and DT08/40 canine xenografts models. Consequently, the significant regression of tumor growth was initiated earlier in STSA-1 tumor xenografts compared to regression in DT08/40 xenografts. The reason for the higher efficacy of GLV-1h109 in STSA-1 xenografts than DT08/40 xenografts was attributed to more efficient replication of virus in STSA-1 cells. In addition, tumor-specific virus infection led to a continued presence of GLAF-1 in peripheral blood, which could be useful as a pharmacokinetic marker to monitor virus colonization and persistence in GLV-1h109- injected xenograft mice. GLAF-1 is a single-chain antibody targeting human and murine VEGF. It was demonstrated that GLAF-1 was functional and recognized both canine and human VEGF with equal efficiency.
Histological analysis of tumor sections 7 days after GLV-1h109 injection confirmed that colonization of VACV and intratumoral expression of GLAF-1 translated into a significant decrease in blood vessel number compared to GLV-1h68 or PBS-treated control tumors. Subsequently, reduction in blood vessel density significantly improved the spread and replication of VACV as observed by FACS analysis and standard plaque assay, respectively. Inhibition of tumor angiogenesis and increased replication of virus further improved the infiltration of innate immune cells mainly granulocytes and macrophages in STSA-1 tumor xenografts. Both the results, i.e. improved virus spread and increased infiltration of innate immune cells in tumor, were explained by a phenomenon called “vascular normalization”, where anti-VEGF therapy normalizes the heterogeneous tumor vasculature thereby improving delivery and spread of VACV. In summary, the effects of inhibition of tumor angiogenesis on virus spread and replication were demonstrated using a vaccinia virus caring an anti- angiogenic payload targeting vascular endothelial growth factor (VEGF) in canine cancer xenografts.
In the final studies, the effects of VACV therapy on modulation of the immune system were analyzed in canine cancer patients enrolled in a phase I clinical trial. V-VET1 (clinical grade LIVP6.1.1 VACV) injection significantly increased the percentages of CD3+CD8+ T lymphocytes at 21 days after initiation of treatment. CD3+CD8+ T lymphocytes are mainly cytotoxic T lymphocytes that have potential to lyse cancer cells. Subsequently, the frequency of immune suppressor cells, mainly MDSCs and Treg was also analyzed in peripheral blood of canine cancer patients. Increase in the MDSC population and decreased CD8/Treg ratio is known to have inhibitory effects on the functions of cytotoxic T cells. We demonstrated that injection of V-VET1 in canine cancer patients significantly reduced the percentages of MDSCs at 21 days post initiation of treatment. Additionally, CD8/Treg ratio was increased 21 days after initiation of V-VET1 treatment. We also showed that changes in the frequency of immune cells neither depends on dose of virus nor depends on tumor type according to the data observed from this clinical trial with eleven analyzed patients.
This preclinical and clinical data have important clinical implications of how VACV therapy can be used for the treatment of canine cancers. Moreover, dogs with natural cancers can be used as an ideal animal model to improve the oncolytic virotherapy for human cancers. Furthermore, modulation of tumor microenvironment mainly tumor angiogenesis and tumor immunity has significant impact on the success of oncolytic virotherapy.
Besides HIV and tuberculosis, malaria still is one of the most devastating infectious diseases especially in developing countries, with Plasmodium falciparum being responsible for the frequently lethal form of malaria tropica. It is a major cause of mortality as well as morbidity, whereby pregnant women and children under the age of five years are most severely affected. Rapidly emerging drug resistances and the lack of an effective and safe vaccine hamper the combat against malaria by chemical and pharmacological regimens, and moreover the poor socio-economic and healthcare conditions in malaria-endemic countries are compromising the extermination of this deadly tropical disease to a large extent. Malaria research is still questing for druggable targets in the parasitic protozoan which pledge to be refractory against evolving resistance-mediating mutations and yet constitute affordable and compliant antimalarial chemotherapeutics.
The parasite kinome consists of members that represent most eukaryotic protein kinase groups, but also contains several groups that can not be assigned to conservative ePK groups. Moreover, given the remarkable divergence of plasmodial kinases in respect to the human host kinome and the fact that several plasmodial kinases have been identified that are essential for the intraerythrocytic developmental cycle, these parasite enzymes represent auspicious targets for antimalarial regimens. Despite elaborate investigations on several other ePK groups, merely scant research has been conducted regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family, PfCLK-1-4. In other eukaryotes, CLKs are involved in mRNA processing and splicing by means of phosphorylation of serine/arginine-rich (SR) proteins, which are crucial components of the splicing machinery in the alternative splicing pathway. All four PfCLKs are abundantly expressed in asexual parasites and gametocytes, and stage-specific expression profiles of PfCLK-1 and PfCLK-2 exhibited nucleus-associated localization and an association with phosphorylation activity. In the course of this study, PfCLK-3 and PfCLK-4 were functionally characterized by indirect immunofluorescence, Western blot analysis and kinase activity assays. These data confirm that the two kinases are primarily expressed in the nucleus of trophozoites and both kinases possess in vitro phosphorylation activity on physiological substrates. Likewise PfCLK-1 and PfCLK-2, reverse genetic studies exhibited the indispensability of both PfCLKs on the asexual life cycle of P. falciparum, rendering them as potential candidates for antiplasmodial strategies. Moreover, this study was conducted to identify putative SR proteins as substrates of all four PfCLKs. Previous alignments revealed a significant homology of the parasite CLKs to yeast SR protein kinase Sky1p. Kinase activity assays showed in vitro phosphorylation of the yeast Sky1p substrate and SR protein Npl3p by precipitated PfCLKs. In addition, four homologous plasmodial SR proteins were identified that are phosphorylated by PfCLKs in vitro: PfASF-1, PFSRSF12, PfSFRS4 and PfSR-1. All four parasite SR splicing factors are predominantly expressed in the nuclei of trophozoites. For PfCLK-1, a co-localization with the SR proteins was verified.
Finally, a library of human and microbial CLK inhibitors and the antiseptic chlorhexidine (CHX) was screened to determine their inhibitory effect on different parasite life cycle stages and on the PfCLKs specifically. Five inhibitors out of 63 compounds from the investigated library were selected that show a moderate inhibition on asexual life cycle stages with IC50 values ranging between approximately 4 and 8 µM. Noteworthy, these inhibitors belong to the substance classes of aminopyrimidines or oxo-β-carbolines. Actually, the antibiotic compound CHX demonstrated an IC50 in the low nanomolar range. Stage-of-inhibition assays revealed that CHX severely affects the formation of schizonts. All of the selected CLKs inhibitors also affect gametocytogenesis as well as gametogenesis, as scrutinized in gametocyte toxicity assays and exflagellation assays, respectively. Kinase activity assays confirm a specific inhibition of CLK-mediated phosphorylation of all four kinases, when the CLK inhibitors are applied on immunoprecipitated PfCLKs. These findings on PfCLK-inhibiting compounds are initial attempts to determine putative antimalarial compounds targeting the PfCLKs. Moreover, these results provide an effective means to generate chemical kinase KOs in order to phenotypically study the role of the PfCLKs especially in splicing events and mRNA metabolism. This approach of functionally characterizing the CLKs in P. falciparum is of particular interest since the malarial spliceosome is still poorly understood and will gain further insight into the parasite splicing machinery.
The role of human Ephrin receptor tyrosine kinase A2 (EphA2) in Chlamydia trachomatis infection
(2015)
Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling.
Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication.
Leishmaniasis is a neglected tropical disease that can be manifested through different clinical forms, ranging from cutaneous to visceral. The host response against Leishmania spp. is greatly dependent on T cell-mediated immunity, in which T helper 1 responses are associated with macrophage activation and elimination of the parasite, while regulatory T cells and T helper 2 responses are correlated with parasite survival and persistence of infection. Leishmania uses different virulence factors as strategies for evading the immune response of the host. One of them are cathepsin-like cysteine proteases, which are currently under extensive investigation as targets for drug development. Previous studies with inhibitors of cathepsins B and L in vivo revealed an outstanding modulation of the host T helper cell response. However, the mechanisms behind these observations were not further investigated. Given the urgent need for better treatments against leishmaniasis, the aim of this study was to investigate the effects that the lack of cathepsin B and L activity have on the signals that dendritic cells use to instruct T helper cell polarization in response to infection with Leishmania major.
The cathepsin inhibitors tested showed low or no cytotoxicity in bone marrow-derived dendritic cells, and dendritic cells and macrophages could be generated from cathepsin B and cathepsin L-deficient mice without apparent alterations in their phenotype in comparison to wild-type controls. Furthermore, lack of cathepsin B and L activity showed no impact in the rate of promastigote processing by dendritic cells. Cathepsin B and cathepsin L-deficient macrophages showed no differences in parasite proliferation and capacity to produce nitric oxide in comparison to wild-type macrophages. In response to the parasite, dendritic cells treated with a cathepsin B inhibitor and dendritic cells from cathepsin B-deficient mice showed higher levels of expression of major histocompatibility complex (MHC) class II molecules than dimethyl sulfoxide (DMSO) or wild-type controls, but it was not accompanied by changes in the expression of costimulatory molecules. Wild-type dendritic cells and macrophages are not able to express the pro-inflammatory cytokine interleukin (IL)-12 in response to promastigotes. However, cells treated with a cathepsin B inhibitor or cells deficient for cathepsin B were able to express IL-12, whilethe expression of other cytokines -including IL-6 and tumor necrosis factor (TNF)-alpha-remained unchanged. These characteristics point towards a more “pro-Th1” profile of dendritic cells in the absence of cathepsin B.
