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Schlagwörter: Salmonella , Salmonella enterica , Salmonella typhimurium , Salmonellose , Escherichia coli , Shigella , Infektion , Bakterielle Infektion , Zellkultur , HeLa-Zelle , Apoptosis , Metabolismus , Stoffwechsel , Glucose , Glucosetransport , Glucosestoffwechsel , Katabolismus , Kohlenstoff , Kohlenstoffbedarf , Kohlenstoffhaushalt , Kohlenstoffstoffwechsel , Kohlenstoff-13 , Kohlenstoffisotop Salmonella Typhimurium und enteroinvasive E. coli (EIEC) sind fakultativ intrazelluläre Bakterien aus der Familie der Enterobacteriaceae. Während erstere sich nach der Internalisierung durch eukaryotische Zellen normalerweise in einem spezialisierten Phagosom, der Salmonella-enthaltenden Vakuole (SCV), vermehren, replizieren EIEC im Zytoplasma der Wirtszellen. In der vorliegenden Arbeit wurde zunächst durch Mikroinjektion die Fähigkeit von S. Typhimurium 14028s untersucht, ebenfalls im Zytoplasma von Caco-2-Zellen replizieren zu können. Dabei wurde festgestellt, daß ein früher als S. Typhimurium 14028s WT bezeichneter Stamm eine Insertion eines Desoxythymidins an Position 76 des offenen Leserasters von rfbP trägt, einem Gen, dessen Protein an der LPS-Synthese beteiligt ist. Weiterhin synthetisierte dieser Stamm ein rauhes LPS. Aufgrund von Agglutination konnte der Rauh-Stamm nur mit geringem Erfolg mikroinjiziert werden. Hingegen lag 5 h nach der Mikroinjektion einer nicht invasiven Mutante von Salmonella mit vollständigem LPS der Anteil an Caco-2-Zellen, die mehr als 32 Bakterien enthielten, bei etwa 30 %. Der Anteil war 2-3 mal höher als bei früheren Mikroinjektionen in HeLa-Zellen. Daher wurde das Verhalten von HeLa-Zellen nach einer Infektion durch S. Typhimurium ΔsifA - einer Mutante, die aus der SCV ins Zytoplasma entkommt - untersucht. Dabei wurde festgestellt, daß die sifA-Mutante 10 h nach der Infektion die Aktivität der Caspasen 9 und 3 in HeLa-Zellen, aber nicht in Caco-2-Zellen induziert. In weiteren Versuchen wurde die Bedeutung von Glukose, Glukose-6-phosphat und Mannose als Kohlenstoffquellen für die extra- und intrazelluläre Replikation zweier Isolate enteroinvasiver E. coli und eines S. Typhimurium Stammes analysiert. Zu diesem Zweck wurden zunächst definierte Mutanten in den beiden wichtigsten Phosphoenolpyruvat-abhängigen Phosphotransferasesystemen (PTS) für die Aufnahme von Glukose und Mannose, ptsG und manXYZ, sowie im Antiporter für die Aufnahme von Glukose-6-phosphat, uhpT, konstruiert. Bei Wachstum im Minimalmedium mit Glukose als einziger C-Quelle waren die Generationszeiten aller ΔptsG- und ΔptsG, manXYZ-Mutanten im Vergleich zu den Wildstämmen deutlich verlängert. Ebenso wuchsen ΔmanXYZ-Mutanten bzw. ΔuhpT-Mutanten deutlich langsamer auf Mannose bzw. Glukose-6-phosphat. Jedoch ergaben sich hierbei Stamm-spezifische Unterschiede. So erreichte EIEC 4608-58 ΔuhpT in der stationären Phase eine ähnliche Zelldichte wie der Wildstamm in Gegenwart von Glukose-6-phosphat und eine ΔptsG, manXYZ-Mutante von S. Typhimurium 14028s konnte immer noch effizient mit Glukose wachsen. Infektionsversuche mit Caco-2-Zellen zeigten weiterhin, daß die Deletion von ptsG zu einer signifikanten Erhöhung der Adhärenz und Invasivität von EIEC 4608-58 führt, während sich die intrazellulären Generationszeiten aller hier untersuchten Mutanten kaum veränderten. Selbst die ΔptsG, manXYZ, uhpT-Dreifachmutanten der drei hier verwendeten Enterobakterien und die ΔptsG, manXYZ, glk-Mutante von S. Typhimurium 14028s konnten immer noch in Caco-2-Zellen replizieren, wenn auch mit Stamm-spezifisch verringerten Geschwindigkeiten. 13C-Markierungsexperimente mit [U-13C6]-Glukose als Substrat ergaben jedoch, daß in der Tat alle hier untersuchten enterobakteriellen Wildstämme Glukose während der Replikation in Caco-2-Zellen unter Zellkulturbedingungen verwerten. Glukose-6-phosphat, Glukonat oder Fettsäuren konnten dagegen als wichtigste Kohlenstoffquellen für das intrazelluläre Wachstum ausgeschlossen werden. EIEC 4608-58 metabolisierte Glukose jedoch weniger effizient als EIEC HN280 und schien zudem noch zusätzlich C3-Substrate aus der Wirtszelle aufzunehmen. Das Markierungsmuster zeigte einen Stamm-spezifischen Kohlenstofffluß durch Glykolyse und/oder Entner-Doudoroff-Weg, Pentosephosphatzyklus, Citratzyklus und den anaplerotischen Reaktionen zwischen PEP und Oxalacetat. Mutanten mit Deletionen in ptsG und manXYZ konnten auf alternative C3-Substrate wechseln und glichen dies durch eine erhöhte Aufnahme von Aminosäuren aus den Wirtszellen aus.
