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Glioblastoma is the most common and most aggressive type of brain tumor in adults. In contrast to epithelial cancers, glioblastomas do not metastasize. While the major treatment challenge in epithelial cancers is not the primary tumor but metastasis, glioblastoma patients die of the primary tumor.
However, there is a common theme which underlies the malignant properties of progressed epithelial cancers and glioblastoma: invasion from the primary tumor into the surrounding tissue. In the case of epithelial cancers this is the first and necessary step to metastasis, whereas invasion leads inevitably to tumor recurrence after resection in the case of glioblastoma, causing it to be incurable.
A cellular program which has been described in detail to promote the invasive phenotype in epithelial tumors, is the epithelial-mesenchymal-transition (EMT). Differentiated neural cells are not epithelial, thus, strictly speaking, EMT does not occur in glioblastoma. However, the traits acquired in the process of EMT, especially invasiveness and stemness, are highly relevant to glioblastoma. One of the key transcription factors known to induce EMT in epithelial cancers is ZEB1, which has been described only marginally in the central nervous system so far. Here, I investigate the expression and function of ZEB1 in glioblastoma and during human fetal neural development.
ZEB1 mRNA was significantly upregulated in all histological types of glioma, including glioblastoma, when compared to normal brain. There was no correlation between ZEB1 mRNA levels and tumor grade. Immunohistochemical staining of glioma samples demonstrated that ZEB1 was highly expressed in the great majority of tumor cells. In the developing human brain, intense staining for ZEB1 could be observed in the ventricular and subventricular zone, where stem- and progenitor cells reside. ZEB1 positive cells included cells stained with stem- and progenitor markers like PAX6, GFAP and Nestin. In contrast, ZEB1 was never found in early neuronal cells as identified by TUBB3 staining.
To gain insight into ZEB1 function I generated a human fetal neural stem cell line and a glioblastoma cell line with ZEB1 knockdown, which were compared with their respective control cell lines. First, I found that ZEB1 does not regulate the micro RNA 200 family in either cell line, which has been described as an essential ZEB1 target in epithelial cancers. Second, regulated target genes were identified with a genome wide microarray. The third approach was to directly identify genomic binding sites of ZEB1 by chromatin immunoprecipitation sequencing (ChIP-seq). All three approaches showed that the ZEB1 transcriptional program is surprisingly similar in the neural stem cell line and the glioblastoma cell line. In contrast, it bears only little resemblance to the program described in epithelial cancers.
The most interesting, previously unrecognized ZEB1 target gene identified in this study is integrin b1. It was regulated after ZEB1 knockdown detected by microarray analysis, and has a ZEB1 binding site in its promoter region detected by ChIP-seq. Finally, I addressed the question whether ZEB1 influences tumor growth and invasiveness in a glioblastoma model. After intracranial xenotransplantation in mice, ZEB1 knockdown glioblastoma cells formed significantly smaller and less invasive tumors than control glioblastoma cells.
This study demonstrates that ZEB1 is widely expressed in glioma and relevant for glioblastoma growth and invasion. In contrast to what is known about ZEB1 function in epithelial cancers, ZEB1 is not associated with glioma progression, but instead seems to be an early and necessary event in tumorigenesis. Also with regard to ZEB1 target genes, ZEB1 functions differently in glioblastoma than in epithelial cancers. The two most important ZEB1 targets in epithelial cancers are E-cadherin and the miR-200 family members. Both are not relevant to ZEB1 function in glioblastoma. Interestingly, while the ZEB1 transcriptional program is different from the one described in epithelial cancers, it is highly similar in glioblastoma cells and fetal neural stem cells. This suggests that an embryonic pathway restricted to stem- and progenitor cells during development is reactivated in glioblastoma.
Previously known ZEB1 target genes were tissue specific and therefore seemed unlikely to mediate ZEB1 function in the central nervous system. However, the newly identified ZEB1 target gene integrin b1 is well known to play pivotal roles in both glioblastoma tumorigenesis and invasion as well as in neural stem cells. Additionally, integrin b1 is widely expressed and seems a likely ZEB1 target in other organs than the brain.
Taken together, I demonstrate that ZEB1 is a new regulator of glioblastoma growth and invasion. The transcriptional program of ZEB1 differs from the one in epithelial cancers but is strikingly similar to the one in neural stem cells. The newly identified ZEB1 target gene integrin b1 is likely to mediate crucial ZEB1 functios. Thus, this study identifies ZEB1 as a yet unrecognized player in glioblastoma and neural development. Furthermore, it sets the stage for more research which will help to deepen our understanding of ZEB1 function in the central nervous system and beyond.
Transylvanian Saxons' migration from Romania to Germany: the formation of a 'return' diaspora?