This data is the first report on IL-12 regulation depending on cathepsin B. The IL-12 up-regulation observed was already present at the transcriptional level. Furthermore, it was also present in macrophages and dendritic cells in response to LPS, and the latter had a higher capacity to induce T cell helper 1 polarization in vitro than wild-type dendritic cells. The activation of different signaling pathways was analyzed, but the up-regulation of IL-12 could not be attributed to modulation of nuclear factor-kappaB (NFkappaB), p38 mitogen activated protein kinase (MAPK) and extra-cellular signal-regulated kinase (ERK)1/2 pathways. Thus, the mechanism behind IL-12 regulation by cathepsin B remains to be elucidated, and the impact of these effects is yet to be confirmed in vivo. Altogether it is tempting to speculate that cathepsin B, in addition to its role in processing endocytosed material, is involved in the modulation of the pro-inflammatory cytokine IL-12.
Desert ants of the genus Cataglyphis have become model systems for the study of insect navigation. An age-related polyethism subdivides their colonies into interior workers and short-lived light-exposed foragers. While foraging in featureless and cluttered terrain over distances up to several hundred meters, the ants are able to precisely return back to their often inconspicuous nest entrance. They accomplish this enormous navigational performance by using a path integration system - including a polarization compass and an odometer - as their main navigational means in addition to landmark-dependent orientation and olfactory cues. C. fortis, being the focus of the present thesis, is endemic to the salt flats of western North Africa, which are completely avoided by other Cataglyphis species. The fact that Cataglyphis ants undergo a behavioral transition associated with drastically changing sensory demands makes these ants particularly interesting for studying synaptic plasticity in visual and olfactory brain centers. This thesis focuses on plastic changes in the mushroom bodies (MBs) - sensory integration centers supposed to be involved in learning and memory presumably including landmark learning - and in synaptic complexes belonging to the lateral accessory lobe (LAL) known to be a relay station in the polarization processing pathway. To investigate structural synaptic plasticity in the MBs of C. fortis, synaptic complexes (microglomeruli, MG) in the visual (collar) and olfactory (lip) input regions of the MB calyx were immunolabeled and their pre- and postsynaptic profiles were quantified. The results show that a volume increase of the MB calyx during behavioral transition is associated with a decrease of MG number - an effect called pruning - in the collar and, less pronounced, in the lip that goes along with dendritic expansion in MB intrinsic Kenyon cells. Light-exposure of dark-reared ants of different age classes revealed similar effects and dark-reared ants age-matched to foragers had MG numbers comparable to those of interior workers. The results indicate that the enormous structural synaptic plasticity of the MB calyx collar is primarily driven by visual experience rather than by an internal program. Ants aged artificially for up to one year expressed a similar plasticity indicating that the system remains flexible over the entire life-span. To investigate whether light-induced synaptic reorganization is reversible, experienced foragers were transferred back to darkness with the result that their MBs exhibit only some reverse-type characteristics, in particular differences in presynaptic synapsin expression. To investigate the structure of large synaptic complexes in the LAL of C. fortis and to detect potential structural changes, pre- and postsynaptic profiles in interior workers and foragers were immunolabeled and quantified by using confocal imaging and 3D-reconstruction. The results show that these complexes consist of postsynaptic processes located in a central region that is surrounded by a cup-like presynaptic profile. Tracer injections identified input and output tracts of the LAL: projection neurons from the anterior optic tubercle build connections with neurons projecting to the central complex. The behavioral transition is associated with an increase by ~13% of synaptic complexes suggesting that the polarization pathway may undergo some sort of calibration process. The structural features of these synaptic contacts indicate that they may serve a fast and reliable signal transmission in the polarization vision pathway. Behavioral analyses of C. fortis in the field revealed that the ants perform exploration runs including pirouette-like turns very close to the nest entrance for a period of up to two days, before they actually start their foraging activity. During these orientation runs the ants gather visual experience and might associate the nest entrance with specific landmarks or get entrained to other visual information like the polarization pattern, and, concomitantly adapt their neuronal circuitries to the upcoming challenges. Moreover, the pirouettes may serve to stimulate and calibrate the neuronal networks involved in the polarization compass pathway. Video recordings and analyses demonstrate that light experience enhanced the ants’ locomotor activity after three days of exposure. The fact that both the light-induced behavioral and neuronal changes in visual brain centers occur in the same time frame suggests that there may be a link between structural synaptic plasticity and the behavioral transition from interior tasks to outdoor foraging. Desert ants of the genus Cataglyphis possess remarkable visual navigation capabilities, but also employ olfactory cues for detecting nest and food sites. Using confocal imaging and 3D-reconstruction, potential adaptations in primary olfactory brain centers were analyzed by comparing the number, size and spatial arrangement of olfactory glomeruli in the antennal lobe of C. fortis, C. albicans, C. bicolor, C. rubra, and C. noda. Workers of all Cataglyphis species have smaller numbers of glomeruli compared to those of more olfactory-guided Formica species - a genus closely related to Cataglyphis - and to those previously found in other olfactory-guided ant species. C. fortis has the lowest number of glomeruli compared to all other species, but possesses a conspicuously enlarged glomerulus that is located close to the antennal nerve entrance. Males of C. fortis have a significantly smaller number of glomeruli compared to female workers and queens and a prominent male-specific macroglomerulus likely to be involved in sex pheromone communication. The behavioral significance of the enlarged glomerulus in female workers remains elusive. The fact that C. fortis inhabits microhabitats that are avoided by all other Cataglyphis species suggests that extreme ecological conditions may not only have resulted in adaptations of visual capabilities, but also in specializations of the olfactory system. The present thesis demonstrates that Cataglyphis is an excellent candidate for studying the neuronal mechanisms underlying navigational features and for studying neuronal plasticity associated with the ant’s lifelong flexibility of individual behavioral repertoires.
Protein kinases as targets for the development of novel drugs against alveolar echinococcosis
(2015)
The metacestode larval stage of the fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most lethal zoonosis of the northern hemisphere. The development of metacestode vesicles by asexual multiplication and the almost unrestricted infiltrative growth within the host organs is ensured from a population of undifferentiated, proliferative cells, so-called germinative cells. AE treatment options include surgery, if possible, as well as Benzimidazole-based chemotherapy (BZ). Given that the cellular targets of BZs, the -tubulins, are highly conserved between cestodes and humans, the chemotherapy is associated with considerable side-effects. Therefore, BZ can only be applied in parasitostatic doses and has to be given lifelong. Furthermore, the current anti-AE chemotherapy is ineffective in eliminating the germinative cell population of the parasite, which leads to remission of parasite growth as soon as therapy is discontinued.
This work focuses on protein kinases involved in the proliferation and development of the parasite with the intention of developing novel anti-AE therapies. Polo-like kinases (Plks) are important regulators of the eukaryotic cell cycle and are involved in the regulation and formation of the mitotic spindles during the M-phase of the cell cycle. Plks have already been shown to be associated with deregulated cellular growth in human cancers and have been investigated as novel drug targets in the flatworm parasite Schistosoma mansoni. In the first part of this work, the characterisation of a novel and druggable parasite enzyme, EmPlk1, which is homologous to the polo-like kinase 1 (Plk1) of humans and S. mansoni (SmPlk1), is presented. Through in situ hybridisation, it could be demonstrated that emplk1 is specifically expressed in the Echinococcus germinative cells. Upon heterologous expression in the Xenopus oocyte system, EmPlk1 induced germinal vesicle breakdown, thus indicating that it is an active kinase. Furthermore, BI 2536, a compound originally designed to inhibit the human ortholog of EmPlk1, inhibited the EmPlk1 activity at a concentration of 25 nM. In vitro treatment of parasite vesicles with similar concentrations of BI 2536 led to the elimination of the germinative cells from Echinococcus larvae, thus preventing the growth and further development of the parasite. In in vitro cultivation systems for parasite primary cells, BI 2536 effectively inhibited the formation of new metacestode vesicles from germinative cells. Thus, BI 2536 has profound anti-parasitic activities in vitro at concentrations well within the range of plasma levels measured after the administration of safe dosages to patients (50 nM after 24 h). This implies that EmPlk1 is a promising new drug target for the development of novel anti-AE drugs that would specifically affect the parasite’s stem cell population, namely the only parasite cells capable of proliferation. In addition to the chemotherapeutic aspects of this work, the inhibitor BI 2536 could be further used to study the function of stem cells in this model organism, utilising a method of injection of parasite stem cells into metacestode vesicles, for instance, as has been developed in this work.
In the second part of this work, a novel receptor tyrosine kinase, the Venus flytrap kinase receptor (EmVKR) of E. multilocularis has been characterised. Members of this class of single-pass transmembrane receptors have recently been discovered in the related trematode S. mansoni and are associated with the growth and differentiation of sporocyst germinal cells and ovocytes. The ortholog receptor in EmVKR is characterised by an unusual domain composition of an extracellular Venus flytrap module (VFT), which shows significant similarity to GABA receptors, such as the GABAB receptor (γ-amino butyric acid type B) and is linked through a single transmembrane domain to an intracellular tyrosine kinase domain with similarities to the kinase domains of human insulin receptors. Based upon the size (5112bp) of emvkr and nucleotide sequence specificities, efforts have been made to isolate the gene from cell culture samples to study the ligand for the activation of this receptor type in Xenopus oocytes. To date, this type of receptor has only been described in invertebrates, thus making it an attractive target for drug screening. In a first trial, the ATP competitive inhibitor AG 1024 was tested in our in vitro cell culture.
In conclusion, the EmVKR represents a novel receptor tyrosine kinase in E. multilocularis. Further efforts have to be made to identify the activating ligand of the receptor and its cellular function, which might strengthen the case for EmVKR as a potential drug target. The successful depletion of stem cells in the metacestode vesicle by the Plk1 inhibitor BI 2536 gives rise to optimising the chemical component for EmPlk1 as a new potential drug target. Furthermore, this inhibitor opens a new cell culture technique with high potential to study the cellular behaviour and influencing factors of stem cells in vitro.