Development of novel Listeria monocytogenes strains as therapeutic agents for targeted tumor therapy
(2010)
Despite marked progress in development and improvement of cancer therapies the rate of cancer related death remained stable over the last years. Especially in treating metastases alternative approaches supporting current therapies are required. Bacterial and viral vectors have been advanced from crude tools into highly sophisticated therapeutic agents detecting and treating neoplastic leasions. They might be potent enough to fill in this therapeutic demand. In this thesis Listeria monocytogenes was investigated as carrier for targeted bacterial cancer therapy. One part of the study focussed on modification of a functional bacterial mRNA delivery system. Genomic integration of T7 RNA polymerase driving mRNA production allowed reduction to an one-plasmid-system and thereby partially relieved the growth retardation exerted by mRNA delivery. Importantly the integration allowed metabolic attenuation of the mRNA delivery mutant potentially enabling in vivo applications. Further expansion of the bacterial RNA delivery system for transfer of shRNAs was examined. Bacterial mutants producing high amounts of RNA containing shRNA sequences were constructed, however a functional proof of gene silencing on delivery in eukaryotic cell lines was not achieved. The second part of this thesis focussed on increasing tumor colonization by Listeria monocytogenes in vivo. Coating bacteria with antibodies against receptors overexpressed on distinct tumor cell lines enabled specific bacterial internalization into these cells in vitro. Optimization of the bacterial antibody coating process resulted in an up to 104-fold increase of intracellular bacteria. Combination of this antibody-mediated targeting with the delivery of prodrug-converting enzymes showed a cytotoxic effect in cell lines treated with the corresponding prodrug. Since incubation in murine serum completely abrogated antibodymediated bacterial internalization the antibodies were covalently linked to the bacteria for application in xenografted tumor mice. Bacteria coated and crosslinked in this manner showed enhanced tumor targeting in a murine tumor model demonstrating antibodymediated bacterial tumor targeting in vivo. Independent of antibody-mediated tumor targeting the intrinsic tumor colonization of different Listeria monocytogenes mutants was examined. Listeria monocytogenes ΔaroA ΔinlGHE colonized murine melanoma xenografts highly efficient, reaching up to 108 CFU per gram of tumor mass 7 days post infection. Taken together the presented data shows highly promising aspects for potential bacterial application in future tumor therapies. Combination of the delivery systems with antibodymediated- and intrinsic bacterial tumor targeting might open novel dimensions utilizing Listeria monocytogenes as therapeutic vector in targeted tumor therapy.
iNKT cells are a population of T cells with unique characteristics. In contrast to most αβ T cells which recognize peptides presented by highly polymorphic MHC molecules, iNKT cells are reactive to glycolipids presented by CD1d, a non-polymorphic MHC-I like molecule. Moreover, whereas MHC-restricted αβ T cells bear highly variable receptors (TCRs) formed after somatic recombination of the V(D)J gene segments, the TCR of iNKT cells is formed by an invariant α chain, which always contains the same gene segments: AV14 and AJ18; and a β chain of limited BV gene usage: BV8S2, BV7 or BV2, in the mouse. This invariant α chain is the reason for which these cells are named “i” and the NK part of their name refers to the expression of receptors typical of natural killer (NK) cells. iNKT cells recognize glycolipids of endogenous and microbial origin. After activation they secrete large amounts of very different cytokines such as IFN-γ and IL-4 and thus influence immune responses and pathological conditions. One of the most potent iNKT cell agonists, recognized by the semi-invariant TCR, is the synthetic glycolipid α-Galactosylceramide (α-Gal). iNKT cells can be visualized using CD1d-multimeric complexes loaded with α-Gal and flow cytometry, since this reagent has enough avidity to stain these cells. Interestingly, mouse iNKT cells can be stained with human α-Gal-loaded CD1d oligomers and human iNKT cells can also be visualized with mouse α-Gal-loaded CD1d oligomers, indicating a high degree of conservation of the recognition of α-Gal presented by CD1d through evolution. Previous studies showed that rats have the genes necessary to build semi-invariant TCRs: They have a CD1d homologue; one or two BV8S2 homologues and interestingly, up to ten AV14 gene segments, which are highly conserved when compared to the mouse genes. Importantly, it has been shown at least for two of these AV14 gene segments that they can produce invariant TCRα chains which, when coexpressed with