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Understanding major causes of biodiversity and range dynamics requires research on evolutionary processes under consideration of environmental changes. In my thesis, I investigated the spatio-temporal evolution of the Neotropical tree genus Cedrela from the Meliaceae family by studying its genetic diversity, taxonomy, colonization history, climatic niche changes and dynamics of species distributions. My results show that climatic and geological changes are major drivers of biological diversification in Cedrela.
In the interest of understanding the development of a multicellular organism, subcellular events must be seen in the context of the entire three-dimensional tissue. In addition, events that occur within a short period of time can be of great importance for the relatively long developmental process of the organ. Thus, it is required to capture subcellular events in a larger spatio-temporal scale context, which has been up to now a technical challenge. In developmental biology, light microscopy has always been an important tool. The dilemma of light microscopy, in particular fluorescence microscopy, is that molecules receive high light intensities that might change the conformation of molecules, which can have signaling or toxic effects. In Light Sheet-based Fluorescence Microscopy (LSFM), the energy required for a single recording is reduced by several orders of magnitude compared to other fluorescence microscopy techniques. During the last ten years, LSFM has emerged as a preferred tool to capture all cells during embryogenesis of the zebrafish Danio rerio, the fruit fly Drosophila melanogaster or recently the red flour beetle Tribolium castaneum for a period of several days. The motivation of this work was to gain new insights in developmental related processes of plant organs. The aim of this work was to establish a protocol for imaging plant growth over a long period of time using LSFM and perform comprehensive analyses at the cellular level. Plants have to cope with a variety of environmental conditions, therefore the conditions inside the microscope chamber had to be brought under control. The sample preparation methods and the standardized conditions at a physiological level allowed the study of gravity response, day-night rhythms, organ shape development as well as the intracellular dynamic events of the cytoskeleton and endosomal compartments in an unprecedented manner. Several of these projects were successfully published in collaborations with Prof. Jozef Šamaj (Palacký University Olomouc, Czech Republic), Prof. Niko Geldner (University of Lausanne, Switzerland), Prof. Malcom Bennett (University of Nottingham, UK) and Dr. Jürgen Kleine-Vehn (University of Natural Resources and Life Sciences, Austria). The main part of my work focused on the formation of lateral roots in Arabidopsis thaliana and was conducted in close collaboration with Dr. Alexis Maizel (University of Heidelberg, Germany). Previously, most experiments that describe lateral root formation have been performed on a small number of cells and for short periods of time. Capturing the complete process of lateral roots is an ambitious goal, because first, the primordium of a lateral root is located deep inside the primary root and imaging quality is impaired due to scattering of the overlaying tissue. Second, the process takes about 48 h, i.e. the plant has to be kept healthy for the whole period. Third, the amount of excitation light required for the spatio-temporal might have phototoxic effects that lead to a stop of growth at least in conventional microscopic techniques. In Arabidopsis embryogenesis, the sequence of cell divisions is relatively invariant. However, whether lateral root organogenesis follows particular cell division patterns has been unknown. The complete process of lateral root formation was captured from the first cell division until after the emergence from the main root. Images of a nuclei marker and a plasmamembrane marker were recorded every 5 min for a time period of up to 64 h. The positions and cell divisions of all cells were tracked manually. In collaboration with Alexander Schmitz (Goethe University Frankfurt am Main, Germany) and Dr. Jens Fangerau (University of Heidelberg, Germany), comprehensive analyses of the data were performed. A lateral root forms from initially 8-15 founder cells, arranged in a patch of 5-8 parallel files. The occurrence of new cell layers by periclinal divisions, as well as the sequence of layer generation was conserved and resembles the sequence suggested by Malamy and Benfey in 1997. Besides this stereotyped occurrence of periclinal divisions, radial divisions were found to appear stochastically, following no particular pattern. A large variability was also found in the contribution of founder cells and cell files to the final lateral root. In summary, the results suggest that a stereotyped pattern of cell divisions at particular developmental stages and a dynamically adapted control of cell divisions exist in parallel. Both properties allow a controlled but flexible development of the organ according to variations in cell topology and mechanical properties of the surrounding tissue. This work shows that LSFM, the sample preparation methods and controlled environmental conditions allow to capture and analyse the development of plants over several days at high resolution in an unprecedented manner.