In this work we wanted to investigate the role of NFATc1 in lymphocyte physiology and in pathological conditions (eg. psoriasis). NFATc1 is part of the signal transduction
pathways that regulates B cells activation and function. NFATc1 has different isoforms that are due to different promoters (P1 and P2), polyadenylation and alternative splicing. Moreover, we tried to elucidate the points of interactions between the NFAT and the NF-κB pathways in
activated B-cell fate. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA binding domain. We used mice which over-express a constitutive active version of NFATc1/α in their B cells with -or without- an ablated IRF4. IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt’s lymphoma cells and
induces terminal differentiation toward plasma cells. Our experiments showed that a ‘double hit’ in factors affecting B cell activation (NFATc1 in this case) and late B cell Differentiation (IRF4 in this case) alter the development of the B cells, lead to increase in their numbers and increase in stimulation induced proliferation. Therefore, the overall picture indicates a link between these 2 genes and probable carcinogenic alterations that may occur in B cells.
We also show that in splenic B cells, c-Rel (of the NF-κB canonical pathway) Support the induction of NFATc1/αA through BCR signals. We also found evidence that the lack of NFATc1 affects the expression of Rel-B (of the NF-κB non-canonical pathway). These data suggest a tight interplay between NFATc1 and NF-κB in B cells, influencing the competence of B cells and their functions in peripheral tissues.
We also used IMQ-induced psoriasis-like inflammation on mice which either lack NFATc1 from B cell. Psoriasis is a systemic chronic immunological disease characterized
primarily by abnormal accelerated proliferation of the skin keratinocytes. In psoriasis, the precipitating event leads to immune cell activation. Our experiments showed that NFATc1 is needed for the development of psoriasis. It also showed that IL-10 is the link that enables NFAT
from altering the B cell compartment (eg Bregs) in order to affect inflammation. The important role of B cell in psoriasis is supported by the flared up psoriasis-like inflammation in mice that lack B cells. Bregs is a special type of B cells that regulate other B cells and T cells; tuning the immunological response through immunomodulatory cytokines.
The learned helplessness phenomenon is a specific animal behavior induced by prior exposure to uncontrollable aversive stimuli. It was first found by Seligman and Maier (1967) in dogs and then has been reported in many other species, e.g. in rats (Vollmayr and Henn, 2001), in goldfishes (Padilla, 1970), in cockroaches (Brown, 1988) and also in fruit flies (Brown, 1996; Bertolucci, 2008). However, the learned helplessness effect in fruit flies (Drosophila melanogaster) has not been studied in detail. Thus, in this doctoral study, we investigated systematically learned helplessness behavior of Drosophila for the first time.
Three groups of flies were tested in heatbox. Control group was in the chambers experiencing constant, mild temperature. Second group, master flies were punished in their chambers by being heated if they stopped walking for 0.9s. The heat pulses ended as soon as they resumed walking again. A third group, the yoked fly, was in their chambers at the same time. However, their behavior didn’t affect anything: yoked flies were heated whenever master flies did, with same timing and durations. After certain amount of heating events, yoked flies associated their own behavior with the uncontrollability of the environment. They suppressed their innate responses such as reducing their walking time and walking speed; making longer escape latencies and less turning around behavior under heat pulses. Even after the conditioning phase, yoked flies showed lower activity level than master and control flies. Interestingly, we have also observed sex dimorphisms in flies. Male flies expressed learned helplessness not like female flies. Differences between master and yoked flies were smaller in male than in female flies. Another interesting finding was that prolonged or even repetition of training phases didn’t enhance learned helplessness effect in flies.
Furthermore, we investigated serotonergic and dopaminergic nervous systems in learned helplessness. Using genetic and pharmacological manipulations, we altered the levels of serotonin and dopamine in flies’ central nervous system. Female flies with reduced serotonin concentration didn’t show helpless behavior, while the learned helplessness effect in male flies seems not to be affected by a reduction of serotonin. Flies with lower dopamine level do not display the learned helplessness effect in the test phase, suggesting that with low dopamine the motivational change in learned helplessness in Drosophila may decline faster than with a normal dopamine level.
Traditionally, ischemic stroke has been regarded as the mere consequence of cessation of cerebral blood flow, e.g. due to the thromboembolic occlusion of a major brain supplying vessel. However, the simple restoration of blood flow via thrombolysis and/or mechanical recanalization alone often does not guarantee a good functional outcome. It appears that secondary detrimental processes are triggered by hypoxia and reoxygenation, which are referred to as ischemia/reperfusion (I/R) injury. During recent years it became evident that, beside thrombosis inflammation and edema formation are key players in the pathophysiology of cerebral ischemia. The contact-kinin system represents an interface between thrombotic, inflammatory and edematous circuits. It connects the intrinsic coagulation pathway with the plasma kallikrein-kinin system (KKS) via coagulation factor FXII.
The serine protease inhibitor C1-inhibitor (C1-INH) has a wide spectrum of inhibitory activities and counteracts activation of the contact-kinin system at multiple levels. The first part of the thesis aimed to multimodally interfere with infarct development by C1-INH and to analyze modes of actions of human plasma derived C1-INH Berinert® P in a murine model of focal cerebral ischemia. It was shown that C57BL/6 mice following early application of 15.0 units (U) C1-INH, but not 7.5 U developed reduced brain infarctions by ~60% and less neurological deficits in the model of transient occlusion of the middle cerebral artery (tMCAO). This protective effect was preserved at more advanced stages of infarction (day 7), without increasing the risk of intracerebral bleeding or affecting normal hemostasis. Less neurological deficits could also be observed with delayed C1-INH treatment, whereas no improvement was achieved in the model of permanent MCAO (pMCAO). Blood-brain-barrier (BBB) damage, inflammation and thrombosis were significantly improved following 15.0 U C1-INH application early after onset of ischemia. Based on its strong antiedematous, antiinflammatory and antithrombotic properties C1-INH constitutes a multifaceted therapeutic compound that protects from ischemic neurodegeneration in ‘clinically meaningful’ settings.
The second part of the thesis addresses the still elusive functional role of macrophages in the early phase of stroke, especially the role of the macrophage-specific adhesion molecule sialoadhesin (Sn). For the first time, sialoadhesin null (Sn-/-) mice, homozygous deficient for Sn on macrophages were subjected to tMCAO to assess the clinical outcome. Neurological and motor function was significantly improved in Sn-/- mice on day 1 after ischemic stroke compared with wildtype (Sn+/+) animals. These clinical improvements were clearly detectable even on day 3 following tMCAO. Infarctions on day 1 were roughly the same size as in Sn+/+ mice and did not grow until day 3. No intracerebral bleeding could be detected at any time point of data acquisition. Twenty four hours after ischemia a strong induction of Sn was detectable in Sn+/+ mice, which was previously observed only on perivascular macrophages in the normal brain. Deletion of Sn on macrophages resulted in less disturbance of the BBB and a reduced number of CD11b+ (specific marker for macrophages/microglia) cells, which, however, was not associated with altered expression levels of inflammatory cytokines. To further analyze the function of macrophages following stroke this thesis took advantage of LysM-Cre+/-/IKK2-/- mice bearing a nuclear factor (NF)-ϰB activation defect in the myeloid lineage, including macrophages. Consequently, macrophages were not able to synthesize inflammatory cytokines under the control of NF-ϰB. Surprisingly, infarct sizes and neurological deficits upon tMCAO were roughly the same in conditional knockout mice and respective wildtype littermates. These findings provide evidence that macrophages do not contribute to tissue damage and neurological deficits, at least, not by release of inflammatory cytokines in the early phase of cerebral ischemia. In contrast, Sn which is initially expressed on perivascular macrophages and upregulated on macrophages/microglia within the parenchyma following stroke, influenced functional outcome.
I. Nowadays, tropical landscapes experience large-scale land use intensification and land conversion driven by increasing demand for resourses. Due to the continuously high demand for tropical timber and politically intended step increase in palm oil production, multiple rounds of logging and subsequent conversion to oil palm plantations became a regionally wide-spread land conversion pattern in Southeast Asia. Although many tree species and some animals are highly threatened by logging, a great number of species groups, such as birds or mammals, have been shown to persist in logged forests. Accordingly, many ecosystem services, such as dung removal, seed dispersal or the activity of scavengers, are functionally maintained in logged forests. In contrast, oil palm plantations have been shown to not only dramatically alter the species composition and reduce biodiversity, but also curtail many crucial biotic and abiotic ecosystem functions. The focus of this dissertation was to investigate the response of anuran species richness and community composition to logging and conversion to oil palm plantation in northern Borneo (chapter II). I analysed the diet of various frog species and their change with habitat degradation. Furthermore, I assessed the shift in the trophic position of the anuran community as well as the response of anuran phylogenetic, dietary, and functional diversity to logging and conversion to oil palm plantations (chapter III). Finally, the resilience of the predator-prey interaction between an ant-specialist toad and its ant prey was analysed using shifts in species-level interactions (chapter IV).
II. This part of the study compares the species richness, relative abundance and community composition of stream anuran assemblages among primary forests, repeatedly logged forests and oil palm plantations. I used a highly standardised sampling setup applying transect-based sampling. Surprisingly, most of the anuran species native to primary forests were able to survive in logged forest streams. In contrast, on average only one third of the forest species richness was found in oil palm plantation streams. However, a high percentage of canopy cover above the plantation streams was able to mitigate this loss substantially. This study demonstrates the high conservation value of logged forests for Southeast Asian anurans. In contrast, the conversion to oil palm plantations leads to a dramatic decline of forest species. However, they have a mainly unused potential to contribute to the protection of parts of the regional anuran biodiversity if conservation-oriented management options are implemented.
III. In this part, I analysed the shifts in trophic position and multiple diversity layers of Southeast Asian stream-dependent anuran species across a gradient of disturbance from primary forest through intensively logged forest to oil palm plantation. For this purpose, I identified the diet composition of 59 anuran species by means of stomach flushing. Furthermore, I use diet composition of frog species as well as species traits to calculate dietary and functional diversity, respectively. I found that the trophic position of the entire anuran community is elevated in heavily disturbed habitats. Furthermore, species diversity, phylogenetic species variation, dietary diversity, and functional diversity were reduced. However, beyond the effect of the decreased species richness, only phylogenetic species variability and functional diversity were significantly impacted by land conversion, indicating a non-random loss of phylogenetic groups and functionally unique species. Overall, the observed changes to species interactions and functional composition suggest a greatly modified role of anurans in altered habitats and major foodweb reorganisation. Such far-reaching changes to the way species groups interact are likely to threaten local biodiversity and ecosystem functioning in natural and particularly modified habitats. However, I could also show, that small-scale habitat quality, provided by riparian reserves, is able to mitigate the negative consequences of land conversion considerably.