BV8-containing β chains, react to α-Gal presented by rat CD1d. Furthermore, ex vivo stimulation of primary splenocytes with α-Gal results in the secretion of IL-4 and IFN-γ. Surprisingly, rat semi-invariant TCRs do not recognize α-Gal presented by mouse CD1d and accordingly, mouse α-Gal-loaded CD1d tetramers failed to stain a discrete population of rat iNKT cells. Taking all together, despite that strong evidence suggested that iNKT cells are present in the rat, the direct identification of such population and the analysis of CD1d-restricted immune responses were still pending for this species. Hence the work presented in this doctoral thesis was aimed to identify iNKT cells, to analyze their phenotype and also to study the distribution and function of CD1d in the rat. For these purposes, we produced essential reagents which were still lacking such as rat specific anti-CD1d monoclonal antibodies and rat CD1d oligomers. Importantly, two of three anti-rat CD1d monoclonal antibodies (all of them generated in our laboratory before this thesis was initiated) also recognized mouse CD1d and therefore allowed a direct comparison of CD1d expression between rat and mouse. Whereas CD1d distribution in the hematopoietic system was found to be extremely similar between these two species; in non-lymphatic tissues important differences were observed. Interestingly, CD1d protein was detected at not yet described sites such as the rat exocrine pancreas and rat and mouse Paneth cells. These monoclonal antibodies did not only allowed the analysis of CD1d expression, but also the first demonstration of the function of rat CD1d as an antigen presenting molecule, since cytokine release in response to α-Gal was blocked when they were added to ex vivo cultures of rat primary cells. Staining of primary rat iNKT cells (possible now with the newly generated rat CD1d oligomers) revealed interesting similarities with human iNKT cells. First, we observed that rat iNKT cells are only a minority among all NKR-P1A/B positive T cells. Human iNKT cells constitute also a very small proportion of NKR-P1A (CD161) expressing T cells, whereas in mice inbred strains which express NKR-P1C (NK1.1), most of NKRP1C expressing T cells are iNKT cells. Second, the majority of rat iNKT cells are either CD4 or DN and only a small proportion expresses CD8β. These findings are similar to humans and different to mice which lack CD8+ iNKT cells. Third, analysis of various inbred rat strains demonstrated different iNKT cell frequencies which correlated with cytokine secretion after α-Gal stimulation of primary cells. In comparison to mice, iNKT cell numbers are markedly reduced in rats. In F344 rats, inbred rat strain which released the highest cytokine amounts after α-Gal stimulation, approximately 0.25% and 0.1% of total liver and spleen lymphocytes, respectively, are iNKT cells. In contrast, in LEW rats iNKT cells were practically absent and neither IL-4 nor IFN-γ were detected after stimulation of primary cells with α-Gal. Once more, these frequencies are very close to those observed in humans. Last, as reported for human peripheral blood cells, rat iNKT cells could be easily expanded in vitro by adding α-Gal to cultures of intrahepatic lymphocytes, whereas the expansion of mouse iNKT cells was not possible using the same protocol. The presence of a multimember AV14 gene segment family in the rat is an intriguing characteristic. These AV14 gene segments are extremely homologous except in the CDR2α region. Based on the amino acid sequence of this region they have been divided into two different types: Type I and II. A specific tissue distribution of the different types was proposed in the first study where the presence of several AV14 gene segments was described. We also analyzed the AV14 gene segment usage in F344 and LEW inbred rat strains. In F344 rats we found no preferential usage of either AV14 gene segment type in the spleen and the liver but type II AV14 gene segments appeared more frequently in the thymus. In contrast, LEW rats show a preferential usage of type I AV14 gene segments in all three compartments analyzed: Thymus, spleen and liver. Taken all together, the usage of newly generated reagents allowed to gain novel insights into CD1d expression in the rat and in the mouse and to directly identify rat iNKT cells for the first time. The phenotypic and functional analysis of rat iNKT cells revealed numerous similarities with human iNKT cells. These are of special interest, since rats serve to investigate several pathological conditions including models for autoimmune diseases. The possibility now to analyze iNKT cells and CD1d-restricted T cell responses in the rat might help to understand the pathogenesis of such diseases. In addition, the uncomplicated in vitro expansion and culture of rat iNKT cells should facilitate the analysis of the immunomoldulatory capacities of these cells.
