Refine
Has Fulltext
- yes (17)
Is part of the Bibliography
- yes (17)
Year of publication
- 2010 (17) (remove)
Document Type
- Doctoral Thesis (17) (remove)
Language
- English (17) (remove)
Keywords
- Dendritische Zelle (2)
- Geruch (2)
- Neurobiologie (2)
- T-Lymphozyt (2)
- social insects (2)
- ADHD (1)
- Acetylcholinrezeptor (1)
- Actinomyceten (1)
- Actinomycetes (1)
- Algorithmus (1)
Institute
- Graduate School of Life Sciences (17) (remove)
This thesis is divided into three parts with the main goal allocating novel antimicrobial compounds that could be used as future antibiotics. The first part aimed to evaluate the potential of plant suspension cultures for the production of antimicrobial proteins. The extracellular, intracellular and cell wall bound fractions of seven heterotrophic and photomixotrophic plant cell suspension cultures treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial proteins. Bioactivities were tested against a selected panel of human isolates including Gram-positive and Gram-negative bacteria as well as fungi using the disc diffusion assay. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while the cell wall bound fractions showed lowest activities. Among the 21 fractions tested, the intracellular fraction of Lavendula angustifolia elicited with DC3000 was most active against Candida maltosa. The second most active fraction was the intracellular fraction of Arabidopsis thaliana elicited with salicylic acid which was moreover active against all test strains. The antimicrobial activity of elicited Arabidopsis thaliana cell cultures was tested by bioautography to locate the antimicrobial proteins in the crude extract. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was selected for further gel filtration chromatography on S-200 column leading to the purification of one 19 kDa antimicrobially active protein, designated, AtAMP. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins. The second part comprises the isolation of actinomycetes associated with marine sponges and testing the bioactivities of new species for further investigations. Actinobacterial communities of eleven taxonomically different sponges that had been collected from offshore Ras Mohamed (Egypt) and from Rovinj (Croatia) were investigated by a culture-based approach using different standard media for isolation of actinomycetes and media enriched with aqueous sponge extract to target rare and new actinomycete species. Phylogenetic characterization of 52 representative isolates out of 90 based on almost complete sequences of genes encoding 16S rRNA supported their assignment to 18 different actinomycete genera. Altogether 14 putatively new species were identified based on sequence similarity values below 98.2% to other strains in the NCBI database. The use of M1 agar amended with aqueous sponge extract yielded a putative new genus related to Rubrobacter which highlighting the need for innovative cultivation protocols. Biological activity testing showed that five isolates were active against Gram-positives only, one isolate was active against Candida albicans only and one isolate showed activity against both groups of pathogens. Moreover, the antiparasistic activity was documented for four isolates. These results showed a high diversity of actinomycetes associated with marine sponges as well as highlighted their potential to produce anti-infective agents. The third part of the thesis focused on the isolation and structure elucidation of new bioactive compounds. Streptomyces strain RV15 recovered from sponge Dysidea tupha, was selected for further chemical analysis by virtue of the fact that it exhibited the greatest antimicrobial potential against Staphylococcus aureus as well as Candida albicans among the all tested strains. Moreover, members of the genus Streptomyces are well known as prolific producers of interesting pharmacologically active metabolites. Chemical analysis of the methanolic crude extract using different chromatographic tools yielded four new compounds. The structures of the new compounds were spectroscopically elucidated to be four new cyclic peptides, namely, cyclodysidins A-D. Their bioactivity was tested against different proteases, bacteria and Candida as well as tumor cell lines. The compounds did not show any significant activities at this point.
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.
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.
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.