IV. Here I assess how logging of rain forest and conversion to oil palm plantations affect the populations of the ant-specialist giant river toad (Phrynoidis juxtaspera), and availability and composition of its ant prey. I measured canopy cover as an estimate for the degree of disturbance. I found that toad abundance decreased with increasing disturbance. At the same time, ant community composition was altered, and local ground-foraging ant species richness increased with disturbance. However, for a given amount of canopy cover, primary forest supported more ant species than altered habitats. Despite these changes, composition of ants consumed by toads was only weakly affected by habitat change, with the exception of the invasive yellow crazy ant (Anoplolepis gracilipes), which was positively selected in oil palm plantations. This suggests that predator-prey interactions can be mostly maintained with habitat disturbance despite shifts in community composition, and even that some predators are capable of exploiting new prey sources in novel ecosystems.
V. I could show that anuran diversity and their trophic interaction is negatively impacted by logging and in particular by conversion to oil palm plantations. From species richness and community composition, my study expanded to phylogenetic, dietary and functional diversity. Furthermore, I investigated the interaction of a particular toad species with its preferred prey (ants), on species level. This increasing degree of detail in my study provided comprehensive results, beyond the detail of many related studies. Overall, conservation of the remaining forest in Southeast Asia is urgently required to protect anuran biodiversity and their trophic interactions.
The Na+-D-glucose cotransporter in small intestine is regulated in response to food composition. Short term regulation of SGLT1 occurs post-transcriptionally in response to changes in luminal glucose. Adaptation to dietary carbohydrate involves long term regulation at the transcriptional level. The intracellular protein RS1 (gene RSC1A1) is involved in transcriptional and post-transcriptional regulation of SGLT1. RS1 contains an N-terminal domain with many putative phosphorylation sites. By Expressing SGLT1 in oocytes of Xenopus laevis it was previously demonstrated that the post-transcriptional down-regulation of SGLT1 by RS1 was dependent on the intracellular glucose concentration and activated by protein kinase C (PKC). The role of RS1 for short term regulation of SGLT1 in mouse small intestine in response to glucose and PKC was investigated comparing effects in RS1-/- mice and wildtype mice. Effects on SGLT1 activity were determined by measuring phlorizin inhibited uptake of α-methylglucoside (AMG). The involvement of RS1 in glucose dependent short term regulation could not be elucidated for technical reasons. However, evidence for RS1 independent short-term downregulation of SGLT1 after stimulation of PKC could be provided. It was shown that this downregulation includes decrease in the amount and/or in turnover of SGLT1 in the brush-border membrane as well as an increase of substrate affinity for AMG transport. Trying to elucidate the role of RS1 in long term regulation of SGLT1 in small intestine in response to glucose and fat content of the diet, wildtype and RS1-/- mice were kept for 2 months on a normo-caloric standard diet with high glucose and low fat content (ND), on a hyper-caloric glucose-galactose reduced diet with high fat content (GGRD) or on a hyper-caloric diet with a high fat and high glucose content (HFHGD). Thereafter the animals were starved overnight and SGLT1 mediated AMG uptake was measured. Independent of diet AMG uptake in ileum was smaller compared to duodenum and jejunum. In jejunum of wildtype and RS1-/- mice kept on the fat rich diets (GGRD and HFHGH) transport activity of SGLT1 was lower compared to mice kept on ND with low fat content. This result suggests an RS1 independent downregulation due to fat content of diet. Different to RS1-/- mice, the duodenum of wildtype mice showed transport activity of SGLT1 smaller in mice kept on glucose galactose reduced diet (GGRD) compared to the glucose galactose rich diets (ND and HFHGG). These data indicate that RS1 is involved in glucose dependent long term regulation in duodenum.
Fear conditioning is an efficient model of associative learning, which has greatly improved our knowledge of processes underlying the development and maintenance of pathological fear and anxiety. In a differential fear conditioning paradigm, one initially neutral stimulus (NS) is paired with an aversive event (unconditioned stimulus, US), whereas another stimulus does not have any consequences. After a few pairings the NS is associated with the US and consequently becomes a conditioned stimulus (CS+), which elicits a conditioned response (CR).
The formation of explicit knowledge of the CS/US association during conditioning is referred to as contingency awareness. Findings about its role in fear conditioning are ambiguous. The development of a CR without contingency awareness has been shown in delay fear conditioning studies. One speaks of delay conditioning, when the US coterminates with or follows directly on the CS+. In trace conditioning, a temporal gap or “trace interval” lies between CS+ and US. According to existing evidence, trace conditioning is not possible on an implicit level and requires more cognitive resources than delay conditioning.
The associations formed during fear conditioning are not exclusively associations between specific cues and aversive events. Contextual cues form the background milieu of the learning process and play an important role in both acquisition and the extinction of conditioned fear and anxiety. A common limitation in human fear conditioning studies is the lack of ecological validity, especially regarding contextual information. The use of Virtual Reality (VR) is a promising approach for creating a more complex environment which is close to a real life situation.
I conducted three studies to examine cue and contextual fear conditioning with regard to the role of contingency awareness. For this purpose a VR paradigm was created, which allowed for exact manipulation of cues and contexts as well as timing of events. In all three experiments, participants were guided through one or more virtual rooms serving as contexts, in which two different lights served as CS and an electric stimulus as US. Fear potentiated startle (FPS) responses were measured as an indicator of implicit fear conditioning. To test whether participants had developed explicit awareness of the CS-US contingencies, subjective ratings were collected.
The first study was designed as a pilot study to test the VR paradigm as well as the conditioning protocol. Additionally, I was interested in the effect of contingency awareness. Results provided evidence, that eye blink conditioning is possible in the virtual environment and that it does not depend on contingency awareness. Evaluative conditioning, as measured by subjective ratings, was only present in the group of participants who explicitly learned the association between CS and US.
To examine acquisition and extinction of both fear associated cues and contexts, a novel cue-context generalization paradigm was applied in the second study. Besides the interplay of cues and contexts I was again interested in the effect of contingency awareness. Two different virtual offices served as fear and safety context, respectively. During acquisition, the CS+ was always followed by the US in the fear context. In the safety context, none of the lights had any consequences. During extinction, a additional (novel) context was introduced, no US was delivered in any of the contexts. Participants showed enhanced startle responses to the CS+ compared to the CS- in the fear context. Thus, discriminative learning took place regarding both cues and contexts during acquisition. This was confirmed by subjective ratings, although only for participants with explicit contingency awareness. Generalization of fear to the novel context after conditioning did not depend on awareness and was observable only on trend level.
In a third experiment I looked at neuronal correlates involved in extinction of fear memory by means of functional magnetic resonance imaging (fMRI). Of particular interest were differences between extinction of delay and trace fear conditioning. I applied the paradigm tested in the pilot study and additionally manipulated timing of the stimuli: In the delay conditioning group (DCG) the US was administered with offset of one light (CS+), in the trace conditioning group (TCG) the US was presented 4s after CS+ offset. Most importantly, prefrontal activation differed between the two groups. In line with existing evidence, the ventromedial prefrontal cortex (vmPFC) was activated in the DCG. In the TCG I found activation of the dorsolateral prefrontal cortex (dlPFC), which might be associated with modulation of working memory processes necessary for bridging the trace interval and holding information in short term memory.
Taken together, virtual reality proved to be an elegant tool for examining human fear conditioning in complex environments, and especially for manipulating contextual information. Results indicate that explicit knowledge of contingencies is necessary for attitude formation in fear conditioning, but not for a CR on an implicit level as measured by FPS responses. They provide evidence for a two level account of fear conditioning. Discriminative learning was successful regarding both cues and contexts. Imaging results speak for different extinction processes in delay and trace conditioning, hinting that higher working memory contribution is required for trace than for delay conditioning.
During development of the nervous system, spontaneous Ca2+ transients are observed that regulate the axon growth of motoneurons. This form of spontaneous neuronal activity is reduced in motoneurons from a mouse model of spinal muscular atrophy and this defect correlates with reduced axon elongation. Experiments from our group demonstrated that voltage-gated sodium channel pore blockers decrease spontaneous neuronal activity and
axon growth in cultured motoneurons, too. In these experiments, saxitoxin was more potent than tetrodotoxin. We identified the saxitoxin-sensitive/tetrodotoxin-insensitive voltage-gated sodium channel NaV1.9 as trigger for the opening of voltage-gated calcium channels. In motoneurons, expression of NaV1.9 was verified via quantitative RT-PCR. Immuno labelling
experiments revealed enrichment of the channel in axonal growth cones and at the nodes of Ranvier of isolated nerve fibres from wild type mice. Motoneurons from NaV1.9 knock-out mice show decreased spontaneous activity and reduced axonal elongation. This growth defect can be rescued by NaV1.9 overexpression. In motoneurons from Smn-deficient mice, NaV1.9 distribution appeared to be normal.
Recently, patients carrying a missense mutation in the NaV1.9-encoding gene SCN11A were identified. These patients are not able to feel pain and suffer from muscular weakness and a delayed motor development. Molecular biological work during this dissertation supported the analysis of this mutation in a mouse model carrying the orthologous alteration in the Scn11a
locus. The cooperation study confirmed that a gain-of-function mechanism underlies the NaV1.9-mediated channelopathy, thus suggesting a functional role of NaV1.9 in human motoneurons.