In dieser Arbeit wurde das PVM-Mausmodell verwendet, um die Bedeutung der Typ I und Typ III Interferonantwort für die Pathogenese einer pneumoviralen Infektion zu analysieren. Hierzu wurden zunächst mit Hilfe der reversen Genetik rekombinante PVM-Mutanten hergestellt, bei denen die Gene für die NS-Proteine, welche vermutlich als Interferonantagonisten fungieren, deletiert sind. Die Charakterisierung der Replikationsfähigkeit der rPVM dNS-Mutanten erfolgte in vitro in Interferon-kompetenten und Interferon-inkompetenten Zelllinien. Ein zentraler Schritt innerhalb dieser Charakterisierung war die Untersuchung der Induktion von Interferonen in vivo und in vitro nach Infektion mit den rPVM dNSMutanten, wobei nachgewiesen wurde, dass die NS-Proteine von PVM als Interferonantagonisten fungieren. In allen Interferon-kompetenten Zellkulturen wurde eine Attenuierung von rPVM dNS1, rPVM dNS2 und rPVM dNS1dNS2 bezogen auf rPVM beobachtet. In allen Interferon-inkompetenten Zellkulturen konnte die Attenuierung der rPVM dNS-Mutanten nahezu vollständig revertiert werden. Nach Infektion mit den rPVM dNS-Mutanten wurde in verschiedenen Zelllinien eine Induktion von Typ I und Typ III Interferonen betrachtet, wobei Unterschiede in der Stärke der Interferon-Induktion nach Infektion mit den rPVM dNS-Mutanten vorhanden waren. Zusammenfassend war es möglich, die NS1- und NS2-Proteine von PVM in Analogie zu RSV eindeutig als Antagonisten der Interferonantwort zu identifizieren. Die Untersuchung der protektiven Rolle von Typ I und Typ III Interferonen für die Replikation und Pathogenität von PVM bildete den zweiten Teil dieser Arbeit. Hierzu wurde die Replikationsfähigkeit und Pathogenität der rPVM dNS-Mutanten in verschiedenen Interferon-defizienten Mausstämmen getestet. Die Untersuchungen ergaben eine protektive Rolle von Typ I und Typ III Interferonen bei einer Infektion mit PVM, wobei den Typ I Interferonen ein effektiverer Einfluss zugeordnet werden konnte. Ein Vergleich von Replikation und Virulenz zwischen den verschiedenen Typ I oder Typ III oder Typ I/Typ III Interferonrezeptor-defizienten Mausstämmen belegte eine erhöhte Suszeptibilität der Typ I/Typ III Interferonrezeptor-defizienten Mäuse gegenüber einer Infektion mit den rPVM dNS-Mutanten. Eine vollständige Aufhebung der Attenuierung wurde auch in den Typ I/Typ III Interferonrezeptor-defizienten Mäusen nicht erlangt. Eine anti-apoptotische Funktion der NS-Proteine zusätzlich zu ihrer Wirkungsweise als Interferonantagonisten wurde aufgrund der unvollständigen Revertierung der Pathogenität der rPVM dNS-Mutanten in Typ I/Typ III Interferonrezeptor-defizienten Mäusen vermutet. Der abschließende Teil dieser Dissertation befasste sich mit der Frage, welche Zellen bei einer natürlichen pulmonalen Infektion Interferone in vivo produzieren. In vitro wurde beobachtet, dass überraschenderweise nur sehr wenige virusinfizierte oder uninfizierte Zellen Typ I Interferone bilden. Der Nachweis darüber, welche Zellen während einer pulmonalen Infektion hauptsächlich Interferone in vivo produzieren, war aufgrund der fehlenden Eignung der kommerziell erhältlichen Interferon-Antikörper für intrazelluläre Gewebefärbungen nicht möglich. Dennoch gelang es abschließend durch eine neue Nachweismethode erstmals Zellen mit rezeptorgebundenen Interferon zu identifizieren, wobei es sich um ziliierte Epithelzellen, Alveolarmakrophagen und vermutlich Clarazellen sowie Typ I und Typ II Pneumozyten handelte.
Cooperation is beneficial for social groups and is exemplified in its most sophisticated form in social insects. In particular, eusocial Hymenoptera, like ants and honey bees, exhibit a level of cooperation only rarely matched by other animals. To assure effective defense of group members, foes need to be recognized reliably. Ants use low-volatile, colony-specific profiles of cuticular hydrocarbons (colony odor) to discriminate colony members (nestmates) from foreign workers (non-nestmates). For colony recognition, it is assumed that multi-component colony odors are compared to a neuronal template, located in a so far unidentified part of the nervous system, where a mismatch results in aggression. Alternatively, a sensory filter in the periphery of the nervous system has been suggested to act as a template, causing specific anosmia to nestmate colony odor due to sensory adaptation and effectively blocking perception of nestmates. Colony odors are not stable, but change over time due to environmental influences. To adjust for this, the recognition system has to be constantly updated (template reformation). In this thesis, I provide evidence that template reformation can be induced artificially, by modifying the sensory experience of carpenter ants (Camponotus floridanus; Chapter 1). The results of the experiments showed that template reformation is a relatively slow process taking several hours and this contradicts the adaptation-based sensory filter hypothesis. This finding is supported by first in-vivo measurements describing the neuronal processes underlying template reformation (Chapter 5). Neurophysiological measurements were impeded at the beginning of this study by the lack of adequate technical means to present colony odors. In a behavioral assay, I showed that tactile interaction is not necessary for colony recognition, although colony odors are of very low volatility (Chapter 2). I developed a novel stimulation technique (dummy-delivered stimulation) and tested its suitability for neurophysiological experiments (Chapter 3). My experiments showed that dummy-delivered stimulation is especially advantageous for presentation of low-volatile odors. Colony odor concentration in headspace was further increased by moderately