(2013)
Processes and patterns of migration on a global scale have changed in profound ways during the last two decades (Smith and King, 2012). In the European context, this is exemplified by transformations to the traditional mobility patterns from East to West Europe (Koser and Lutz, 1998), with migrants more likely to be involved in temporary circular and transnational mobility (Favell, 2008). Since the end of the Second World War, historical and political events in Europe have facilitated the mobility of ethnic Germans from Eastern Europe to Germany. Subsequently, the fall of the Iron Curtain has permitted unrestrained East-West movements, which resulted in mass migrations towards the West and diaspora fragments in the East. However, after settlement in the West, ethnic Germans have also been absorbed within wider temporary and transnational movements (Koser, 2007). Within this context, this thesis examines the post-migratory lives of three generations of Transylvanian Saxons in Germany by exploring the cultural, social, economic and political dimensions of this community. This thesis aims to contribute to on-going academic debates about diasporas by explicitly responding to Hoerder s (2002) call for more studies on ethnic German diasporas. It shows that Transylvanian Saxons, who relocated to the ancestral homeland, do not disrupt identities and lives forged in diaspora, but rather, they negotiate complex identities and belongings in relation to both home and homeland . It reveals a double diaspora and the necessity to perceive identity and diaspora as dynamic processes and constantly evolving in relation to time, space and place. This double diasporic allegiance in the case of the Transylvanian Saxons suggests interrogating the formation of a return diaspora and its importance for processes of international migration.
Endocannabinoids (eCB) are signaling lipids and became known for their importance in the central nervous system as well as in immune defense. Beneficial effects of eCB are shown in processes of excitotoxic lesion, secondary damage and neuronal plasticity throughout the last years. Two canabinoid receptors, type 1 (CB1) and type 2 (CB2) as the respective endogenous ligands belong to the endocannabinoid system (eCBS). In 1990, the CB1 could be cloned and was localised mainly on neurons. Shortly thereafter in 1993, the CB2 was characterised and found primarily on cells belonging to the immune system. N-arachidonoylethanolamide (AEA), often called anandamide, and 2-arachidonoylglycerol (2-AG) are the best characterised eCB. N-palmitylethanolamide (PEA) and N-oleoylethanolamide (OEA) have no or only low affinity to CB1 but enhance the affinity of AEA significantly. This group is therefore often summarized as N-ethanolamides (NEA). ECB are derivates of arachidonic acid and are stored in membranes where they become hydrolysed on demand by specific enzymes. Traumatic brain injury altered the levels of eCB in the blood in vivo and when applied in vitro after neuronal damage, eCB could reduce the damaging burden. Further studies demonstrated that eCB are potent to down-regulate pro-inflammatory cytokines and most important to decrease neuronal excitation.
In the present study, the intrinsic regulation of the endocannabinoid system after neuronal damage over time was investigated in rat Organotypic Hippocampal Slice Cultures (OHSC). Temporal and spatial dynamics of eCB levels were analysed after transection of the perforant pathway (PPT) in originating neurons (enthorhinal cortex, EC), areas of deafferentiation/anterograde axonal degeneration (dentate gyrus, DG) and of the synaptically linked cornu ammonis region 1 (CA1) as well as after excitotoxic lesion in the respective regions.
A strong increase of all eCB was observed only in the denervation zone of the DG 24 hours post PPT. In excitotoxic lesioned OHSC all eCB were elevated, in the investigated regions up to 72 hours post lesion (hpl). The responsible enzyme for biosynthesis of the NEA, NAPE-PLD protein, was increased during the early timepoints of measurement (1-6 hpl). The responsible catabolizing enzyme, FAAH, and the CB1 receptor were up-regulated at a later timepoint, 48 hpl, explaining the eCB levels. In the present model, the inhibition of the enzyme responsible for 2-AG hydrolysis (MAGL) was neuroprotective as previously shown and a re-distribution within neurons and astrocytes during neuronal damage could be observed. In primary cell cultures microglia expressed the regulating enzymes of 2-AG and the enzyme responsible for NEA down-regulation, FAAH. Astrocytes expressed mainly the catalyzing enzymes, indicating the role for eCB break-down. All these findings together demonstrate the great capacity of the eCBS to control inflammatory processes and consequently neuronal cell death.