Integrating neurobiological markers of depression: an fMRI-based pattern classification approach
(2010)
While depressive disorders are, to date, diagnosed based on behavioral symptoms and course of illness, the interest in neurobiological markers of psychiatric disorders has grown substantially in recent years. However, current classification approaches are mainly based on data from a single biomarker, making it difficult to predict diseases such as depression which are characterized by a complex pattern of symptoms. Accordingly, none of the previously investigated single biomarkers has shown sufficient predictive power for practical application. In this work, we therefore propose an algorithm which integrates neuroimaging data associated with multiple, symptom-related neural processes relevant in depression to improve classification accuracy. First, we identified the core-symptoms of depression from standard classification systems. Then, we designed and conducted three experimental paradigms probing psychological processes known to be related to these symptoms using functional Magnetic Resonance Imaging. In order to integrate the resulting 12 high-dimensional biomarkers, we developed a multi-source pattern recognition algorithm based on a combination of Gaussian Process Classifiers and decision trees. Applying this approach to a group of 30 healthy controls and 30 depressive in-patients who were on a variety of medications and displayed varying degrees of symptom-severity allowed for high-accuracy single-subject classification. Specifically, integrating biomarkers yielded an accuracy of 83% while the best of the 12 single biomarkers alone classified a significantly lower number of subjects (72%) correctly. Thus, integrated biomarker-based classification of a heterogeneous, real-life sample resulted in accuracy comparable to the highest ever achieved in previous single biomarker research. Furthermore, investigation of the final prediction model revealed that neural activation during the processing of neutral facial expressions, large rewards, and safety cues is most relevant for over-all classification. We conclude that combining brain activation related to the core-symptoms of depression using the multi-source pattern classification approach developed in this work substantially increases classification accuracy while providing a sparse relational biomarker-model for future prediction.
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.
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.
Resin, a sticky sap emitting terpenoids and other volatiles, is produced by various plant species to seal wounds and protect themselves against herbivores and microbes. Among several other insects, bees have evolved the surprising ability to handle the repellent plant sap and use it to construct and defend their nests. Whereas the collection of pollen and nectar has been intensively studied in bees, resin collection has received only little attention. The aim of this dissertation was to better understand how the physiological and chemical properties of resin and resin-derived compounds (terpenes) affect the ecology of stingless bees. I therefore asked why, where and how stingless bees of Borneo (seven study-species), Australia (eight) and Costa Rica (27) collect and process plant resins, addressing the importance of a largely neglected resource not only for building and defensive properties, but also for the bees’ chemical diversity. Stingless bees are highly opportunistic resin foragers with all species collecting resin from a similar set of tree species. They locate and/or recognize resin sources on the basis of several volatile mono- and sesquiterpenes. I found that different bee species and even colonies significantly varied in the amount of resin collected. Predator attack (e.g., by ants) had the strongest affect on resin intake, whereas manual nest destruction only slightly increased the number of resin foragers. Resin is used to build, maintain and defend nests, but also as source for chemical compounds (terpenes) which stingless bees include in their surface profiles (chemical profiles). They directly transfer resin-derived compounds to their body surfaces (cuticular terpenes), but only include a subset (8 %) of the large number (>> 1000) of terpenes found in tree resins. This phenomenon can only be explained by a hitherto unknown ability to filter environmentally derived compounds which results in species-specific terpene profiles and thus in an increased chemical heterogeneity among species. Moreover, due to the addition of resin-derived substances the diversity of compounds on the bees’ body surfaces by far exceeds the chemical diversity of profiles in other hymenopterans. Because stingless bees filter but do not modify resin-derived compounds, species from Borneo, Australia and Costa Rica all resemble the characteristic resin of typical trees in their regions of origin. This chemical similarity reveals a strong correlation between the diversity of tree resins and the diversity of cuticular terpenes among stingless bees in a given habitat. Because different tree species are found in different tropical regions, the chemical composition of tree resins varies between tropical regions as does the composition of cuticular terpenes in bee species from these regions. Cuticular terpenes are however most common among stingless from Borneo, with 100 % of species studied having resin-derived terpenes in their chemical profiles. They are least common in Costa Rica, with only 40 % of species having terpenes. Likewise, resin collection was found to be highest in Tetragonilla collina colonies of Borneo where occasionally up to 90 % of foragers collected resin. By contrast, resin collection was only performed by 10 % of foragers of a given colony in Australia and by a maximum of 40 % in Costa Rica. The dominance of resin and resin-derived compounds in the chemical ecology of bees from Borneo may mirror the dominance of a particular Southeast Asian tree family: the highly resinous dipterocarps. Such a correlation between the chemistry of bees and the chemistry of tree resins therefore underlines the close relationship between stingless bees and the trees of their habitat. Cuticular terpenes are assumed to protect bees against predators and/or microbes. Sesquiterpenes, a specific group of terpenes, most vary between species and impair inter-specific aggression by reducing aggressive behavior in species without sesquiterpenes, thereby providing a novel mechanism to achieve interspecific tolerance among insects. Reduced interspecific aggression may also be an important factor enabling the non-aggressive aggregation of nests from stingless bee colonies of up to four different species, because such aggregations frequently comprise both species with and species without sesquiterpenes. Given its various functions, resin represents a highly important resource for stingless bees which directly affects their chemical ecology, defensive properties and inter-specific communication. It remains to be investigated how the bees influence the resin-derived terpene profiles on their body surface and in their nests, particularly how they manage to exclude entire groups of terpenes. Whether bees actually need a high diversity of different resin sources and therefore tree species to maintain the homeostasis of their colonies or whether they would do equally well with a limited amount of resin sources available, should also be addressed in future studies. Answers to this question will directly impair bee and forest management in (sub)tropical regions.