An earlier study showed in hippocampal neurons that the receptor tyrosine kinase tropomyosin receptor kinase B (TrkB) can open the NaV1.9 channel. TrkB is localized in
growth cones of motoneurons and subsequently found in close proximity to NaV1.9. In order to proof whether TrkB is involved in spontaneous excitability in motoneurons, TrkB knock-out mice were analysed. Isolated motoneurons from TrkB knock-out mice show a reduced spontaneous activity and axon elongation. It remains to be studied whether TrkB and NaV1.9 are functionally connected.
Calcium ions can activate intracellular signalling cascades that control key functions in all types of neurons. These functions include neuronal excitability and excitation, synaptic plasticity, cell migration, transmitter release, gene transcription, and apoptosis. The major intracellular neuronal store for calcium is the endoplasmic reticulum (ER), a continuous and dynamic, membranous organelle that extends through all parts of neurons, from axons to dendrites. The calcium concentration in the ER is appr. one thousand fold higher than in the cytosol and this calcium gradient is built up by the sarco-/endoplasmic reticulum calcium ATPase (SERCA) pump that pumps calcium from the cytosol into the ER.
Despite detailed knowledge about various induced calcium signals within neurons, it was still elusive, how resting neurons maintain their ER calcium content at rest. In order to shed light on the calcium homeostasis at rest, the targeted-esterase induced dye loading (TED) technique was improved. TED allows the direct and non-disruptive visualization of ER calcium in presence of extracellular calcium, thus enabling to visualize the dynamic flow of ER calcium. TED is based on the overexpression of an ER-targeted mouse carboxylesterase. Inside the ER the carboxylesterase cleaves the acetoxymethyl ester calcium dye Fluo5N, AM, thereby converting this dye into a calcium sensitive, low-affinity, cell membrane impermeable calcium indicator that is trapped in the ER. When bound to calcium ions and excited by fluorescent light, its fluorescence intensity increases one hundredfold compared to the calcium-free state.
It was observed that calcium withdrawal from resting neurons led to a rapid loss of calcium from both the ER and the cytosol, which recovered upon calcium re-addition. It was concluded that a strong calcium influx and efflux must exist under resting conditions that maintain a constant calcium concentration in neurons at rest. TED calcium imaging could visualize this resting calcium influx event. When the inhibitor of store-operated calcium entry (SOCE), SKF-96365, was acutely added to neurons an immediate decline in ER calcium levels was observed, whereas cytosolic calcium levels remained constant. Based on these findings, a novel calcium homeostasis model is proposed in which a strong SOCE-like calcium influx and a corresponding calcium efflux maintain the ER calcium levels at rest. These fluxes are adapted to disturbances in order to maintain a constant calcium level in resting neurons.
This study visualizes for the first time the resting calcium flow into the ER. The calcium enters the neurons via a store-operated calcium entry-like mechanism, a form of calcium influx that was thought to be induced by signalling events.
While TGF-β is able to regulate miRNA expression in numerous cell types, TGF-β-dependent changes in the miRNA profile of CD8+ T cells had not been studied before. Considering that TGF-β suppresses CD8+ T cell effector functions in numerous ways, we wondered whether induction of immune-regulatory miRNAs could add to the known transcriptional effects of TGF-β on immune effector molecules. In this study, we used miRNA arrays, deep sequencing and qRT-PCR to identify miRNAs that are modulated by TGF-β in human CD8+ T cells. Having found that the TGF-β-dependent downregulation of NKG2D surface expression in NK cells and CD8+ T cells does not go along with a corresponding reduction in mRNA levels, this pathway appeared to be a possible target of TGF-β-inducible miRNAs. However, this hypothesis could not be confirmed by miRNA reporter assays. Instead, we observed that DAP10 transcription is suppressed by TGF-β which in turn negatively affects NKG2D surface expression. In spite of promising preliminary experiments, technical difficulties associated with the transfection of primary NK cells and NK cell lines unfortunately precluded the final proof of this hypothesis.
Instead, we focused on the TGF-β-induced changes in the miRNome of CD8+ T cells and confirmed the induction of the miR-23a cluster members, namely miR-23a, miR-27a and miR-24 by three different techniques. Searching for potential targets of these miRNAs which could contribute to the immunosuppressive action of TGF-β in T cells, we identified and confirmed a previously unknown regulation of IFN-γ mRNA by miR-27a and miR-24. Newly generated miRNA reporter constructs further revealed that LAMP1 mRNA is a target of miR-23a. Upon modulation of the miR-23a cluster in CD8+ T cells by the respective miRNA antagomirs and mimics, significant changes in IFN-γ expression confirmed the functional relevance of our findings. Effects on CD107a/LAMP1 expression were, in contrast, rather minimal. Still, overexpression of the miR-23a cluster attenuated the cytotoxic activity of antigen-specific CD8+ T cells. Taken together, these functional data reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8+ T-cell effector functions, even in the absence of TGF-β signaling.
Cardiac healing after myocardial infarction (MI) represents the cardinal prerequisite for proper replacement of the irreversibly injured myocardium. In contrast to innate immunity, the functional role of adaptive immunity in postinfarction healing has not been systematically addressed. The present study focused on the influence of CD4+ T lymphocytes on wound healing and cardiac remodeling after experimental myocardial infarction in mice. Both conventional and Foxp3+ regulatory CD4+ T cells (Treg cells) became activated in heart draining lymph nodes after MI and accumulated in the infarcted myocardium. T cell activation was strictly antigen-dependant as T cell receptor-transgenic OT-II mice in which CD4+ T cells exhibit a highly limited T cell
receptor repertoire did not expand in heart-draining lymph nodes post-MI. Both OT-II and major histocompatibility complex class II-deficient mice lacking a CD4+ T cell compartment showed a fatal clinical postinfarction outcome characterized by disturbed scar tissue construction that resulted in impaired survival due to a prevalence of left-ventricular ruptures. To assess the contribution of anti-inflammatory Treg cells on wound healing after MI, the Treg cell compartment was depleted using DEREG mice that specifically express the human diphtheria toxin receptor in Foxp3-positive cells, resulting in Treg cell ablation after diphtheria toxin administration. In a parallel line of experiments, a second model of anti-CD25 antibody-mediated Treg cell immuno-depletion was used. Treg cell ablation prior to MI resulted in adverse postinfarction left-ventricular dilatation associated with cardiac deterioration. Mechanistically, Treg cell depletion resulted in an increased recruitment of pro-inflammatory neutrophils and Ly-6Chigh monocytes into the healing myocardium. Furthermore, Treg cell-ablated mice exhibited an adverse activation of conventional non-regulatory CD4+ and CD8+ T cells that
showed a reinforced infiltration into the infarct zone. Increased synthesis of TNFα and IFNγ by conventional CD4+ and CD8+ T cells in hearts of Treg cell-depleted mice provoked an M1-like macrophage polarization characterized by heightened expression of healing-compromising induced NO synthase, in line with a reduced synthesis of healing-promoting transglutaminase factor XIII (FXIII), osteopontin (OPN) and transforming growth factor beta 1 (TGFβ1).
Therapeutic Treg cell activation by a superagonistic anti-CD28 monoclonal antibody stimulated Treg cell accumulation in the infarct zone and led to an increased expression of mediators inducing an M2-like macrophage polarization state, i.e. interleukin-10, interleukin-13 and TGFβ1. M2-like macrophage differentiation in the healing infarct was associated with heightened expression of scar-forming procollagens as well as scar-stabilizing FXIII and OPN, resulting in improved survival due to a reduced incidence of left-ventricular ruptures. Therapeutic Treg cell activation and the induction of a beneficial M2-like macrophage polarization was further achieved by employing a treatment modality of high clinical potential, i.e. by therapeutic administration of IL-2/ anti-IL-2 monoclonal antibody complexes. The findings of the present study suggest that therapeutic Treg cell activation and the resulting improvement of healing may represent a suitable strategy to attenuate adverse infarct expansion, left-ventricular remodeling, or infarct ruptures in patients with MI.
1. Since the early nineteenth century describing (and understanding) patterns of distribution of biodiversity across the Earth has represented one of the most significant intellectual challenges to ecologists and biogeographers. Among the most striking patterns of species richness are: the latitudinal and elevational gradients, with peaks in number of species at low latitudes and somewhere at mid altitudes, although other patterns, e.g. declines with increasing elevation, are often observed. Even in highly diverse tropical regions, species richness is not evenly distributed but there are “hotspots” of biodiversity where an exceptional number of species, especially endemics, are concentrated. Unfortunately, such areas are also experiencing dramatic loss of habitat. Among vertebrate taxa, amphibians are facing the most alarming number of extinctions. Habitat destruction, pollution and emergence of infectious diseases such as chytridiomycosis, are causing worldwide population declines. Responses to these drivers can be multidirectional and subtle, i.e. they may not be captured at the species but at the genetic level. Moreover, present patterns of diversity can result from the influence of past geological, climatic and environmental changes.
In this study, I used a multidisciplinary and multilevel approach to understand how and to which extent the landscape influences amphibian diversity. Mount Kilimanjaro is an exceptional tropical region where the landscape is rapidly evolving due to land use changes; additionally, there is a broad lack of knowledge of its amphibian fauna. During two rainy seasons in 2011, I recorded anurans from the foothills to 3500 m altitude; in addition, I focused on two river frog species and collected tissue samples for genetic analysis and swabs for detection of chytridiomycosis, the deadly disease caused by Batrachochytrium dendrobatidis (Bd).
2. I analyzed how species richness and composition change with increasing elevation and anthropogenic disturbance. In order to disentangle the observed patterns of species diversity and distribution, I incorporated inferences from historical biogeography and compared the assemblage of Mt. Kilimanjaro and Mt. Meru (both recent volcanoes) with those of the older Eastern Arc Mountains. Species richness decreased with elevation and locally increased in presence of water bodies, but I did not detect effects of either anthropogenic disturbance or vegetation structure on species richness and composition. Moreover, I found a surprisingly low number of forest species. Historical events seem to underlie the current pattern of species distribution; the young age of Mt. Kilimanjaro and the complex biogeographic processes which occurred in East Africa during the last 20 million years prevented montane forest frogs from colonizing the volcano.