Tympanal hearing organs of insects emit distortion-product otoacoustic emissions (DPOAEs) which are indicative of nonlinear mechanical sound processing. General characteristics of insect DPOAEs are comparable to those measured in vertebrates, despite distinct differences in ear anatomy. DPOAEs appear during simultaneous stimulation with two pure tones (f1<f2) as additional spectral peaks at frequencies of nf1-(n-1)f2 and nf2-(n-1)f1, with the 2f1-f2 emission being the most prominent one. Insect DPOAEs are highly vulnerable to manipulations that interfere with the animal's physiological state and disappear after death. First evidence from locusts suggested that scolopidial mechanoreceptors might play a role in frequency-specific DPOAE generation (Möckel et al. 2007). The overall aim of this thesis was to determine the source of sensitive, nonlinear hearing at high frequencies and of DPOAE generation in tympanal organs of insects.
The first project of the present thesis involved general characteristics of DPOAE generation in the bushcricket Mecopoda elongata and the selective exclusion of the scolopidial mechanoreceptors using the neuroactive insectizide pymetrozine (Möckel et al. 2011). Pymetrozin appears to act highly effective and selectively on chordotonal organs, without affecting other sensory organs that lack scolopidial receptors. Pymetrozine solutions were applied as closely as possible to the scolopidia via a cuticle opening in the tibia, distally to the organ. Applications at concentrations between 10-3 and 10-7 M led to a pronounced and irreversible decrease of DPOAE amplitudes. Both this study on bushcrickets (Möckel et al. 2011) and an earlier one on locusts (Möckel et al. 2007) hence indicate the involvement of scolopidia in DPOAE generation in insects, by using complementary methods (pharmacological versus mechanical manipulation) and different animal models.
The second project of the present thesis investigated the temperature-dependence of DPOAEs in the locust Locusta migratoria (Möckel et al. 2012). The suggested biological origin of acoustic two-tone distortions in insects should involve metabolic processes, whose temperature-dependence would directly affect the DPOAE generation. Body temperature shifts resulted in reversible, level- and frequency-dependent effects on the 2f1–f2 emission. Using low f2 frequencies of up to 10 kHz, a body temperature increase (median +8–9°C) led to an upward shift of DPOAE amplitudes of approximately +10 dB, whereas a temperature decrease (median –7°C) was followed by a reduction of DPOAE amplitudes by 3 to 5 dB. Both effects were only present in the range of the low-level component of DPOAE growth functions below f2 stimulus levels of approximately 30-40 dB SPL. Emissions induced by higher stimulus levels and frequencies (e.g. 12 and 18 kHz) remained unaffected by any temperature shifts. The Arrhenius activation energy of the underlying cellular component amounted to 34 and 41 kJmol-1 (for growth functions measured with 8 and 10 kHz as f2, respectively). Such activation energy values provide a hint that an intact dynein-tubulin system within the scolopidial receptors could play an essential part in the DPOAE generation in tympanal organs.
The third project of this thesis demonstrated mechanical DPOAE analogs in the tympanum's vibration pattern during two-tone stimulation in the locust Schistocerca gregaria, using laser Doppler vibrometry (Möckel et al. 2014). DPOAE generation crucially relies on the integrity of the scolopidial mechanoreceptors (Möckel et al. 2007, 2011), which in locusts, directly attach to the tympanal membrane. During two-tone stimulation, DPOAEs were shown to mechanically emerge at the tympanum region where the auditory mechanoreceptors are attached. Those emission-coupled vibrations differed remarkably from tympanum waves evoked by external pure tones of the same frequency, in terms of wave propagation, energy distribution, and location of amplitude maxima. In contrast to traveling wave-like characteristics of externally evoked vibrations, intrinsically generated waves were locally restricted to the region around the high frequency receptors’ attachment position. The mechanical gradient of the tympanal membrane that leads to direction-dependent properties probably avoids the spreading of these locally evoked waves, which are then reflected and occur only in restricted areas as standing waves. Selective inactivation of mechanoreceptors by mechanical lesions did not affect the tympanum's response to external pure tones, but abolished the emission's displacement amplitude peak. These findings provide evidence that tympanal auditory receptors, comparable to the situation in mammals, comprise the required nonlinear response characteristics, which during two-tone stimulation lead to additional, highly localized deflections of the tympanum.
By far not all genetic information is expressed by mRNA coding regions of the DNA. 98% of the human genome is not encoding for proteins. Therefore, these non-coding regions have been considered as “junk DNA” for a long time [1, 2]. The last years, new high throughput sequencing techniques have allowed the elucidation of the heterogeneous population of non-coding RNAs (ncRNAs, Table 1). RNAs longer than 200 nucleotides (nt) belong to the family of long non-coding RNAs (lncRNAs). They can exhibit numerous functions: The biggest family of RNAs is represented by the ribosomal RNAs (rRNAs). Together with the transfer RNAs (tRNAs) they are essential for the translation of mRNA into an amino acid sequence.