This study focuses on phosphoantigen specific Vg9Vd2 T cells which only exist in human and non-human primates. This population accounts for 1%-5% of peripheral blood T-lymphocytes but their frequency can rise to 50% of total blood T cells upon infection. Vg9Vd2 T cells can be activated by nonpeptide compounds with critical phosphate moieties which are termed as phosphoantigens. These include isopentenyl pyrophosphate (IPP), a key compound of isoprenoid synthesis in all organisms, and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a direct precursor of IPP in DOXP pathway which only exist in eubacteria, plants, apicomplexaen parasites. Its activity as phosphoantigen is at least 1000 fold higher than that of IPP. However, direct structural evidence of phosphoantigen binding to the TCR is missing so far. Moreover, Vg9Vd2 T cells have potent anti-tumor activity e.g. against the B-cell lymphoma Daudi, whose Vg9Vd2 T cell activating properties have been suggested to result from sensing of abnormal intracellular IPP levels by the Vg9Vd2 TCR or Vg9Vd2 TCR binding to other postulated ligands such as an ectopically expressed F1-ATPase or UL-16 binding protein 4 (ULBP4). Aminobisphosphonates and alkymines were hypothesized to activate Vg9Vd2 T cells indirectly by inhibiting the IPP consuming enzyme farnysyl pyrophosphates synthesis (FPPS) although off target effects of these drugs or a direct interaction with the Vg9Vd2 TCR could not be excluded. This thesis presents new approaches for the mechanistic analysis of Vg9Vd2 T cell activation. By employing retroviral transduction of FPPS specific shRNA, it shows that specific shRNA reduces expression of FPPS and is sufficient to convert hematopoietic and non-hematopoietic tumor cell lines into Vg9Vd2 T cell activators. FPPS knockdown cells activated Vg9Vd2 T cells as measured by increased levels of CD69 and CD107a, kill of FPPS knockdown cells and induction of IFN-γ secretion. The IPP-synthesis-inhibiting drug mevastatin reduced Vg9Vd2 T cell activation by FPPS knockdown cells or aminobisphosphonate treated cells but not activation by the phosphoantigen bromohydrin pyrophosphate (BrHPP). A reduced growth of the FPPS knockdown cells has not been observed which is different to what has been reported for aminobisphosphonate treated cells. Finally, the human B-cell lymphoma RAJI has been transduced with Tetracyclin-inducible FPPS specific shRNA and proven to gain and loose the capacity to activate Vg9Vd2 TCR transductants upon doxycylin provision or removal. Another approach for the analysis of Vg9Vd2 T cell activation is Vg9Vd2 TCR transduced mouse cell lines with specificity for phosphoantigens. In contrast to the previously used Vg9Vd2 TCR transduced Jurkat cells, these cells do not present phosphoantigens, and are therefore specially suited for analysis of phosphoantigen presentation. The response of the new TCR transductants to presumed Vg9Vd2 TCR ligands/activators such as phosphoantigens, aminobisphosphonates or FPPS knockdown cells, depended strongly on the expression of a rat/mouse CD28 molecule by the transductants and its ligation by the (CD80) counter receptor on the ligand-presenting cell. The response is likely to reflect recognition of cognate Vg9Vd2 TCR antigens since mutations in the TCR-δ chain CDR2 and 3 abolished this response but activation by TCR or CD3 specific antibodies. A major difference between TCR transductants and primary gd T cells, was the lacking response of TCR transductants to Daudi or IPP. In addition their sensitivity to other soluble phosphoantigens was about 100 fold weaker than that of primary cells, stimulation of both cell type to CD80 expressing FPPS knock down or aminobisphosphonates was similar. Finally, the transductants have also been used to analyze effects of over-expression or knockdown of enzymes of isoprenoid synthesis such as 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGR), mevalonate-5-pyrophosphate decarboxylase (MVD), isopentenyl pyrophosphate isomerase (IDI), geranyl-geranyl pyrophosphate synthase (GGPPS) but no clear effects have been found. In conclusion, this thesis supports the concept of Vg9Vd2 T cells being sensors of a dysregulated isoprenoid metabolism and established new tools to study ligand recognition and TCR mediated activation of this T cell population. These tools will be most useful to address following questions: 1) How does the dysregulation of isoprenoid metabolism affect tumor growth? 2) What is the correlation between the modulation of IPP levels and the Vg9Vd2 TCR binding or expression of other postulated ligands? 3) Are there any mevalonate pathway enzymes other than FPPS and HMGR, which play an important role in Vg9Vd2 T cells activation? 4) What is/are the putative phosphoantigen-presenting molecule(s)?