heating the dummies, and this allowed me to measure neuronal correlates of colony odors in the peripheral and the central nervous system using electroantennography and calcium imaging, respectively (Chapter 4). Nestmate and non-nestmate colony odor elicited strong neuronal responses in olfactory receptor neurons of the antenna and in the functional units of the first olfactory neuropile of the ant brain, the glomeruli of the antennal lobe (AL). My results show that ants are not anosmic to nestmate colony odor and this clearly invalidates the previously suggested sensory filter hypothesis. Advanced two-photon microscopy allowed me to investigate the neuronal representation of colony odors in different neuroanatomical compartments of the AL (Chapter 5). Although neuronal activity was distributed inhomogeneously, I did not find exclusive representation restricted to a single AL compartment. This result indicates that information about colony odors is processed in parallel, using the computational power of the whole AL network. In the AL, the patterns of glomerular activity (spatial activity patterns) were variable, even in response to repeated stimulation with the same colony odor (Chapter 4&5). This finding is surprising, as earlier studies indicated that spatial activity patterns in the AL reflect how an odor is perceived by an animal (odor quality). Under natural conditions, multi-component odors constitute varying and fluctuating stimuli, and most probably animals are generally faced with the problem that these elicit variable neuronal responses. Two-photon microscopy revealed that variability was higher in response to nestmate than to non-nestmate colony odor (Chapter 5), possibly reflecting plasticity of the AL network, which allows template reformation. Due to their high variability, spatial activity patterns in response to different colony odors were not sufficiently distinct to allow attribution of odor qualities like ‘friend’ or ‘foe’. This finding challenges our current notion of how odor quality of complex, multi-component odors is coded. Additional neuronal parameters, e.g. precise timing of neuronal activity, are most likely necessary to allow discrimination. The lower variability of activity patterns elicited by non-nestmate compared to nestmate colony odor might facilitate recognition of non-nestmates at the next level of the olfactory pathway. My research efforts made the colony recognition system accessible for direct neurophysiological investigations. My results show that ants can perceive their own nestmates. The neuronal representation of colony odors is distributed across AL compartments, indicating parallel processing. Surprisingly, the spatial activity patterns in response to colony are highly variable, raising the question how odor quality is coded in this system. The experimental advance presented in this thesis will be useful to gain further insights into how social insects discriminate friends and foes. Furthermore, my work will be beneficial for the research field of insect olfaction as colony recognition in social insects is an excellent model system to study the coding of odor quality and long-term memory mechanisms underlying recognition of complex, multi-component odors.
The saprophytic filamentous fungus Aspergillus fumigatus has been gaining importance as an opportunistic human pathogen over the past decades. Advances in modern medicine have created a growing group of patients susceptible to infection with A. fumigatus, often contracting potentially deadly invasive aspergillosis. The virulence of this pathogen appears to be a multifactorial trait, a combination of physiological characteristics that enables the fungus to infect immunocompromised humans. This work concentrates on the nitrogen metabolism of A. fumigatus, which is essential for meeting the nutritional needs inside the human host. Using DNA microarrays, the transcriptional response during growth on three different secondary nitrogen sources was examined, which revealed the metabolic versatility of A. fumigatus, especially when challenged with proteins as the sole source of nitrogen. In-depth transcriptional profiling of the eight-member oligopeptide transporter (OPT) gene family underlined the importance of oligopeptide transport for growth on complex nitrogen sources like BSA or collagen. Heterologous expression of the opt genes in Saccharomyces cerevisiae showed their functionality as oligopeptide transporters, and characterized their substrate specificity. Using a Cre/loxP based genetic tool, a complete deletion of all opt genes in A. fumigatus was achieved. The resultant strain exhibited diminished growth on medium where the oligopeptide GPGG was the sole nitrogen source, but did not show any other in vitro phenotype. The opt deletion strain was not attenuated in virulence in a murine model of pulmonary aspergillosis, suggesting that the OPT gene family is not necessary for successful infection. The connection of oligopeptide transport and extracellular proteolytic activity was investigated by deleting the genes encoding Dpp4 and Dpp5, two dipeptidyl peptidases, or PrtT, the transcriptional regulator of major secreted proteases, in the complete opt deletion background. In contrast to the deletion of dpp4 and dpp5, which did not result in any additional phenotype, the absence of prtT led to a drastic growth defect on porcine lung agar. This suggests a synergistic action of extracellular proteolytic digest of proteins and transport of oligopeptide degradation products into the cell. Finally, this work established the bacterial β-Rec/six site-specific recombination system as a novel genetic tool for targeted gene deletion in A. fumigatus.