Die soziale Arbeitsteilung bei Honigbienen ist ein komplexes selbstorganisatorisches System, welches auf zwei Ebenen der biologischen Organisation zu verorten ist: dem Individuum und der Kolonie. Die Regulation der Bruttemperatur ist ebenfalls diesen Gesetzmäßigkeiten unterworfen. Die Arbeits-bereitschaft einzelner Bienen bildet die Grundlage für die Temperaturregulierung des kolonialen Brutnestes.
In dieser Arbeit wird dieses Zusammenspiel aus individuellen Beteiligungen der Arbeiterinnen sowie der erbrachten Gesamtleistung der Kolonie während des Brutwärmens untersucht. Dazu wird eine kleine Bienengruppe auf einer Brutwabe einer thermischen Belastung ausgesetzt. Ein speziell für diese Untersuchungen entwickelter Versuchsaufbau integriert erstmals die Infrarot-Thermografie mit den Temperaturmessungen einer Brutfläche. Somit ist es möglich, die Thoraxtemperaturen der einzelnen, am Brutwärmen beteiligten Arbeiterinnen störungsfrei zu messen und gleichzeitig das erzeugte räumliche und zeitliche Temperaturmuster der Brutwabe zu ermitteln. Zusätzlich wird der Temperaturverlauf der Außentemperatur sowie der zellumgebenden Luft untersucht.
Es kann gezeigt werden, dass die Lufttemperatur im Innenraum eines Bienenstocks ein wichtiger Faktor in der Temperaturregulierung des Brutnestes ist, da sie die untere Temperaturgrenze im Bienenstock bildet. Weiterhin wird der Einfluss der brutwärmenden Arbeiterinnen auf die Temperaturentwicklung einer Brutfläche sichtbar. Durch das flexible Verhalten der Arbeiterinnen kann einer Brutfläche bei thermischer Belastung durch lokal wechselndes Brutwärmen optimal Wärme zugeführt werden. Es gibt es Hinweise auf eine zyklische Periodizität im zeitlichen Temperaturverlauf der Brutzellen, welche auf einen Brutwärmrhythmus durch die Bienen schließen lässt. Durch den Einsatz zweier Unterarten (Apis mellifera carnica & Apis mellifera mellifera) wird sichtbar, dass es zwischen den Gruppen Unterschiede in der Aufrechterhaltung der Lufttemperatur über der Wabe gibt.
Myxobacteria are on order of Gram-negative, soil dwelling bacteria that feature an impressive number of properties: they can glide on solid surfaces by using two different motility motors, subsist by preying on other microorganisms, are often producers of multiple natural products, and upon adverse environmental conditions, they are able to form multicellular structures called “fruiting bodies”. The process, in which these macroscopically visible structures arise from independent single cells, has been the predominant subject of myxobacterial research for many decades. More precisely, researchers have strived for the discovery of genes, proteins and small molecules that act as signals, receivers or modulators of this complex process. In this regard, the species Myxococcus xanthus has evolved into the model organism due to its relatively simple and reliable handling in a laboratory environment. The research underlying this thesis focused on the identification and biosynthesis of lipids that may act as intercellular signaling molecules during the course of fruiting body formation of the myxobacterium Myxococcus xanthus as part of the “E-signal” system. In general, lipids containing branched-chain fatty acids with an uneven number of carbon atoms were found to be important players in this particular process. Nevertheless, their exact roles remain largely unknown as of this day. The first publication that is part of this thesis deals with an aspect that even strengthened the importance of role of iso-branched compounds in myxobacteria: myxobacterial metabolism is able to transform precursors of iso-lipids to isoprenoids. It addresses the question whether isoprenoids in general are important for fruiting body formation. Phenotypic analysis of mutants impaired in the biosynthesis of the central isoprenoid precursor 3-hydroxymethylglutaryl-Coenzyme A (3-HMG-CoA) from acetate and/or branched chain keto acids and their genetic and metabolic complementation clearly showed that isoprenoids are essential for fruiting body formation and confirmed that leucine derived isovalerate is an important source for isoprenoid precursors in myxobacteria. The second, and by far and away most tedious and sophisticated study, addressed the question as to how myxobacteria form fatty acid derived iso-branched ether lipids and to what extent they are important for fruiting body formation and sporulation. In a previous study, those unusual lipids were identified as specific biomarkers for myxobacterial development. No biochemical pathways to ether lipids specific for prokaryotes were known by then. In this study, a putative candidate gene that may be in involved in ether lipid biosynthesis was