Characterization of allosteric mechanisms on the M2 and M4 mACh receptor using the FRET-technique
(2010)
Allosteric modulators have been proposed as promising new compounds to modify protein function. Allosteric binding sites have been discovered for several G-protein-coupled receptors, including M1-5 muscarinic receptors. Since these receptors play a pivotal role in the regulation of a plethora of organ functions, it is particularly important to investigate the mechanisms of allosteric modulation. To study molecular mechanisms of allosteric modulation in the M2 muscarinic receptor, a new FRET-based sensor was designed. CFP fused to the C-terminus of the receptor and a small fluorescent compound FlAsH, which labels a specific binding sequence in the third intracellular loop, were used as donor and acceptor fluorophores, respectively. The first part of the study was to design a functional FRET receptor sensor. After several optimization steps the constructs FLAG-M2-sl3-FlAsH-GSGEG-CFP and HA-FLAG-M2-sl3-FlAsH-GSGEG-CFP were generated which showed good cell-surface expression, robust changes in FRET and the ability to deliver reproducible data. The second part of this thesis sought to elucidate the mechanisms of the allosteric ligand binding and their effects on the receptor conformation. The described modifications, which were introduced in the wild type M2 mAChR to create the FRET sensor can alter receptor functionality and influence receptor expression. Radioligand binding studies revealed that the used transfection method provided sufficient receptor expression but, unfortunately, about 60 % of the FLAG-M2-sl3-FlAsH-GSGEG-CFP receptor remains in the cytosol. However, this was sufficient to perform FRET experiments. Patch clamp GIRK-measurements with acetylcholine evinced that the new M2-sensor was able to activate Gi-proteins. Also, radioligand-binding assays with the second construct HA-FLAG-M2-sl3-FlAsH-GSGEG-CFP showed ligand affinity comparable to the wildtype receptor. Furthermore inhibition of forskolin-stimulated cAMP production was indistinguishable from the behaviour of the wildtype receptor. According to that, the full functionality of both receptor constructs could be confirmed. FRET measurements with the full muscarinic receptor agonists carbachol and acetylcholine confirmed that the FLAG-M2-sl3-FlAsH-GSGEG-CFP receptor construct showed rapid changes in FRET upon addition of both ligands, which were concentration-dependent. Concentration response curves and the resulting EC50 values of both agonists were similar to those already published in literature. In addition, the orthosteric antagonists atropine and methoctramine inhibited the FRET changes induced by the agonists. This inhibition was significantly faster than the washout kinetics, pointing to an active displacement of the agonists by the antagonists. Allosteric ligands gallamine, tacrine and dimethyl-W84 did not alter receptor conformation when added without an orthosteric ligand. However, when applied in addition to muscarinic agonists, all three substances inhibited the FRET-signal. The extent of this inhibition was dependent on the used concentration of the allosteric ligands. These results reveal that conformational changes brought about by allosteric ligands can be measured with the FRET technique. Furthermore real-time FRET-based kinetic measurements could be performed in living cells and showed that the allosteric ligands gallamine and dimethyl-W84 alter receptor conformation significantly faster than the antagonists atropine and methoctramine. This data indicate that allosteric ligands actively induce the conformational changes in the receptor.