Reactive oxygen species (ROS) are continuously generated in cells and are involved in physiological processes including signal transduction but also their damaging effects on biological molecules have been well described. A number of reports in the literature implicate excessive oxidative stress and/or inadequate antioxidant defense in the pathogenesis of cancer, atherosclerosis, chronic and age related disorders. Several studies have indicated that activation of the renin-angiotensin-aldosterone-system can lead to the formation of ROS. Epidemiological studies have revealed higher renal cell cancer incidences and also higher cancer mortalities in hypertensive individuals. Recently, our group has shown that perfusion of the isolated mouse kidney with Ang II or treatment of several cell lines with Ang II leads to formation of DNA damage and oxidative base modifications. Here, we tried to scrutinize the pathway involved in genotoxicity of Ang II. We confirmed the genotoxicity of Ang II in two kidney cell lines of human origin. Ang II treatment led to the production of superoxide anions which we could hinder when we used the membrane permeable superoxide dismutase (SOD) mimetic TEMPOL. One of the enzymes which is activated in the cells after Ang II treatment and is able to produce ROS is NADPH oxidase. We demonstrated the activation of NADPH oxidase in response to Ang II by upregulation of its p47 subunit using RT-PCR. Also, pPhosphorylation of p47 subunit of NADPH oxidase after Ang II treatment was enhanced. Using two inhibitors we showed that NADPH oxidase inhibition completely prevents DNA damage by Ang II treatment. To differentiate between Nox2 and Nox4 isoforms of NADPH oxidase subunits in the genotoxicity of Ang II, we performed siRNA inhibition and found a role only for Nox4, while Nox2 was not involved. Next, we investigated PKC as a potential activator of NADPH oxidase. We showed that PKC becomes phosphorylated after Ang II treatment and also that inhibition of PKC hinders Ang II from damaging the cells. Our results from using several inhibitors of different parts of the pathway revealed that PKC activation in this pathway is dependent on the action of PLC on membrane phospholipids and production of IP3. IP3 binds to its receptor at endoplasmic reticulum (ER), opening a channel which allows calcium efflux into the cytoplasm. In this manner, both ER calcium stores and extracellular calcium cooperate so that Ang II can exert its genotoxic effect. PLC is activated by AT1R stimulation. We could also show that the genotoxicity of Ang II is mediated via AT1R signaling using the AT1R antagonist candesartan. In conclusion, here we have shown that Ang II is able to damage genomic damage in cell lines of kidney origin. The observed damage is associated with production of ROS. A decrease in Ang II-induced DNA damage was observed after inhibition of G-proteins, PLC, PKC and NADPH oxidase and interfering with intra- as well as extracellular calcium signaling. This leads to the following preliminary model of signaling in Ang II-induced DNA damage: binding of Ang II to the AT1 receptor activates PLC via stimulation of G-proteins, resulting in the activation of PKC in a calcium dependent manner which in turn, activates NADPH oxidase. NADPH oxidase with involvement of its Nox4 subunit then produces reactive oxygen species which cause DNA damage. Dopamine content and metabolism in the peripheral lymphocytes of PD patients are influenced by L-Dopa administration. The PD patients receiving a high dose of L-Dopa show a significantly higher content of dopamine in their lymphocytes compared to PD patients who received a low dose of L-Dopa or the healthy control. Central to many of the processes involved in oxidative stress and oxidative damage in PD are the actions of monoamine oxidase (MAO), the enzyme which is responsible for the enzymatic oxidation of dopamine which leadsing to production of H2O2 as a by-product. We investigated whether dopamine oxidation can cause genotoxicity in lymphocytes of PD patents who were under high dose L-Dopa therapy and afterward questioned the occurrence of DNA damage after dopamine treatment in vitro and tried to reveal the mechanism by which dopamine exerts its genotoxic effect. The frequency of micronuclei in peripheral blood lymphocytes of the PD patients was not elevated compared to healthy age-matched individuals, although the formation of micronuclei revealed a positive correlation with the daily dose of L-Dopa administration in patients who received L-Dopa therapy together with dopamine receptor agonists. In vitro, we describe an induction of genomic damage detected as micronucleus formation by low micromolar concentrations in cell lines with of different tissue origins. The genotoxic effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea which proved the involvement of ROS production in dopamine-induced DNA damage. To determine whether oxidation of dopamine by MAO is relevant in its genotoxicity, we inhibited MAO with two inhibitors, trans-2-phenylcyclopropylamine hydrochloride (PCPA) and Ro 16-6491 which both reduced the formation of micronuclei in PC-12 cells. We also studied the role of the dopamine transporter (DAT) and dopamine type 2 receptor (D2R) signaling in the genotoxicity of dopamine. Inhibitors of the DAT, GBR-12909 and nomifensine, hindered dopamine-induced genotoxicity. These results were confirmed by treatment of MDCK and MDCK-DAT cells, the latter containing the human DAT gene, with dopamine. Only MDCK-DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of D2R with quinpirole in the absence of dopamine did not induce genotoxicity in PC-12 cells, interference with D2R signaling using D2R antagonist and inhibition of G-proteins, phosphoinositide 3 kinase and extracellular signal-regulated kinases reduced dopamine-induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D2R antagonist sulpiride inhibited the dopamine-induced migration of DAT from cytosol to cell membrane. Overall, the neurotransmitter dopamine causes DNA damage and oxidative stress in vitro. There are also indications that high dose L-Dopa therapy might lead to oxidative stress. Dopamine exerts its genotoxicity in vitro upon transport into the cells and oxidization oxidation by MAO. Transport of dopamine by DAT has the central role in this process. D2R signaling is involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake. We provided evidences for receptor-mediated genotoxicity of two compounds with different mechanism of actions. The involvement of these receptors in many human complications urges more investigations to reveal whether abnormalities in the endogenous compounds-mediated signaling can play a role in the initiation of new conditions like carcinogenesis.