investigated. A combination of gene disruption and complementation experiments, phenotypic analysis and monitoring of ether lipid formation by means of GC-MS demonstrated its involvement in myxobacterial ether lipid biosynthesis and the importance of these lipids for the developmental process. Heterologous expression and biochemical testing of this gene together with in-silico sequence analysis and docking experiments confirmed the functions of its predicted domains. The discussion section provides an additional suggestion on how the ether bond formation is performed. Furthermore and most importantly, iso-branched ether lipids were found to be essential for sporulation but not for fruiting body formation. In summary, one or several molecules derived from an iso-branched alkylglycerol seem to play a role during sporulation in M. xanthus and a multidomain enzyme unique for myxobacteria is involved in their biosynthesis. The last manuscript addresses the complexity of lipid metabolism in myxobacteria. Prior to this work, there was limited knowledge about the exact composition of the myxobacterial lipidome and no method was available to monitor putative changes in the myxobacterial lipidome down to the single molecular species for studying lipid biosynthesis or regulation. An ultra-performance liquid chromatography coupled with mass spectrometry based method with electrospray ionization (UPLC-ESI-MS) utilizing standard equipment and a water/acetonitrile/isopropanol based eluent system proved to be geared for the construction of lipid profiles for wild type and mutant cells of M. xanthus and to show their differences. Fragmentation spectra based structure elucidation of lipid molecular species resulted in the identification of 99 molecular species comprising glycerophosphoethanolamines, glycerophosphoglycerols, glycerolipids, ceramides and ceramide phosphoinositols. The latter have never been described for any prokaryotes before. Three dimensional plots were created from the relative intensity differences of the single molecular ion species between the different samples to provide an efficient and versatile visualization of the data and enable the researcher to quickly detect differences.
RNA modifications are present in all three kingdoms of life and detected in all classes of cellular RNAs. RNA modifications are diverse, with more than 100 types of chemical modifications identified to date. These chemical modifications expand the topological repertoire of RNAs and are expected to fine-tune their functions. Ribosomal RNA (rRNA) contains two types of covalent modifications, either methylation on the sugar (Nm) or bases (mN), or base isomerization (conversion of uridine into pseudouridines, "). Pseudouridylations and ribose methylations are catalyzed by site-specific H/ACA and C/D box snoRNPs, respectively. The RNA component (snoRNA) of both types of snoRNPs is responsible for the site selection by base pairing with the rRNA substrate, whereas the protein component catalyzes the modification reaction: Nop1 in C/D box and Cbf5 in H/ACA box snoRNPs. Contrastingly, base methylations are performed by snoRNA independent, ‘protein-only’, methyltransferases (MTases). rRNA modifications occur at highly conserved positions, all clustering around functional ribosomal sites. Mutations in factors involved in rRNA modification have been linked to severe human diseases (e.g. X-linked Dyskeratosis congenita). Emerging evidences indicate that heterogeneity in RNA modification prevails, i.e. not all positions are modified at all time, and the concept of ‘specialized ribosomes’ has been coined. rRNA modification heterogeneity has been correlated with disease etiology (cancer), and shown to play a role in cell differentiation(hematopoiesis). Remarkably, alteration in rRNA modification patterns profoundly affects the preference of ribosomes for cap- versus IRESdependent translation initiation, with major consequences on cell physiology.
In den vergangenen Jahren haben ökologische Fragen in der Naturstoffforschung mehr und mehr an Bedeutung gewonnen. Naturstoffe bilden dabei einen wichtigen Aspekt in der Aufrechterhaltung symbiotischer Systeme.
Symbiosen stellen eine der treibenden Kräfte der Evolution dar. Diese artenübergreifende Interaktion zweier Organismen ermöglicht die Evolution in wechselseitiger Anpassung, wobei per Definition in die Kategorien Mutualismus, Kommensalismus und Parasitismus unterschieden wird. Teilweise führt die obligatorische Abhängigkeit eines Organismus zum partiellen Merkmals- und Stoffwechselwegverlust, der durch seinen Symbiose-Partner kompensiert wird. In den meisten Fällen stellt Symbiose ein komplexes Netzwerk aus mehr als zwei Lebewesen dar.