All effects of the known eCB could not be clarified by CB1/CB2 deficient mice. Several G-protein coupled receptors (GPR) are recently in discussion whether they might and should belong to the endocannabinoid system. The GPR55, the not yet cloned abnormal cannabidiol receptor and further GPRs are candidates as potential endocannabinoid receptors. Recently GPR55 has been discussed as a putative cannabinoid receptor type 3 (CB3). Quantitative PCR revealed that Gpr55 is present in primary microglia and the brain, but the exact regional and cellular distribution and the physiological/pathological effects downstream of GPR55 activation in the CNS still remain open. Therefore, the excitotoxic rat OHSC model, previously used to investigate the neuroprotective potency of eCB, was now used to investigate the neuroprotective potency of GPR55. Activation of GPR55 protected dentate gyrus granule cells in vitro after excitotoxic lesion, induced by NMDA. In parallel, GPR55 activation was able to reduce the number of microglia in the dentate gyrus. These neuroprotective effects vanished however in microglia depleted OHSCs as well as in OHSC transfected with Gpr55 siRNA, indicating a strong involvement of microglia in GPR55 mediated neuroprotection.
In summary, the present study found a strong time-dependent and anterograde mechanism of action of eCB after long-range projection damage and provided further evidence for the neuroprotective properties of eCB. The potential cannabinoid receptor 3 (GPR55) mediates neuronal protection on behalf of microglia.
The TolC protein of E. coli is a versatile OMF which is involved in secretion of antibiotics, heavy metal ions, secondary metabolites and proteins. These individual tasks are accomplished by a dynamic formation of different secretion complexes which comprising a plasma membrane transporter, a Membrane Fusion Protein and TolC as the outer membrane channel-tunnel. The TolC-like protein HgdD of the cyanobacterium Anabaena sp. PCC 7120 was previously described as an indispensable OMF involved in formation of the heterocyst-specific glycolipid layer which is needed to sustain the microoxic environment that allows nitrogen fixation in heterocysts of filamentous cyanobacteria. Here I show that HgdD is involved in macrolide antibiotic resistance and ethidium efflux, which is used as a model substrate for cytotoxic compounds and secondary metabolites. It can be shown that ethidium uptake is a passive and porin-dependent process, while multidrug efflux is performed together with the RND efflux pump All3143 (and the MFP All3144). In contrast to HgdD, All3143 can complement the function of its homologue AcrB in E. coli and was suggested to be named anaAcrB. Multidrug efflux is assisted by SmsA and SchE, two secondary transporters of the MFS-type, which facilitate the transport of cytoplasmatic ethidium to the periplasmic space prior to the All3143- and HgdD-dependent efflux. Moreover, it can be demonstrated that SchE and HgdD are involved in secretion of the metal ion-chelating siderophore schizokinin, which functions in iron(III) acquisition. However, a physical interaction of SchE and HgdD is unlikely since SchE does not possess an OMF interacting domain. In addition, both RND efflux pumps All3143 and Alr1656 are needed for the homeostasis of the photosystems during diazotrophic growth. Although a direct involvement in heterocyst development or metabolism cannot be discounted at this stage, it is speculated that both RND transporters are involved in detoxification of reactive nitrogen species, similar to the function of MexF and MdtF of P. aeruginosa and E. coli respectively. In addition to its function in multidrug efflux, HgdD has been shown to be involved in protein secretion. By comparative analysis of the Anabaena sp. wild type and hgdD mutant secretome it was possible to identify eight putative HgdD protein substrates. The localization of four proteins was exemplary demonstrated by secretome isolation and cell fractionation of hemagglutinin-tagged mutant strains. The absence of detectable protein in the hgdD mutant strain suggests a highly efficient secretion system which is quality controlled by proteolysis of mislocalized proteins.
Cell-cell adhesion is an essential process during the development of multicellular organisms. It is based on various cellular junctions and ensures a tight contact between neighboring cells, enabling interactive exchanges necessary for morphological and functional differentiation and maintaining the homeostasis of healthy tissue organization. Two important types of cell-cell adhesions are the adherens junction (AJ) and the desmosome which link the actin cytoskeleton and intermediate filaments to cadherin-based adhesion sites. The core of these structures is composed of single-span transmembrane proteins of the cadherin superfamily which include, among other members, the classical cadherins, e.g. E-cadherin, as well as the desmosomal cadherins, e.g. desmoglein-3. The cytoplasmic domains of the desmosomal and classical cadherins enable interactions with proteins of the catenin family. Classical cadherins preferentially associate with β-catenin and p120-catenin, whereas desmosomal cadherins bind to γ-catenin and plakophilins. Intriguingly, γ-catenin, also known as plakoglobin, is so far the only protein known to be present both in the AJ and the desmosome.
In this study, we showed that the two homologous, membrane raft-associated proteins flotillin-1 and flotillin-2 associate with core proteins of the AJ and the desmosome in vitro and in vivo. In confluent human, non-malignant epithelial MCF10A cells and human skin cryosections, flotillin-2 colocalized with E-cadherin, desmoglein-3 and γ-catenin at cell-cell contact sites, whereas flotillin-1 showed barely any overlap with these proteins. In addition, we detected a colocalization of both flotillins with the actin-binding protein α-actinin in membrane ruffles in subconfluent and at cell-cell contact sites in confluent MCF10A cells as well as in human skin cryosections. The interaction with α-actinin was later shown to be flotillin-1 dependent by performing indirect GST pulldown experiments with purified α-actinin-1-GST in MCF10A cell lysates.