The genus Borrelia belongs to the Spirochaetes phylum which is far related to Gram negative bacteria. This phylum possesses a characteristic long helically coiled shape with lengths that vary from 5 to 250 μm. Other pathogens as Treponema and Leptospira which cause syphilis and leptospirosis, also belong to the Spirochaetes. Borrelia itself is the causative agent of two human diseases, the Lyme disease and relapsing fever. Borreliae are pathogenic bacteria which cycle between their arthropod vector, in most cases a tick, and a mammal host, very often small rodents. This complex life cycle requires an extraordinary protein up- and down-regulation in order to survive in such different organisms and avoid their immunologic systems. Lyme disease is a multisystemic disease that can affect different organs like skin, joints and nervous system. A red rash with concentric rings, called erythema migrans is a distinctive manifestation that allows clinical diagnosis. It appears after the bite of an infected tick and spreads out to diameters that can reach 15 cm. Relapsing fever is characterized by sudden recurrent fever peaks accompanied with chills, headache, muscle and joint pain and nausea. Both diseases are easily treated with antibiotics in early infection stages. Borrelia species possess a small genome. Many of their genes are related with virulence and the adaptation to the different hosts. The absence of genes in Borrelia involved in the biosynthesis of amino acids, fatty acids or nucleotide is very remarkable. This metabolic deficiency makes Borrelia species dependent on substances produced by the host. The first step in nutrient uptake is accomplished by porins. Bacterial porins are water-filled channels that facilitate the transport of essential molecules through the outer membrane. Four porins have been described in Borrelia up to this point. P66, P13 and Oms28 have been found in Borrelia burgdorferi while Oms38 was discovered in relapsing fever spirochetes. P66 is a singular porin with an extremely high single channel conductance of 11 nS. P13 is a small protein with an α-helical secondary structure which does not fit into the general porin model. The function of Oms28 as a porin has been questioned recently due to its periplasmic membrane-associated location. Finally, Oms38 is a specific porin for dicarboxilates with homologues in Lyme disease species. The aim of this thesis was to broaden the knowledge of the P66 and P13 porins described in the genus Borrelia. Both differ in structure and size from the general Gram negative porin model and could be highly involved in specific tasks in the genus Borrelia. In the first project of this thesis, the presence and pore forming capacity of P66 was studied in several Borrelia species including members of the relapsing fever group. P66 is the best studied porin in Borrelia with a dual function as porin and adhesin. This knowledge is restricted to B. burgdorferi and little or nothing is known about homologues in other Borrelia species. Therefore, three Lyme disease and three relapsing fever species were chosen as representative agents of the genus and the pore forming activity of their P66 homologues was studied. Five out of the six homologues exhibited a similar single channel conductance in a range from 9 to 11 nS. All of them showed no selectivity for cations or anions, and they were voltage dependent starting at different voltages from 30 to 70 mV. Only in the case of the B. hermsii homologue no pore forming activity could be established. It remains unclear if the lack of activity was due to an evolutionary loss of its porin function or to a higher sensibility to the detergents used for purification. In another project, the controversial P66 pore diameter of B. burgdorferi was analyzed with an empirical method. In a former study, the diameter of the P66 channel was estimated to be 2.6 nm based on theoretical considerations. This diameter is rather large and could impair the outer membrane protective function. Different non-electrolytes were used to study the P66 pore diameter indicating a 1.8 nm entrance diameter and a 0.8 nm inner constriction. In addition, the blockage of the channel with some of those non-electrolytes disclosed an oligomeric organization formed by approximately eight independent channels. Such a structure has not been observed so far in any other living organism and could be exclusive of Borrelia or spirochetes. The third project of this thesis deal with the recombinant production of a B. burgdorferi protein with immunogenic potential. This protein might be used to develop new diagnosis tests and therapeutic treatments. P13 is an outer membrane protein present in LD and RF species and it does not have any other known bacterial homologue. These facts make of P13 a good candidate to be used as a therapeutic target. For such purpose, P13 was cloned in two organisms. First, in Escherichia coli were two different constructs were designed to establish the role of a periplasmic cleaved C-terminus. Second, in a virus based vector delivered by Agrobacterium tumefaciens into tobacco plant cells. The vector replicates inside the plant cells spreading the infection to adjacent cells and at the same time producing the recombinant protein. This second expression method should enable the production of large amounts of the recombinant protein reducing time and costs. The last project of this thesis looked into the outer membrane complexome of B. burgdorferi focusing on the P13 and P66 porin complexes. Blue Native Page and second dimension SDS Page were the technique chosen for this purpose. P66 could be shown to be the only protein involved in the formation of the 11 nS pore which complex is probably formed by eight monomers. It was also possible to divide this complex in two halves with approximately half the molecular weight and a conductance of 5.5 nS. In the case of the P13 complex, a possible association with the lipoprotein OspC was revealed. The gel extraction of the P13 complex and its test with the Back Lipid Bilayer assay exhibited a 0.6 nS activity. This is in high contrast with the 3.5 nS activity previously described for this protein. To sum up, P66 is a porin present in many Borrelia species including not only LD but also RF species and which homologues show similar biophysical properties. The diameter of this pore is smaller than previously thought and it has molecular weight sieving properties. In the case of P13, its recombinant procurement will allow the use of P13 as a diagnostic and therapeutic target. The possible association with OspC could facilitate to unravel in future experiments the function of this intriguing protein.