Diese Arbeit beschreibt die Anwendung der Klonierungsmethode ExRec ("overlap extension PCR-yeast homologous recombination") für die vereinfachte Bereitstellung von Naturstoffen. Es konnte ein 45 kb großes Gencluster erfolgreich kloniert und zwei neue Peptide Ambactin und Xenolindicin aus Xenorhabdus charakterisieren werden, wobei letztgenanntes von einem stillen Gencluster stammt. ExRec stellt eine sehr effiziente und wichtige Methode für die Klonierung großer Gencluster als auch für die Klonierung aus Metagenombibliotheken und RNA Pools dar...
Fungal organisms, including the most common human pathogens Candida spp., are commensal organisms that are widely present as part of the human flora. Fungal infections are, most frequently, local infections that do not compromise the life of patients. However, mycotic diseases can be life-threatening if they become systemic infections. Systemic fungal infections have risen over the last three decades in parallel to the increased immune-compromised population as a consequence of diseases (e.g. HIV/AIDS) or therapeutic interventions that affect the immune system (e.g. chemotherapy for cancer treatment and immunosuppressors used for patients with organ transplants). This has resulted in the demand of new antifungal drugs that can eradicate the new infections caused by these opportunistic fungal pathogens. However, most of the current compounds have poor pharmaceutical properties such as narrow spectrum of activity, susceptibility to be extruded by efflux pumps or lack of specificity, which make them not suitable for human clinical applications. The treatment of fungal and parasitic infections has been traditionally difficult because the infective organisms are eukaryotic cells that share most of the pathways and enzymes with human cells. To avoid side effects and to develop a targeted therapy, the research has traditionally been centered on the very few enzymes and pathways existing in the infectious organism but absent in humans. Until now, antifungal therapeutic options are limited and are almost dominated by azole class of sterol biosynthesis inhibitors affecting the synthesis of ergosterol, a major constituent of the fungal cell membrane. Because human cells do not have a cell wall, the development of effective and safe antifungal agents has also been directed to enzymes required for the synthesis of the cell wall. Alternatively, it is theoretically possible to target enzymes that are present in fungal organisms and in humans, when: 1) sufficient selectivity can be achieved, and 2) inhibition of the fungal enzyme is lethal to the fungus but does not produce major side effects to humans. In this line, it would be ideal to evaluate the development of selective inhibitors of enzymes which are already known to be drug targets, like protein kinases.
Cancer is a disease characterized by uncontrolled cell growth and the capacity to disseminate to distant organs. The properties of cancers are caused by genetic and epigenetic alterations when compared to their normal counterparts. Genetic mutations occur in oncogenes and tumor suppressor genes and are the initial drivers of cellular transformation (Lengauer et al., 1998; Vogelstein and Kinzler, 2004). In addition, epigenetic alterations, which influence the expression of oncogenes and tumor suppressor genes independently from sequence alterations, are also involved in the transformation process (Esteller and Herman, 2001; Sharma et al., 2010). Genetic alterations and epigenetic regulatory signals cooperate in tumor etiology. Glioblastoma multiforme (GBM) is a frequent and aggressive malignant brain tumor in humans. The median survival of GBM patients is about 15 months after diagnosis. Like in other cancers, genetic and epigenetic alterations can be detected in GBM. Genetic alterations in GBM affect cell growth, apoptosis, angiogenesis, and invasion; however, epigenetic alterations in GBM also affect the expression of oncogenes or tumor suppresser genes that increase tumor malignancy (Nagarajan and Costello, 2009).
Reprogramming is a cellular process in which somatic cells can be induced to assume the properties of less differentiated stem cells. This process can be mediated through epigenetic modifications of the genome of somatic cells by the action of four defined transcription factors (Oct4, Sox2, Klf4 and Myc) or by the action of the miR 302/367 cluster (Anokye-Danso et al., 2011; Takahashi and Yamanaka, 2006; Takahashi et al., 2007) and result in the generation of induced pluripotent stem cells (iPS cells). Reprogramming of somatic cells by the miR 302/367 cluster can generate nontumorigenic iPS cells through the inhibition of the epithelial to mesenchymal transition (EMT), cell cycle regulatory genes and epigenetic modifiers (Lin and Ying, 2013).