Since flotillin-2 strongly colocalized with cell-cell junctions, this suggested that flotillins might be found in complex with cell adhesion proteins. Thus, we performed coimmunoprecipitation experiments in murine skin lysates and various cell lines of epithelial origin, such as human breast cancer MCF7 cells, human keratinocyte HaCaT cells and primary mouse keratinocytes. These experiments demonstrated that flotillins, especially flotillin-2, coprecipitated with E-cadherin, desmosomal cadherins and γ-catenin in relation to the respective cell type and the maturation status of these cell-cell adhesion structures. However, since γ-catenin is so far the only protein known to be present in the AJ and the desmosome, we further assumed that the complex formation of flotillins with cell adhesion structures is mediated by γ-catenin. For this, we performed indirect GST pulldown experiments in MCF10A cell lysates with bacterially expressed, purified flotillin-1-GST, flotillin-2-GST and γ-catenin-GST and were able to verify the complex formation of adhesion proteins and flotillins in vitro. To further test if the interaction of γ-catenin and flotillins is a direct one, we used purified flotillin-1-GST or flotillin-2-GST and γ-catenin-MBP fusion proteins. Both flotillins directly interacted with γ-catenin in this in vitro assay. In addition, mapping of the interaction domains in γ-catenin by using GST fusion proteins carrying different parts of γ-catenin suggested that flotillins bind to a discontinuous γ-catenin binding domain which consists of a Major determinant around ARM domains 6-12, most likely with a major contribution of the ARM domain 7, and possibly including the NT part of γ-catenin.
To study the effect of flotillin depletion on cell-cell adhesion, we generated stable MCF10A cell lines in which flotillins were knocked down by means of lentiviral shRNAs. Staining of E-cadherin and γ-catenin in these cells showed that the localization at the cell-cell borders was significantly altered after flotillin-2 depletion, which pointed to a role for flotillin-2 in the formation of cell-cell adhesion structures in epithelial cells. Furthermore, isolation of detergent resistant membranes (DRMs) from these cells demonstrated that upon depletion of flotillin-2, a significant amount of E-cadherin and γ-catenin shifted into raft fractions. On the contrary, no change was detected in flotillin-1 knockdown cells. These observations point to a functional role of flotillin-2 in the regulation of raft association of cell-cell adhesion proteins. To gain more insight into the in vivo relevance of our findings, we next studied the function of flotillins in the skin of Flot2-/- knockout mice. Analysis of lysates prepared from the skin of one year old female animals revealed an increased expression of E-cadherin, desmoglein-1 and γ-catenin but not β-catenin, implicating that specific adhesion proteins are upregulated in flotillin-2 knockout skin.
Since flotillins are tightly associated with membrane microdomains we next studied the interaction of flotillin-2 with membrane cholesterol. Using the photoreactive cholesterol analog azocholestanol, we were able to show that flotillin-2 and cholesterol directly interacted. In addition, previous studies speculated that flotillin-2 interacts with cholesterol via two putative cholesterol recognition/interaction amino acid consensus (CRAC) motifs. Analysis of the flotillin-2 sequence revealed that flotillin-2 actually contains four putative CRAC motifs. However, using various flotillin-2 CRAC mutant GFP fusion proteins, we were able to show that none of the putative CRAC motifs is functional, which suggested that flotillin-2 interacts with membrane cholesterol, e.g., via posttranslational modifications, such as myristoylation and palmitoylation which were previously shown to be essential for membrane association of flotillin proteins.
Die Dissertation stellt das Machtgeflecht in der Islamischen Stadt Marawi City (Mindanao, Philippinen) dar, in die die dortigen Gender-Debatten involviert sind. In einer Umgebung, die als Konsequenz des Mindanao Konfliktes als “no war, no peace”-Umgebung definiert werden kann, gibt es drei Hauptdarsteller: die nationale Regierung des mehrheitlich christlichen Staates der Philippinen (GRP), die Autonome Regierung im Muslimischen Mindanao (ARMM), zu der auch Marawi City zählt, und die islamische Rebellengruppe Moro Islamic Liberation Front (MILF), die einen islamischen (unter-)Staat fordert. Die GRP unterstützt Re-Islamisierungs- und Re-Traditionalisierungsbewegungen in der ARMM, um die Opposition zur MILF zu stärken. Die Konsequenz ist jedoch keine Kollaboration zwischen der GRP und der ARMM. Stattdessen nutzen Politiker ihre Privilegien aus, um ihren eigenen Absichten zu folgen. Sei dies, um politische Gegner auszuspielen oder das traditionelle Sultanatssystem zu fördern. Für Gender-Debatten gibt es in diesem Kontext der ungelösten nationalen Frage kaum Spielraum außerhalb einer Islamischen Narrative; dies bedeutet jedoch nicht, dass Gender nicht debattiert wird, sondern, dass die Debatten inner-Islamisch sind, hauptsächlich zwischen Repräsentanten des traditionell synkretistischen Islam und Vertretern Islamischer Revitalisierungsbewegungen. Speziell erstere erscheinen sehr einflussreich bezüglich Gender Strategien in der Region. Dies ist nur teilweise auf die Unterstützung der nationalen Regierung zurückzuführen, sondern ist vor allem eine Frage von Identität. Diese wiederum wird nicht vorranging über Religion, sondern nach ethnischen Maßstäben und im Speziellen im Rahmen von Clanstrukturen definiert.