3. I focused on the genetic level of biodiversity and investigated how the landscape, i.e. elevation, topographic relief and land cover, influence genetic variation, population structure and gene flow of two ecologically similar and closely related river frog species, namely Amietia angolensis and Amietia wittei. I detected greater genetic differentiation among populations in the highland species (A. wittei) and higher genetic variation in the lowland species (A. angolensis), although genetic diversity was not significantly correlated with elevation. Importantly, human settlements seemed to restrict gene flow in A. angolensis, whereas steep slopes were positively correlated with gene flow in A. wittei. This results show that even ecologically similar species can respond differently to landscape processes and that the spatial configuration of topographic features combined with species-specific biological attributes can affect dispersal and gene flow in disparate ways.
4. River frogs of the genus Amietia seem to be particularly susceptible to chytridiomycosis, showing the highest pathogen load in Kenya and other African countries. In the last study, I collected swab samples from larvae of A. angolensis and A. wittei for Bd detection. Both species resulted Bd-positive. The presence of Bd on Mt. Kilimanjaro has serious implication. For instance, Bd can be transported by footwear of hikers from contaminated water and soil. Tourists visiting Mt. Kilimanjaro may translocate Bd zoospores to other areas such as the nearby Eastern Arc Mts. where endemic and vulnerable species may still be naïve to the fungus and thus suffer of population declines.
5. My study significantly contributed to the knowledge of the amphibian fauna of Mt. Kilimanjaro and of East Africa in general, and it represents a valuable tool for future conservation actions and measures. Finally, it highlights the importance of using a multidisciplinary (i.e. community ecology, historical biogeography, landscape genetics, disease ecology) and multilevel (i.e. community, species, population, gene) approach to disentangle patterns of biodiversity.
The number of newly detected autoantibodies (AB) targeting synaptic proteins in neurological disorders of the central nervous system (CNS) is steadily increasing. Direct interactions of AB with their target antigens have been shown in first studies but the exact pathomecha-nisms for most of the already discovered AB are still unclear. The present study investigates pathophysiological mechanisms of AB-fractions that are associated with the enigmatic CNS disease Stiff person syndrome (SPS) and target the synaptically located proteins amphiphysin or glutamate decarboxylase 65 (GAD65).
In the first part of the project, effects of AB to the presynaptic endocytic protein amphiphysin were investigated. Ultrastructural investigations of spinal cord presynaptic boutons in an es-tablished in-vivo passive-transfer model after intrathecal application of human anti-amphiphysin AB showed a defect of endocytosis. This defect was apparent at high synaptic activity and was characterized by reduction of the synaptic vesicle pool, clathrin coated vesi-cles (CCVs), and endosome like structures (ELS) in comparison to controls. Molecular inves-tigation of presynaptic boutons in cultured murine hippocampal neurons with dSTORM microscopy after pretreatment with AB to amphiphysin revealed that marker proteins involved in vesicle exocytosis (synaptobrevin 2 and synaptobrevin 7) had an altered expression in GA-BAergic presynapses. Endophilin, a direct binding partner of amphiphysin also displayed a disturbed expression pattern. Together, these results point towards an anti-amphiphysin AB-induced defective organization in GABAergic synapses and a presumably compensatory rearrangement of proteins responsible for CME.
In the second part, functional consequences of SPS patient derived IgG fractions containing AB to GAD65, the rate limiting enzyme for GABA synthesis, were investigated by patch clamp electrophysiology and immunohistology. GABAergic neurotransmission at low and high activity as well as short term plasticity appeared normal but miniature synaptic potentials showed an enhanced frequency with constant amplitudes. SPS patient IgG after preabsorption of GAD65-AB using recombinant GAD65 still showed specific synaptic binding to neu-rons and brain slices supporting the hypothesis that additional, not yet characterized AB are present in patient IgG responsible for the exclusive effect on frequency of miniature potentials.
In conclusion, the present thesis uncovered basal pathophysiological mechanisms underlying paraneoplastic SPS induced by AB to amphiphysin leading to disturbed presynaptic architec-ture. In idiopathic SPS, the hypothesis of a direct pathophysiological role of AB to GAD65 was not supported and additional IgG AB are suspected to induce distinct synaptic malfunction.
Background: Food craving refers to an intense desire to consume a specific kind of food of which chocolate is the most often craved one. It is this intensity and specificity that differentiates food craving from feelings of hunger. Although food craving and hunger often co-occur, an energy deficit is not a prerequisite for experiencing food craving, that is, it can also occur without being hungry. Food craving often precedes and predicts over- or binge eating which makes it a reasonable target in the treatment of eating disorders or obesity. One of the arguably most extensively validated measures for the assessment of food craving are the Food Cravings Questionnaires (FCQs), which measure food craving on a state (FCQ-S) and trait (FCQ-T) level. Specifically, the FCQ-S measures the intensity of current food craving whereas the FCQ-T measures the frequency of food craving experiences in general. The aims of the present thesis were to provide a German measure for the assessment of food craving and to investigate cognitive, behavioral, and physiological correlates of food craving. For this purpose, a German version of the FCQs was presented and its reliability and validity was evaluated. Using self-reports, relationships between trait food craving and dieting were examined. Cognitive-behavioral correlates of food craving were investigated using food-related tasks assessing executive functions. Psychophysiological correlates of food craving were investigated using event-related potentials (ERPs) in the electroencephalogram and heart rate variability (HRV). Possible intervention approaches to reduce food craving were derived from results of those studies.
Methods: The FCQs were translated into German and their psychometric properties and correlates were investigated in a questionnaire-based study (articles #1 & #2). The relationship between state and trait food craving with executive functioning was examined with behavioral tasks measuring working memory performance and behavioral inhibition which involved highly palatable food-cues (articles #3 & #4). Electrophysiological correlates of food craving were tested with ERPs during a craving regulation task (article #5). Finally, a pilot study on the effects of HRV-biofeedback for reducing food craving was conducted (article #6).
Results: The FCQs demonstrated high internal consistency while their factorial structure could only partially be replicated. The FCQ-T also had high retest-reliability which, expectedly, was lower for the FCQ-S. Validity of the FCQ-S was shown by positive relationships with current food deprivation and negative affect. Validity of the FCQ-T was shown by positive correlations with related constructs. Importantly, scores on the subscales of the FCQ-T were able to discriminate between non-dieters and successful and unsuccessful dieters (article #1). Furthermore, scores on the FCQ-T mediated the relationship between rigid dietary control strategies and low dieting success (article #2). With regard to executive functioning, high-calorie food-cues impaired working memory performance, yet this was independent of trait food craving and rarely related to state food craving (article #3). Behavioral disinhibition in response to high-calorie food-cues was predicted by trait food craving, particularly when participants were also impulsive (article #4). Downregulation of food craving by cognitive strategies in response to high-calorie food-cues increased early, but not later, segments of the Late Positive Potential (LPP) (article #5). Few sessions of HRV-biofeedback reduced self-reported food cravings and eating and weight concerns in high trait food cravers (article #6).
Conclusions: The German FCQs represent sound measures with good psychometric properties for the assessment of state and trait food craving. Although state food craving increases during cognitive tasks involving highly palatable food-cues, impairment of task performance does not appear to be mediated by current food craving experiences. Instead, trait food craving is associated with low behavioral inhibition in response to high-calorie food-cues, but not with impaired working memory performance. Future studies need to examine if trait food craving and, subsequently, food-cue affected behavioral inhibition can be reduced by using food-related inhibition tasks as a training. Current food craving and ERPs in response to food-cues can easily be modulated by cognitive strategies, yet the LPP probably does not represent a direct index of food craving. Finally, HRV-biofeedback may be a useful add-on element in the treatment of disorders in which food cravings are elevated. To conclude, the current thesis provided measures for the assessment of food craving in German and showed differential relationships between state and trait food craving with self-reported dieting behavior, food-cue affected executive functioning, ERPs and HRV-biofeedback. These results provide promising starting points for interventions to reduce food craving based on (1) food-cue-related behavioral trainings of executive functions, (2) cognitive craving regulation strategies, and (3) physiological parameters such as HRV-biofeedback.
Staphylococcus aureus is a major threat to public health systems all over the globe. This second most cause of nosocomial infections is able to provoke a wide variety of different types of infection in humans and animals, ranging from superficial skin and skin structure infections to invasive disease like sepsis or pneumonia. But not enough, this pathogen is also notorious in acquiring and/or developing resistance to antimicrobial compounds, thus limiting available treatment options severely. Therefore, development of new compounds and strategies to fight S. aureus is of paramount importance. But since only 1 out of 5 compounds, which entered clinical trials, becomes a drug, the preclinical evaluation of promising compounds has to be reconsidered, too. The aim of this thesis was to address both sides of this problem: first, to improve preclinical testing by incorporating in vivo imaging technologies to the preclinical testing procedure in order to acquire additional and clearer data about efficacy of promising compounds and second, by evaluating lysostaphin, which is a promising, new option to fight S. aureus infections.
The first aim of this thesis focused on the establishment of a dual modality in vivo imaging platform, consisting of Bioluminescence Imaging (BLI) and Magnetic Resonance Imaging (MRI), to offer detailed insights into the course and gravity of S. aureus infection in the murine thigh infection model. Since luciferase-expressing S. aureus strains were generated in former studies and enabled thus bioluminescence imaging of bacterial infection, this technology should be implemented into the compound evaluation platform in order to non-invasively track the bacterial burden over time. MRI, in contrast, was only rarely used in earlier studies to visualize and measure the course of infection or efficacy of anti-bacterial therapy. Thus, the first set of experiments was performed to identify benefits and drawbacks of visualizing S. aureus infections in the mouse model by different MR methods. Native, proton-based MR imaging showed in this regard increased T2 relaxation times in the infected thigh muscles, but it was not possible to define a clear border between infected and uninfected tissue. Iron oxide nanoparticles and perfluorocarbon emulsions, two MR contrast agents or tracer, in contrast, offered this distinction. Iron oxide particles were detected in this regard by their distortion of 1H signal in proton-based MRI, while perfluorocarbon emulsion was identified by 19F MRI. Mammals do not harbor sufficient intrinsic amounts of 19F to deliver specific signal and therefore, 19F MR imaging visualizes only the signal of administered perfluorocarbon emulsion. The in vivo accumulation of perfluorocarbon emulsion can be imaged by 19F MRI and overlayed on a simultaneously acquired 1H MR image, which shows the anatomical context in clear detail. Since this is advantageous compared to contrast agent based MR methods like iron oxide particle-based MRI, further experiments were performed with perfluorocarbon emulsions and 19F MRI.