In this study I analysed past and recent Daphnia populations from Lake Constance and Greifensee. Herefore, I first established a set of microsatellite markers applicable to European Hyalodaphnia species (chapter 1). Primers were also identified for species specific fragment lengths. 32 markers were then available to characterize the resting egg banks of Daphnia galeata and D. hyalina. Chapter 2 presents the reconstruction of the taxonomic composition in these two ecologically different lakes. This part of my work shows that the eutrophication that occurred in both lakes in the mid of the last century has strongly influenced the Daphnia populations. In both lakes Daphnia galeata established and hybridized with the indigenous D. hyalina. Interspecific hybridization resulted in introgression on the mitochondrial and nuclear level. In chapter 3 resting eggs from the sediments of the 1960s, 1970s, 1980s, 1990s and 2000s were characterized with microsatellite markers. The aim was to specify the extent of interspecific hybridization and nuclear introgression assuming that the genetic exchange between both species has an impact on their adaptation to their habitat. In life history experiments D. galeata and D. galeata x hyalina clones hatched from different time periods showed significant differential responses to food quality. Therefore, the question had to be answered how the Daphnia resting egg bank and the planktonic population are connected. In chapter 4 hatching experiments were conducted to bridge this gap of scientific knowledge in the life cycle of cyclic parthenogenetic waterfleas. Only D. galeata individuals were able to establish a clonal lineage after maturity. All observed recombinant individuals did not reproduce at all or firstly went through another sexual phase of reproduction i.e. produced resting eggs. In order to compare the findings of chapter 4 with the taxon composition of the recent planktonic population of Daphnia in Lake Constance, samples were taken over one season (between May 2005 and September 2006). During the season, the taxonomic composition of Daphnia changes severely with D. galeata being most abundant during the warm season and D. hyalina in the cold season. Moreover, some individuals were detected, that did not follow this pattern. With mitochondrial analysis those individuals were identified as mitochondrial introgressants and processed to life history experiments. Significant differences in the somatic growth rate under different temperatures (5°C, 12.5°C and 20°C) were related to the origin of the mitochondrial genome rather than the nuclear taxonomic assignment of the individual.
The findings of this study show that all organisms exposed to rapid ecological changes and their microevolutionary reaction to those.
Alzheimer’s disease (AD) is a common, age associated neurodegenerative disease that manifests as progressive dementia and is characterized by accumulation of the amyloid beta (Aβ) peptide which is a processing product of a transmembrane protein termed Alzheimer Amyloid Precursor Protein (APP). The Aβ peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed β- and γ-secretase. The β-secretase, also called BACE-1 or memapsin 2, belongs to the family of aspartyl proteases. BACE-1 evidently cleaves APP in an acidic endosomal compartment after endocytosis of APP, thereby facilitating Aβ peptide generation.
Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. The short cytoplasmic tail of BACE-1 with 23 amino acids contains a sorting signal of the acidic cluster, di-leucine (ACDL) type. The two Leu residues in this determinant are important for the clathrin mediated endocytosis of BACE-1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE-1 back to the plasma membrane. The ACDL motif binds to the members of the GGA (Golgi-localized γ ear-containg ARF- binding proteins) family (GGA1-GGA3) that are involved in the sorting of BACE-1.
One of the major aims of this study was to address the role of flotillins in the intracellular sorting of BACE-1. This study shows that flotillin-1 directly binds to the di-leucine motif in the cytoplasmic tail of BACE-1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillin-1 competes with GGA2 for the binding to BACE-1 tail, and thus influences the endosomal sorting of BACE-1. Importantly, depletion of flotillins results in an altered localization of the wildtype BACE-1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. Flotillin knockdown results in an accumulation of BACE-1, implicating reduced degradation and enhanced stability of this protease. Thus, flotillins appear to be important for the cellular targeting of BACE-1 and also influence the amyloidogenic processing of APP, as demonstrated by an increase in the amyloidogenic C-99 processing fragments.
When flotillin depleted cells were subjected to apoptotic stresses including Aβ25-35 synthetic peptide (inducer of the extrinsic apoptosis pathway) or several chemotherapeutic agents (staurosporine, brefeldin A, doxorubicin, carboplatin and paclitaxel: intrinsic apoptosis pathway) and cytotoxicity was determined, various apoptotic markers were activated in flotillin depleted cells. Caspase-3 and GGA3 are well accepted apoptosis markers and an enhanced caspase-3 cleavage was detected upon STS induced apoptosis in SH-SY5Y, HeLa, and HaCaT cell lines and increased GGA3 cleavage was observed in MCF7 cell line.
One of the major reasons for the apoptotic sensitivity in the absence of flotillins was a PI3K/Akt signaling defect. Neuroblastoma cells depleted of flotillins showed diminished levels of total Akt, phospho-Akt and phospho-ERK upon STS induced apoptosis. Since PI3K/Akt was the primary survival pathway affected upon STS induced apoptosis, ectopic expression of Akt in neuroblastoma cell line reduced caspase-3 cleavage and retarded apoptosis.