During the height of the Second World War pressure from Great Britain resulted in the transfer of thousands of German prisoners of war (PoWs) from British to Canadian control. To house them, Canada built a system of PoW camps, including Riding Mountain Camp in southwestern Manitoba. The PoWs sent there soon realized their good fortune: they lived in warm barracks, ate abundant food, and were able to purchase goods from a mail order catalog. But while the PoWs were well treated, they were at the same time subjected to a concerted reeducation campaign organized by the Allies. This reveals that these Canadian camps were not merely warehouses for the PoWs, but in fact, classic reforming institutions.
Initially subjected to ideological training under Nazism, the PoWs were next subjected to another kind of education under the Canadians. Evidence collected from oral history interviewing, archival research, and three seasons of field archaeology combine to reveal that material culture was a key nexus in this competition for the minds of the PoWs. In addition to providing books and teaching courses on history and political science, the Canadians introduced the PoWs to a democratic, capitalistic way of life by familiarizing them with North American consumer goods and by allowing them to fraternize with Canadian civilians. The Nazi bureaucracy, in turn, used material things to try to keep the PoWs from turning to the other side. For example, by sending them crisp new Wehrmacht uniforms from Germany, heartening Christmas cards, and packages filled with German goods adorned with Nazi symbolism.
The spider genus Eusparassus Simon, 1903 (Araneae: Sparassidae: Eusparassinae; stone huntsman spider) is revised worldwide to include 30 valid species distributed exclusively in Africa and Eurasia. The type species E. dufouri Simon, 1932 is redescribed and a neotype is designated from Portugal. An extended diagnosis for the genus is presented. Eight new species are described: Eusparassus arabicus Moradmand, 2013 (male, female) from Arabian Peninsula, E. educatus Moradmand, 2013 (male, female) from Namibia, E. reverentia Moradmand, 2013 (male, female) from Burkina Faso and Nigeria, E. jaegeri Moradmand, 2013 (male, female) from South Africa and Botswana, E. jocquei Moradmand, 2013 (male, female) from Zimbabwe, E. borakalalo Moradmand, 2013 (female) from South Africa, E. schoemanae Moradmand, 2013 (male, female) from South Africa and Namibia and E. mesopotamicus Moradmand and Jäger, 2012 (male and female) from Iraq, Iran and Turkey. 22 species are re-described six of them are transferred from the genus Olios Walckenaer, 1837. Six species-groups are proposed: the dufouri-group [8 species: E. dufouri, E. levantinus Urones, 2006, E. barbarus (Lucas, 1846), E. atlanticus Simon, 1909, E. syrticus Simon, 1909, E. oraniensis (Lucas, 1846), E. letourneuxi (Simon, 1874), E. fritschi (Koch, 1873); Iberian Peninsula to parts of north-western Africa], walckenaeri-group [3 species: E. walckenaeri (Audouin, 1826), E. laevatus (Simon, 1897), E. arabicus; eastern Mediterranean to Arabia and parts of north-eastern Africa], doriae-group [7 species: E. doriae (Simon, 1874), E. kronebergi Denis, 1958, E. maynardi (Pocock, 1901), E. potanini (Simon, 1895), E. fuscimanus Denis, 1958, E. oculatus (Kroneberg, 1846) and E. mesopotamicus; Middle East to Central and South Asia], vestigator-group (3 species: E. vestigator (Simon, 1897), E. reverentia, E. pearsoni (Pocock, 1901); central to eastern Africa and an isolated area in NW India], jaegeri-group [4 species: E. jaegeri, E. jocquei, E. borakalalo, E. schoemanae; southern and south-eastern Africa], tuckeri-group [2 species: E. tuckeri (Lawrence, 1927), E. educatus; south-western Africa). Two species, E. pontii Caporiacco, 1935 and E. xerxes (Pocock, 1901) cannot be placed in any of the above groups. Two species are transferred from Eusparassus to Olios: O. flavovittatus (Caporiacco, 1935) and O. quesitio Moradmand, 2013. 14 species are recognized as misplaced in Eusparassus, thus nearly half of the described species prior to this revision were placed mistakenly in this genus. Neotypes are designated for E. walckenaeri from Egypt, E. barbarus, E. oraniensis and E. letourneuxi (all three from Algeria) to establish their identity. The male and female of Cercetius perezi Simon, 1902, which was known only from the immature holotype, are described for the first time. It is recognized that the monotypic and little used generic name Cercetius Simon, 1902 — a species, which had been known only from the immature holotype — as a synonym of the widely used name Eusparassus. The case proposal 3596 (conservation of name Eusparassus) is under consideration by ICZN.