Experimental studies to elucidate the accumulation of perfluorocarbon emulsion at the site of infection showed robust 19F MR signals after administration between day 2 and at least day 8 p.i.. Perfluorocarbon emulsion accumulated in all investigated mice in the shape of a ‘hollow sphere’ at the rim of the abscess area and the signal remained stable as long as the infection prevailed. In order to identify the mechanism of accumulation, flow cytometry, cell sorting and histology studies were performed. Flow cytometry and cell sorting analysis of immune cells at the site of infection showed that neutrophils, monocytes, macrophages and dendritic cells carried contrast media at the site of infection with neutrophils accounting for the overwhelming portion of perfluorocarbon signal. In general, most of the signal was associated with immune cells, thus indicating specific immune cell dependent accumulation. Histology supported this observation since perfluorocarbon emulsion related fluorescence could only be visualized in close proximity to immune cell nuclei.
After establishing and testing of 19F MRI with perfluorocarbon emulsions as infection imaging modality, the effects of antibiotic therapy upon MR signal was investigated in order to evaluate the capability of this modality for preclinical testing procedure. Thus, the efficacy of vancomycin and linezolid, two clinically highly relevant anti - S. aureus compounds, were tested in the murine thigh infection model. Both of them showed reduction of the colony forming units and bioluminescence signal, but also of perfluorocarbon emulsion accumulation strength and volume at the site of infection, which was visualized and quantified by 19F MRI. The efficacy pattern with linezolid being more efficient in clearing bacterial infection was shown similarly by all three methods. In consequence, 19F MRI with perfluorocarbon emulsion as MR tracer proved to be capable to visualize antibacterial therapy in preclinical testing models.
The next step was consequently to evaluate a promising new compound against S. aureus infections. Thus, lysostaphin, an endo-peptidase that cleaves the cell wall of S. aureus, was tested in different concentrations alone or in combination with oxacillin for efficacy in murine thigh and catheter associated infection models. Lysostaphin only in the concentration of 5 mg/kg body weight or combined with oxacillin in the concentration of 2 mg/kg showed strong reduction of bacterial burden by colony forming unit determination and bioluminescence imaging in both models. The perfluorocarbon accumulation was investigated in the thigh infection model by 19F MRI and was strongly reduced in terms of volume and signal strength in both above-mentioned groups. In general, lysostaphin showed comparable or superior efficacy than vancomycin or oxacillin alone. Therefore, further development of lysostaphin for the treatment of S. aureus infections is recommended by these experiments. Overall, the antibiotic efficacy pattern of all applied antibiotic regimens was similar with all three applied methods, demonstrating the usefulness of MRI for antibiotic efficacy testing. Importantly, treatment with oxacillin either alone or in combination with lysostaphin resulted in stronger perfluorocarbon emulsion accumulation at the site of infection than expected compared to the results from bioluminescence imaging and colony forming unit determination. This might be an indication for immunomodulatory properties of oxacillin.
Further murine infection experiments demonstrated in this context a differential release of cytokine and chemokines in the infected thigh muscle in dependence of the applied antibacterial therapy. Especially treatment with oxacillin, but to a less degree with minocycline or linezolid, too, exhibited high levels of various cytokines and chemokines, although they reduced the bacterial burden efficiently. In consequence, possible immunomodulatory effects of antibacterial compounds have to be taken into account for future applications of imaging platforms relying on the visualization of the immune response. However, this observation opens a new field for these imaging modalities since it might be extraordinary interesting to study the immunomodulatory effects of compounds or even bacterial factors in vivo. And finally, a two modality imaging platform which combines methods to visualize on the one hand the bacterial burden and on the other hand the immune response offers an innovative, new platform to study host-pathogen interaction in vivo in a non-invasive fashion.
In summary, it could be shown that perfluorocarbon emulsions accumulate in immune cells at the site of infection in the murine S. aureus thigh infection model. The accumulation pattern shapes a ‘hollow sphere’ at the rim of the abscess area and its size and perfluorocarbon content is dependent on the severity of disease and/or efficacy of antibiotic therapy. Thus, 19F MRI with perfluorocarbon emulsions is a useful imaging modality to visualize sites and course of infection as well as to evaluate promising antibacterial drug candidates. Furthermore, since the accumulation of tracer depends on immune cells, it might be additionally interesting for studies regarding the immune response to infections, auto-immune diseases or cancer, but also to investigate the efficacy of immunomodulatory compounds and immunization.
Motivation and Aim:
Cardiovascular disease has been the leading cause of mortality and morbidity throughout the world. In developed countries, cardiovascular diseases are already responsible for a majority of deaths and will become the pre-eminent health problem worldwide (1,2). Rupture of atherosclerotic plaque accounts for approximately 70% of fatal acute myocardial infarction and sudden heart deaths. Conventional criterias for the diagnosis of “vulnerable plaques” are calcified nodules, yellow appearance of plaque, a thin cap, a large lipid core, severe luminal stenosis, intraplaque hemorrhage, inflammation, thrombogenicity, and plaque injury (3-5).
Noninvasive diagnosis of vulnerable plaque still remains a great challenge and a huge research prospect, which triggered us to investigate the feasibility of PET imaging on the evaluation of atherosclerosis. Nuclear imaging of atherosclerosis, especially co-registered imaging modalities, could provide a promising diagnostic tool including both anatomy and activities to identify vulnerable atherosclerotic plaque or early detection of inflammatory endothelium at risk. Furthermore, the development of specific imaging tracers for clinical applications is also a challenging task. The aim of this work was to assess the potential of novel PET imaging probes associated with intra-plaque inflammation on animal models and in human respectively.
Methods
In this work, several molecular imaging modalities were employed for evaluation of atherosclerosis. They included Positron emission tomography / Computed tomography (PET/CT) for human studies, and micro-PET, autoradiography and high-resolution magnetic resonance imaging (MRI) for animal studies. Radiotracers for PET imaging included the glucose analogue 18F-Fluorodeoxyglucose (18F-FDG), the somatostatin receptor avide tracer 68Ga-DOTATATE, and the Gallium-68 labeled fucoidan (68Ga-Fucoidan), which was developed as a PET tracer to detect endothelial P-selectin, which overexpressed at early stage of atherosclerosis and endothelial overlying activated plaque. Tracer’s capabilities were firstly assessed on cellular level in vitro. Subsequently, Animal studies were conducted in two animal models: 1, Apolipoprotein E (ApoE-/-) mice having severe atherosclerotic plaque; 2, Lipopolysaccharide (LPS) -induced mice for receiving acute vascular inflammation. Corresponding analyses on protein and histological level were conducted as well to confirm our results.
In human study, 16 patients with neuroendocrine tumors (NETs) were investigated on imaging vascular inflammation. These patients had undergone both 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT for staging or restaging within 6 weeks. 16 patients were randomized into two groups: high-risk group and low-risk group. Uptake ratio of both tracers from two groups were compared and correlated with common cardiovascular risk factors.
Results and Conclusion
In murine study, the expression of somatostatin receptor 2, which is the main bio-target of 68Ga-DOTATATE on macrophage/monocyte was confirmed by flow cytometry and immunohistochemistry. Prospectively, high specific accumulation of 68Ga-DOTATATE to the macrophage within the plaques was observed in aorta lesions by autoradiography and by micro-PET. In study with 68Ga-fucoidan, a strong expression of P-selectin on active endothelium overlying on inflamed plaque but weaker on inactive plaques was confirmed. Specific focal uptake of 68Ga-fucoidan were detected at aorta segments by micro-PET, and correlated with high-resolution magnetic resonance imaging (MRI), which was used to characterize the morphology of plaques. 68Ga-fucoidan also showed a greater affinity to active inflamed plaque in comparison of inactive fibrous plaque, which was assessed by autoradiography. Specificity of 68Ga-DOTATATE and 68Ga-fucoidan were confirmed by ex-vivo blocking autoradiography and in vivo blocking PET imaging respectively.
In human study, focal uptake of both 18F-FDG and 68Ga-DOTATATE was detected. Analyzing concordance of two tracers’ uptake ratio, Out of the 37 sites with highest focal 68Ga-DOTATATE uptake, 16 (43.2%) also had focal 18F-FDG uptake. Of 39 sites with highest 18F-FDG uptake, only 11 (28.2%) had a colocalized 68Ga-DOTATATE accumulation. Correlated tracers’ uptake and calcium burden and risk factors, Mean target-to-background ratio (TBR) of 68Ga-DOTATATE correlated significantly with the presence of calcified plaques (r=0.52), hypertension (r=0.60), age (r=0.56) and uptake of 18F-FDG (r=0.64). TBRmean of 18F-FDG correlated significantly only with hypertension (r=0.58; p<0.05). Additionally, TBRmean of 68Ga-DOTATATE is significant higher in the high risk group while TBRmean of 18F-FDG is not.