The direct downstream target of Akt is FOXO3a, whose localization was investigated in flotillin depleted cells. A major proportion of FOXO3a was localized in the nucleus of flotillin knockdown cells, implicating that FOXOs are active in these cells and subsequently trigger the transcription of death genes. Strikingly, an essential anti-apoptotic molecule and a major cancer target, Mcl-1, was inherently downregulated in flotillin knockdown cells. Mcl-1 is a chief member of the Bcl-2 family as it plays a pivotal role in cell survival and it is a critical protein in cancer therapeutics as suppression of Mcl-1 protein can curtail the survival and growth of tumorous cells.
Neuroblastoma cells were rescued from undergoing permanent damage due to STS induced apoptosis by overexpression of anti-apoptotic Bcl-2. Phorbol esters are well known PKC activators, and pre-treatment of neuroblastoma cells with phorbol esters along with staurosporine reduced caspase-3 cleavage.
These results demonstrate that absence of flotillins can sensitize cellular systems to apoptosis induction. The two main characteristics of cancer cells include resistance to apoptosis and unresponsiveness to chemotherapeutic agents. It is a well established fact that impaired apoptosis is central to tumour development. This study implicates that the downregulation of flotillin function can trigger cellular susceptibility and enhances apoptosis in response to conventional chemotherapeutic agents. Therefore, flotillins can serve as vital regulators in providing a more rational approach in molecular-targeted therapies for receding cancer growth and survival.
Wie andere Vögel auch, verfügen Hühner über zwei verschiedene Magnetfeldrezeptoren. In der vorliegenden Arbeit werden diese beiden Rezeptoren, vor allem unter dem Aspekt Verhaltensontogenie eingehender untersucht. Meine Ergebnisse werden durch histologische Untersuchungen gestützt. Ich untersuchte zwei Hühnerrassen, einen braunen und einen weißen Legehuhn Stamm. Mit der Standardmethode konnte ich die Befunde der Literatur bestätigen. Zur Untersuchung des Magnetkompasses im Auge, habe ich Hühner darauf trainiert einen roten Tischtennisball, auf den sie geprägt wurden, in einer bestimmten magnetischen Richtung zu suchen. Im unbelohnten“ Test ist das Magnetfeld um 90 Grad gedreht, so dass der magnetische Norden nun im geographischen Osten liegt. Die braunen Hühner benutzen den Magnetkompass zum Lösen der gestellten Aufgabe, die weißen Hühner wählen zufällig eine Richtung. Eine Veränderung der Trainingsmethode, ein Training im gedrehten Magnetfeld und eine „Bestrafung“, haben das Ergebnis verändert. Die weißen Hühner sind nun in der Lage, die magnetisch richtige Richtung zu finden, die braunen Hühner reagieren verängstigt und wählen nur zufällig eine Richtung. Beide Hühnerrassen können also - unter verschiedenen Voraussetzungen - einen magnetischen Kompass für die Orientierung benutzen.
Die Funktion der äußeren Haarsinneszellen geht weit über die normale Rezeptoreigenschaft der Kategorie Mechanorezeptor hinaus. Äußere Haarzellen mit ihrer reichhaltigen efferenten Innervierung sind nicht nur für die sensorische Aufnahme mechanischer Bewegung zuständig, sondern ermöglichen aufgrund ihrer motorischen Funktionen die mechanische Verstärkung der Wanderwelle in der Cochlea. Äußere Haarzellen sind eine maßgebliche Komponente des ´cochleären Verstärkers` und ihr Ausfall führt zur Schwerhörigkeit. Beiprodukte des cochleä-ren Verstärkers sind otoakustische Emissionen, deren Messung Aufschluss über aktive mechanische Prozesse im Innenohr gibt.
Die äußeren Haarsinneszellen bilden Synapsen mit dem olivo-cochleären efferenten System, welches im Zentrum der vorliegenden Untersuchung steht. Es vermittelt den Einfluss des Zentralnervensystems auf das Corti-Organ des Innenohrs. Über die akustische Reizung des olivo-cochleären Reflexbogens ist man in der Lage, das efferente System zu aktivieren und gleichzeitig die Antworteigenschaften der Cochlea zu verändern. Efferente Modulationen des cochleären Verstärkers können sich z. B. in einer Veränderung des Emissionspegels bemerk-bar machen. Die Fledermausspezies Carollia perspicillata ist aufgrund ihres Echoortungs-systems mit einem sehr sensitiven und hochauflösenden Hörvermögen ausgestattet und eignet sich hervorragend als Modelltier in der Hörforschung, insbesondere auch deshalb, da oto-akustische Emissionen sehr gut messbar sind.