Semaphorin receptors in the immunological synapse: regulation and measles virus-driven modulation
(2010)
Measles virus (MV) infection causes approximately 164,000 deaths per year worldwide (WHO, 2008). The main cause of death is MV-induced immunosuppression but the underlying mechanisms are not fully understood. It has been suggested that MV renders T cells dysfunctional by disrupting the integrity of actin dynamics while MV infection of dendritic cells results in their inability to sustain T cell activation. During neuronal development, semaphorins (SEMAs), especially SEMA3A, induce a collapse of growing dendrites via the binding to plexin-A1 (plexA1) and its coreceptor neuropilin-1 (NP-1). The collapse results from a disruption of actin dynamics. In this study, the roles of these three molecules were investigated in human immune cells and their possible role in MV induced immunosuppression. The present data have shown that plexA1 is an important component of human immunological synapse (IS). It translocated transiently to the surface of T cells after CD3/28 ligation and accumulated at the stimulatory interface between T cells and DCs (or CD3/28 coated beads). When plexA1 expression was inhibited (RNAi) or its function was disrupted (exogenous blocking or dominant negative expression), T cell expansion was reduced. Upon MV exposure, translocation of plexA1 and NP-1, another important component of IS, towards the stimulatory interface in T cells was abrogated. Moreover, MV infection interfered with plexA1/NP-1 turnover in maturing DCs and promoted early and substantial release of SEMA3A from these cells, particularly in the presence of allogenic T cells. As revealed by scanning electron microscopy, the release of SEMA3A caused a transient loss of actin-based protrusions on T cells. SEMA3A affected chemotactic migration of T cells and DCs, and reduced formation of allogenic DC/T cell conjugates. In conclusion, MV targeted SEMA receptor function both by disrupting their recruitment to the IS and by promoting a premature release of their repulsive ligand, SEMA3A. Both of which could contribute to MV-induced immunosuppression.
Stem cells with the particular potential to self renew and to differentiate into multiple cell lineages are fascinating cell types for basic and applied research. Pluripotent embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation embryos. Upon differentiation ES cells can give rise to cells of ecto-, meso- and endoderm including germ cells. In contrast, multipotent adult stem cells are more restricted in their differentiation outcomes,they differentiate into cells of their tissue of origin. For example, hematopoietic stem cells (HSCs) that reside in hemogenic tissues such as the bone marrow (BM) differentiate into hemato-/lymphoid cell lineages. Upon differentiation of stem cells not the genome, but the epigenetic regulation changes. Differentiation-associated epigenetic changes generate cell types with distinct phenotypes and functions. For stem cell-based therapies it is important to deeper understand the relation between epigenome and cellular function. In the scope of this thesis I aimed to analyze cultures of differentiating stem cells with respect to gene expression, chromatin regulation and differentiation potential. For the analysis of global histone modification levels, which represent one mechanism for epigenetic regulation, fow cytometric protocols were established that allow single cell measurements. By applying this methodology decreased histone acetylation levels were shown in differentiated ES cell populations. In contrast, comparable histone acetylation levels were observed in differentiated and undifferentiated BM cells. In addition, I investigated effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on murine BM cells, comprising also HSCs. Upon TSA treatment the frequency of cells with in vitro and in vivo hematopoietic activity was increased, while lineage committed cells underwent apoptosis. Next, the loss of pluripotency was assessed in differentiating ES cell cultures. Using short-term in vitro differentiation protocols marker-based analyses and functional assays were performed.Functionally pluripotency was diminished after 2 days of differentiation as assessed by colony formation, embryoid body (EB) formation and cardiomyogenic differentiation approaches. In contrast, pluripotency marker expression was reduced at later time points. Further, the application of distinct differentiation systems (aggregation EB, clonal EB or monolayer (ML) culture) had an impact on the progression and homogeneity of differentiation cultures. To further study the end of pluripotency, differentiated ES cells were placed under ES cell culture conditions. The data suggest that 3 days differentiated ES cells had passed a point of no return and failed to regain Oct4-eGFP expression and that HDAC inhibitor treatment selectively killed differentiated ES cells. Finally, I aimed to study the effect of EED - a core subunit of the histone methylating Polycomb repressive complex 2 (PRC2) - on ES cell chromatin and function. ES cells lacking EED showed loss of histone H3 lysine 27 trimethylation (H3K27me3) accompanied by increased histone acetylation and reduced H3K9me3 levels. Despite typical ES cell morphology and pluripotency marker expression, EED knockout (KO) ES cells exhibited altered nuclear heterochromatin organization, delayed chromatin mobility and a failure in proper differentiation. Conclusively, my data provide insights into the epigenetic regulation of stem cells. Particularly, the results suggest that HDAC inhibitor treatment was detrimental for differentiated BM as well as for differentiated ES cells and that ES cells after 3 days of differentiation had lost pluripotency. Further, the data demonstrate that EED KO ES cells self renewed, exhibited morphology and pluripotency marker expression similar to wild type ES cells, but failed to differentiate. This indicates an important role of EED not only for undifferentiated but also for differentiating ES cells.