In conclusion, we evaluated vascular inflammation of atherosclerosis non-invasively using the two PET tracers: 68Ga-DOTATATE and 68Ga-Fucoidan. 68Ga-DOTATATE show specific affinity to infiltrated macrophage within the plaques. 68Ga-Fucoidan may hold the potential to discriminate between active and inactive atherosclerotic plaques in terms of variant accumulation on different-types of plaques. PET as leading molecular imaging technique provides superiority in assessing cellular activity, which is pivotal for understanding internal activity of atherosclerotic plaques. Since diagnosis of atherosclerosis is a complex and multi-dimensional task. More integrated imaging technology such as PET/MRI, faster imaging algorithm, more efficient radiotracer are required for further development of atherosclerosis imaging,
Marine sponges (phylum Porifera) are simple, sessile, filter-feeder animals. Microbial symbionts are commonly found in the sponge internal tissue, termed the mesohyl. With respect to the microbial content, sponges are classified as either low-microbial abundance sponges (LMA), or high-microbial abundance sponges (HMA). The HMA/LMA dichotomy was explored in this Thesis using the Red Sea sponges as experimental models. A range of methods encompassing transmission electron microscopy, 16S rRNA gene deep sequencing, and metatranscriptomics was employed towards this goal. Here, particular emphasis was placed on the functional analysis of sponge microbiomes.
The Red Sea sponges Stylissa carteri, Xestospongia testudinaria, Amphimedon ochracea, and Crella cyathophora were classified as HMA or LMA sponges using transmission electron microscopy. The diversity, specificity, and transcriptional activity of microbes associated with the sponges S. carteri (LMA) and X. testudinaria (HMA) and seawater were investigated using 16S rRNA amplicon pyrosequencing. The microbial composition of S. carteri was more similar to that of seawater than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly seawater sequences (~24%) than the X. testudinaria data set (~6%). The most abundant operational taxonomic units (OTUs) were shared between all three sources (S. carteri, X. testudinaria, seawater), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. S. carteri microbiomes were enriched of Gammaproteobacteria and members of the genus Synechococcus and Nitrospira. Enriched members of X. testudinaria microbiomes included Chloroflexi, Deferribacteres, and Actinobacteria. The transcriptional activity of sponge-associated microorganisms was assessed by comparing 16S rRNA gene with transcript amplicons, which showed a good correlation.
The microbial functional gene repertoire of sponges and seawater from the Red Sea (X. testudinaria, S. carteri) and the Mediterranean (Aplysina aerophoba, Dysidea avara) were investigated with the environmental microarray GeoChip 4. Amplicon sequencing was performed alongside in order to assess microbial diversity. The typical microbial diversity patterns characteristic of HMA (abundance of Gammaproteobacteria, Chloroflexi, Acidobacteria, Deferribacteres, and others) and LMA sponges (abundance of Alpha-, Beta-, Gammaproteobacteria, Cyanobacteria, and Bacteroidetes) were confirmed. The HMA/LMA dichotomy was stronger than any possible geographic pattern based on microbial diversity (amplicon) and functional genes (GeoChip). However upon inspection of individual genes detected by GeoChip, very few specific differences were discernible, including differences related to microbial ammonia oxidation, ammonification (higher gene abundance in sponges over seawater) as well as denitrification (lower gene abundance). Furthermore, a higher abundance of a gene, pcc, representative of archaeal autotrophic carbon fixation was noted in sponges over seawater. Thirdly, stress-related genes, in particular those related to radiation, were found in lower abundances in sponge microbiomes than in seawater. With the exception of few documented specific differences, the functional gene repertoire between the different sources appeared largely similar.
The most actively expressed genes of S. carteri microbiomes were investigated with metatranscriptomics. Prokaryotic mRNA was enriched from sponge total RNA, sequenced using Illumina HiSeq technology, and annotated with the metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline. High expression of archaeal ammonia oxidation and photosynthetic carbon fixation by members of the genus Synechococcus was detected. Functions related to stress response and membrane transporters were among the most highly expressed by S. carteri symbionts. Unexpectedly, gene functions related to methylotrophy were highly expressed by gammaproteobacterial symbionts. The presence of seawater-derived microbes is indicated by the phylogenetic proximity of organic carbon transporters to orthologs of members from the SAR11 clade. In summary, the most expressed functions of the S. carteri-associated microbial community were revealed and linked to the dominant taxonomic members of the microbiome.
In conclusion, HMA and LMA Red Sea sponges were used as models to gain insights into relevant themes in sponge microbiology, i.e. diversity, specificity, and functional activities. Overall, my Thesis contributes to a better understanding of sponge-associated microbial communities, and the implications of this association to marine ecology.
Atherosclerosis is an active and progressive condition where the vascular cell adhesion molecules as VCAM-1 play a vital role controlling the recruitment of immune cells within the early and advanced plaques. Therefore targeting of VCAM-1 molecules with specific contrast agent bears the possibility to monitor the VCAM-1 expression, visualize the plaque progression starting at the early alterations, and help to establish early prevention of atherosclerosis before the origin of the thrombus formation, of which late recognition leads to myocardial infarction. Furthermore noninvasive magnetic resonance imaging (MRI) offers the benefit of combining the molecular and anatomic data and would thus enable specific detection of VCAM-1 targeted iron oxide contrast agent within inflammatory process of atherosclerosis. This thesis exactly presents the VCAM-1 concept as a suitable molecular approach and the potential of specific ultrasmall superparamagnetic iron oxide (USPIO) conjugated to the VCAM-1 binding peptide over unspecific non-targeted USPIO particles for evaluation of atherosclerosis. This work firstly demonstrated that selection of VCAM-1 molecules offers a good and potential strategy for imaging of atherosclerosis, as these vascular cell adhesion molecules are highly expressed in the early phase of inflammation and also continuously up-regulated within the advanced plaques. Secondly, this thesis showed the proof of principle and capability of the newly designed USPIO contrast agent conjugated to the specific cyclic peptide for VCAM-1 recognition. The experimental studies including ultra-high field MRI enabled further ex vivo and in vivo detection of applied USPIO-VCAM-1 particles within the aortic root region of early and advanced atherosclerotic plaques of 12 and 30 week old apolipoprotein E deficient (ApoE-/-) mice. Using a combination of histology and electron microscopy, this study for the first time pointed to distribution of targeted USPIO-VCAM-1 particles within plaque cells expressing VCAM-1 not only in luminal regions but also in deeper medial smooth muscle cell areas. Hence functionalized USPIO particles targeting VCAM-1 molecules allow specific and sensitive detection of early and advanced plaques at the molecular level, giving the new possibilities for early recognition of atherosclerotic plaques before the appearance of advanced and prone to rupture lesions. In contrast to the functionalized USPIO-VCAM-1, utilized non-targeted USPIO particles did not succeed in early plaque 6 identification limiting visualization of atherosclerosis to advanced forms in atherosclerotic ApoE-/- mice.
Alveolar echinococcosis (AE) is a severe and life-threatening disease caused by the metacestode larva of the fox-tapeworm Echinococcus multilocularis. Parasite entry into the host evokes an early and potentially parasiticidal Th1 immune response that is gradually replaced by a permissive Th2 response. An immunoregulatory environment has also been reported in the host as the disease progresses. As a result of immunomodulation, E. multilocularis larvae persist in the host for decades without being expelled, and thus almost act like a perfect transplant. Very little is currently known on the molecular basis of the host immunomodulation by E. multilocularis. In this work, in vitro cultivation systems were used to assess the influence of metabolites released by the parasite larvae (E/S products) on host immune effector cells. E/S products of cultivated larvae that respresent the early (primary cells) and chronic (metacestode vesicles) phase of AE induced apoptosis and tolerogenic properties (poor responsiveness to LPS stimulation) in host dendritic cells (DC) whereas those of control larvae (protoscoleces) failed to do so. These findings show that the early infective stage of E. multilocularis induces tolerogenicity in host DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. Interestingly, metacestode E/S products promoted the conversion of naïve CD4+ T-cells into Foxp3+ regulatory T-cells in vitro, whereas primary cell and protoscolex E/S products failed to do it. Since Foxp3+ regulatory T-cells are generally known to mediate immunosuppression, the present finding indicates that Foxp3+ regulatory T-cells, expanded by E/S products of the metacestode larva, could play a role in the parasite-driven immunomodulation of the host observed during AE. Furthermore, a substantial increase in number and frequency of suppressive Foxp3+ regulatory T-cells could be observed within peritoneal exudates of mice following intraperitoneal injection of E. multilocularis metacestodes, indicating that Foxp3+ regulatory T-cells could also play an important role in E. multilocularis-driven immunomodulation in vivo. Interestingly, a parasite activin ortholog, EmACT, secreted by metacestodes, was shown to expand host regulatory T-cells in a TGF-β-dependent manner, similarly to mammalian activin A. This observation indicated that E. multilocularis utilizes evolutionarily conserved TGF-β superfamily ligands, like EmACT, to expand host regulatory T-cells. Taken together, the present findings suggest EmACT, a parasite activin secreted by the metacestode and capable of expanding host regulatory T-cells, as an important player in the host immunomodulation by E. multilocularis larvae. Another parasite factor EmTIP, homologous to mammalian T-cell immunomodulatory protein (TIP) was characterized in this work. EmTIP could be detected in the secretions of the parasite primary cells and localized to the intercellular space within the parasite larvae. EmTIP blockade inhibited the proliferation of E. multilocularis primary cells and the formation of metacestode vesicles indicating a major role for parasite development. Furthermore, EmTIP evoked a strong release of IFN-γ by CD4+ T-cells hence suggesting that the secretion of this factor as a result of its role in parasite development could “secondarily” induce a potentially protective Th1 response. In conclusion, this work identified two molecules, EmACT and EmTIP, with high immunomodulatory potential that are released by E. multilocularis larvae. The data presented do provide insights into the mechanisms of parasite-driven host immunomodulation during AE that are highly relevant for the development of anti-parasitic immune therapies.
A subtly regulated and controlled course of cellular processes is essential for the healthy functioning not only of single cells, but also of organs being constituted thereof. In return, this entails the proper functioning of the whole organism. This implies a complex intra- and inter-cellular communication and signal processing that require equally multi-faceted methods to describe and investigate the underlying processes. Within the scope of this thesis, mathematical modeling of cellular signaling finds its application in the analysis of cellular processes and signaling cascades in different organisms. ...