The first comprehensive molecular phylogeny of the family Sparassidae with focus on the genus Eusparassus is investigated using four molecular markers (mitochondrial COI and 16S; nuclear H3 and 28S). The monophyly of Eusparassus and the dufouri, walckenaeri and doriae species-groups are recovered with the latter two groups more closely related. The monophyly of the tuckeri-group is not supported and the position of E. jaegeri as the only available member of the jaegeri-group is not resolved within the Eusparassus clade. DNA samples of the vestigator-group were not accessible for this study. The origination of the genus Eusparassus around 70 million years ago (MA) is estimated according to molecular clock analyses. Using this recent result in combination with some biogeographic and geological data, the Namib Desert is proposed as the place of ancestral origin for Eusparassus and putative Eusparassinae genera.
Further analyses are done on the phylogenetic relationships of Sparassidae and its subfamilies. The Eusparassinae are not confirmed as monophyletic, with the two original genera Eusparassus and Pseudomicrommata in separate clades and only the latter clusters with most other assumed Eusparassinae, here termed the "African clade". Monophyly of the subfamilies Sparianthinae, Heteropodinae sensu stricto, Palystinae and Deleninae is recovered. The Sparianthinae are supported as the most basal clade, diverging considerably early (143 MA) from all other Sparassidae. The Sparassinae and genus Olios are found to be polyphyletic. The Sparassidae are confirmed as monophyletic and as most basal group within the RTA-clade. The divergence time of Sparassidae from the RTA-clade is estimated with 186 MA in the Jurassic. No affiliation of Sparassidae to other members of the "Laterigradae" (Philodromidae, Selenopidae and Thomisidae) is observed, thus the crab-like posture of this group was proposed a result of convergent evolution. Only the families Philodromidae and Selenopidae are found members of a supported clade. Including a considerable amount of RTA-clade representatives, the higher-level clade Dionycha is not but monophyly of the RTA-clade itself is supported.
Lipid mediators have been referred as bioactive lipids, whose change in lipid levels resulted in functional or pathophysiological consequences. They are in the focus of biological research, nevertheless this is a late recognition due to the many difficulties of working with bioactive lipids due to their properties: hydrophobic, unstable and they occur in only in small quantities. Liquid chromatography and mass spectrometry have facilitated the work with them. Especially in this field, cardiovascular diseases and inflammatory mediated diseases and cancer are pathophysiological events where LMs are deregulated. Additionally, if the modulation of one LM pathway is not sufficient to overcome a disease, the combination of targeting two or more pathways could be effective. Needless to say, lipid signaling cascades are complicated pathways and possible shunting into other pathways when inhibiting or genetically deleting enzymes should be taken into consideration.
The first part of this work has focused on enzymes that metabolize eicosanoids, like mPGES-1 and 5-LO. mPGES-1 is an important enzyme metabolizing PGH2 and one of the key players of the AA cascade. Its product, PGE2 plays an important role in different inflammatory processes. Inhibition of the mPGES-1 might be a promising step to circumvent COX dependent side effects of NSAIDs. The class of quinazoline compounds around the lead structure FR20 has been investigated on isolated human and murine enzyme, in HeLa cells and in different human whole blood (HWB) settings to establish the possible effects of these compounds on eicosanoid profiling. Novel compounds with inhibitory activities in the submicromolar range (IC50: 0.13 µM - 0.37 µM on isolated enzyme) were obtained which were also effective in cells and HWB. Furthermore, pharmacological profiling of toxicity and lipid screening with LC/MS-MS revealed that compounds also reduce PGE2 levels in intact cells and whole blood; they do not impair cell viability but lack the ability to inhibit the murine mPGES-1 enzyme. This problem could be overcome by means of chemical synthesis varying the scaffold (quinoline, quinazoline) or introducing biosteric replacement in the phenyl moieties.
5-LO is a relevant enzyme that plays an important role in eicosanoid signaling in particular in leukotriene biosynthesis. Leukotrienes are involved in asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, sepsis, cancer and atherosclerosis. Moreover, genetic variants in the genes of the 5-LO pathway have been associated with the risk of development of acute myocardial infarction and stroke. Eicosanoids are increased in infectious exacerbations of chronic obstructive pulmonary disease (COPD). They are also elevated in the airways of stable COPD patients compared to healthy subjects. Therefore, 5-LO has attired the scientific community as a possible therapeutic target to treat the several disease conditions listed before. In this study an extensive evaluation of imidazo[1,2-a]pyridines as a suitable lead structure for novel 5-LO targeting compounds was presented Within the three publications, 5-LO inhibitory activity of synthesized compounds was investigated in intact PMNL, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and compounds were in vitro pharmacological evaluated. Chemical modifications for lead optimization via straight forward synthesis were used to combine small polar groups (hydroxy, and methoxy groups) which led to a suitable candidate with desired in vitro pharmacokinetic profile in terms of solubility and intrinsic clearance without showing any cytotoxicity. More than 70 imidazo[1,2-a]pyridine derivatives have been synthesized, resulting in more than 50 active compounds. Although it was not possible to introduce a solubility group without impairing the 5-LO inhibitory activity, combination of small polar groups lead to a more favorable solubility and in vitro metabolic stability. Overall, the development of 5-LO inhibitors with high efficacy and selectivity in vivo will provide a possible treatment for patients having one of the diseases where leukotriene biosynthesis plays an important role.
Other types of 5-LO inhibitors have been synthesized during this work, NO-NSAIDs can be postulated as novel 5-LO inhibitors that could circumvent the undesired side-effects of inhibiting COX isoforms (ulcer perforation, gastrointestinal bleeding and in some cases death). It is suggested that NO group is released in situ or after compounds are metabolized. NO-NSAIDs maintain the same anti-inflammatory properties by inhibiting 5-LO in clinical relevant concentrations. NO-NSAIDs are currently under clinical trial for the treatment of diseases where inflammation plays an important role. Synthesis of NO-NSAIDs is straightforward and can be applied for most NSAIDs recently published. Among them, the most promising candidate is NO-sulindac that was able to inhibit 5-LO product formation in intact PMNL, purified 5-LO and HWB in micromolar concentration. Additional experiments regarding their mechanism are currently being performed.
The present study could show that dual inhibitors are an interesting approach that is practicable. It has been used in the recent years to overcome side-effects and diseases concerning more pathophysiological conditions. MetS is an example of a conjunction of symptoms: hyperglycemia, hypertriglyceridemia, hypertension and obesity. Due to its complex nature, the current treatment strategies of MetS require multiple pharmacological compounds regulating lipid and glucose homeostasis as well as blood pressure and coagulation. This study describes the first synthesis of dual sEH/PPAR modulators as potential agents for treatment of MetS. Following a combinatorial approach, an acidic head group known as a pharmacophore important for PPARα/γ dual agonistic activity was combined with different hydrophobic urea derivatives in order to introduce an epoxide mimetic (sEH pharmacophore). The resulting compounds yielded high inhibition of sEH and different patterns of PPAR agonistic activity. This study demonstrates that the pharmacophores of PPAR agonists and sEH inhibitors can be easily combined, resulting in a simplified blueprint of a dual sEH/PPAR modulator. Further in vivo pharmacological evaluation studies are needed in order to evaluate, which pattern of PPAR activation shows the most promising profile for treatment of metabolic syndrome.
Another example of dual pharmacology has been presented in this work. Natural products derived compounds were able to target sEH and exhibit promising antiproliferative properties. The principle of addressing multiple targets by natural products can be transferred to synthetic multi-target ligands. In conclusion, several (E)-styryl-1H-benzo[d]imidazoles were synthesized and evaluated on recombinant sEH after an initial hit (IPS) that lead to potent sEH inhibitors exhibiting antiproliferative activities. Following the natural product-inspired design, the desired biological activity from a bacterial secondary metabolite has been enhanced and transferred to a synthetic compound series. The resulting compounds were accessible via an easy synthetic route and offered a possibility to investigate the structure-activity relationships. The natural product inspired drug design extends the valuable role of natural products as drugs and drug precursors to templates for fully synthetic bioactive molecules. Simplification of natural products by means of chemical synthesis could lead to an interesting field in the treatment of cancer.
Affinity chromatography has been used to unravel unknown- and off-target effects which either contribute to the biological effect of the inhibitor or that counteract or lead to undesired side-effects. During this PhD work, two main projects related to this technique have been established. In the first one, related to an imidazo[1,2-a]pyridine inhibitor (EP6), it has been shown that epoxide-sepharose is a reliable material in order to couple compounds bearing an alcohol. Coupling of an analogue of EP6 to the sepharose has been accomplished and affinity towards 5-LO was demonstrated. The challenging step is to discern from unspecific protein binders and analysis via SDS-PAGE separation and mass spectrometry. Further experiments using other cell types or improving SDS-PAGE analysis (e.g. 2D gel analysis) should be useful to unravel EP6 off-target effect. During the second project related to off-target effects of celecoxib and DMC, the main problem was the coupling of the functional group to the sepharose. Affinity towards COX-2 could not be demonstrated pointing out the inefficient coupling method. Higher pH values during coupling reaction should be tested in further experiments. Nevertheless, affinity chromatography is a useful technique to unravel cellular mechanisms.
Sphingolipid metabolism is also a recent area that attired the attention of cancer researchers, due to their important roles in cell proliferation and apoptosis. Ceramide metabolism inhibitors were synthesized and evaluated on different assay systems in order to assess their efficacy on several cancer lines. Remarkably, 2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (32) was a useful scaffold to mimic the sphingoid base. This key intermediate was used to produce ceramide analogues that could enter the cell and target apoptosis machinery. EB143 (38) increased ceramide levels in an in vitro ceramide synthase assay in a dose-response manner meaning that ceramide synthase was not inhibited but the ceramide de novo synthesis was activated. This effect was due to the fact that EB143 is a cytotoxic compound with an interesting antiproliferative profile. Further chemical modifications should be carried out to modulate this effect.
COX and LO inhibitors are cancer-preventive not only by inhibiting specific antiapoptotic AA metabolites but also by facilitating accumulation of AA which promotes neutral SMase activity and increases the proapoptotic ceramide. Several 5-LO inhibitors have been evaluated on several cancer lines and sphingolipid levels were measured in order to obtain a relationship. A549, Capan-2 and MCF-7 cells line were incubated with synthetic 5-LO inhibitors and zileuton. Compounds were cytotoxic to all cancer cell lines except from A549. Needless to say, zileuton did not exhibit a cytotoxic profile. Synthetic 5-LO inhibitors were able to modify ceramide levels but were useless when coincubating with sphingolipid metabolism inhibitors (myoricin, amitryptiline etc.) and inconsistent results were obtained. On the contrary, zileuton selectively increased Cer-C16 levels and in less extend Cer-C24:1. When using a SPT inhibitor (myoricin) alone was able to reduce C24:1 and Cer-C16:0 levels below the control, a similar effect occurred when incubation the cells with zileuton and myriocin. Interestingly, treatment of zileuton together with either amitryptiline or desipramine led to a decrease in Cer-C24:1 and levels Cer-C16:0 but the inhibition was not complete indicating that probably the de novo pathway has an important role. Further investigations on mRNA level should be carried out in order to discern which CerS is activated.
The main objective of the present thesis was the synthesis of lipid signaling modulators and their evaluation in vitro as therapeutic strategy to overcome pathophysiological conditions (cancer, metabolic syndrome, etc). It has been accomplished on many relevant targets like 5-LO, mPGES-1, sEH and PPAR and these lipid signaling modulators could be used in the treatment of diseases conditions where lipid mediators play an important role.
Studies on the focusing performance of a Gabor lens depending on nonneutral plasma properties
(2013)
The concept of the Gabor lens goes back to an idea by Dennis Gabor, who proposed a magnetron-type trap as an effective diverging lens for electron beams (collecting lens for positive ion beams).
Electrons confined inside the lens volume by orthogonal magnetic and electric fields, create an electric space charge field that causes a radial symmetric focusing force on an ion beam passing through the lens volume.
Since the beginning of the 1990s, a new design of this lens type as well as numerical models to describe the confined plasma cloud have been developed at the Institute for Applied Physics (IAP, Johann Wolfgang Goethe-University Frankfurt).
Thanks to an improved understanding of the plasma confinement as a function of the external fields, two lenses have successfully been tested for low beam currents and remain in operation.
In the scope of this work, the performance of a prototype Gabor lens for the transport of intense, i.e. space charge dominated ion beams, was investigated at the High Current Test Injector (HOSTI) of GSI Helmholtzzentrum für Schwerionenforschung GmbH for the first time.
To ensure an optimal focusing performance of the Gabor lens a homogeneous and stable electron confinement is required. Therefore, new non-interceptive diagnostic methods were developed to investigate the parameters and state of the confined nonneutral plasma column as a function of the external fields.
An essential part of the studies was the time-resolved diagnostic of an occurring plasma instability and the determination of the electron temperature via optical spectroscopy. The latter necessitated the detailed investigation of atomic excitation as well as the measurement of optical-emission cross sections.
A comparison of the results from both experiments i.e. the beam transport measurements at GSI and the diagnostic experiments performed at IAP concerning the plasma state, gave first indications of possible interaction processes between the nonneutral plasma and the ion beam.