Das efferente System von C. perspicillata wurde in dieser Untersuchung durch akustische Stimulation der kontralateralen Cochlea angeregt. Die Stimuli, die nicht nur in ihrem Pegel sondern auch in ihrer Bandbreite und in der Mittelfrequenz in Relation zu den ipsilateralen Stimulusfrequenzen variierten, beeinflussten dabei die Generierung der otoakustischen Emis-sionen (DPOAE, engl: distortion product otoacoustic emissions) im ipsilateralen Ohr: akustische Stimulation der kontralateralen Cochlea bewirkte zuverlässig eine Änderung der DPOAE- Amplitude im kontralateralen Ohr. Vor allem eine Suppression des cochleären Verstärkers in Form von DPOAE-Pegelverminderungen wurde beobachtet. Die supprimieren-den Effekte erreichten trotz leiser bis moderater kontralateraler Rauschpegel (bis maximal 54 dB SPL) Werte von bis zu 14, 17.1 und 13.9 dB SPL (bei f2 = 20, 40 und 60 kHz und effek-tivstem kontralateralen Rauschstimulus) und waren damit deutlich größer als in vorangegang-enen Studien an anderen Spezies. Die DPOAE-Pegelverminderungen waren positiv mit dem x Pegel der kontralateralen akustischen Stimulation, ebenso wie seiner Bandbreite und der Mittelfrequenzen in Relation zu den ipsilateralen Stimulusfrequenzen korreliert. Es gab keinen absoluten Frequenzbereich, in dem die efferenten Effekte am größten gewesen wären. Vielmehr traten maximale Effekte immer durch etwas oberhalb der ipsilateralen Stimulusfre-quenzen gelegene kontralaterale Rauschstimuli auf. Die Effekte waren auch abhängig von der Bandbreite des kontralateralen Rauschstimulus und maximal bei einer relativen Bandbreite von 1.5 Oktaven. Die Verschiebung des efferenten Effekts hin zu hohen Frequenzen und die Bandbreitenabhängigkeit sind vereinbar mit den anatomischen Eigenschaften der Projektio-nen der medialen olivo-cochleären Efferenzen in der Säugetiercochlea. Kontralaterale akusti-sche Reizung bewirkte auch eine Verschiebung der Wachstumsfunktionen der 2f1-f2 -DPOAE in einen unsensitiven Bereich und außerdem eine Verformung der Wachstumsfunktion. Bei-des könnte durch Beeinträchtigung des cochleären Verstärkers verursacht sein. Eine Beteili-gung des Mittelohrmuskels an den Effekten kann nahezu ausgeschlossen werden und die beobachteten Effekte sind höchstwahrscheinlich dem olivo-cochleären System zuzuschreiben.
Funktionell ist denkbar, dass bei C. perspicillata das mediale olivo-cochleäre System im Kontext einer Frequenzverschärfung bei der cochleären Verstärkung der Basilarmembranbe-wegung aktiv wird. Aus diesem Grund wurden ipsilateral sogenannte DPOAE-Suppressions- Abstimmkurven gemessen, welche die mechanische Abstimmschärfe im Innenohr beschrei-ben. Während und nach kontralateraler Reizung kam es zu Veränderungen der Abstimmkur-ven. Signifikante Effekte konnten allerdings nicht festgestellt werden, da die Veränderungen der Suppressions-Abstimmkurven variabel und schlecht kategorisierbar war.
Die vorliegenden Ergebnisse unterstützen weit verbreitete Hypothesen zur Funktion der medialen olivo-cochleären Effernzen in Bezug auf mechanische Suppression, Verbesserung des cochleären Signal-Rauschverhältnisses und einer generellen frequenzspezifischen Wirkung.
Protein quality control systems (PQC), i.e. UPS and aggresome-autophagy pathway, have been suggested to be a promising target in cancer therapy. Simultaneous pharmacological inhibition of both pathways have shown increase efficacy in various tumors, such as ovarian and colon carcinoma. Here, we investigate the effect of concomitant inhibition of 26S proteasome by FDA-approved inhibitor Bortezomib, and HDAC6, as key mediator of the aggresome-autophagy system, by the highly specific inhibitor ST80 in rhabdomyosarcoma (RMS) cell lines. We demonstrated that simultaneous inhibition of 26S proteasome and selective aggresome-autophagy pathway significantly increases apoptosis in all tested RMS cell lines. Interestingly, we observed that a subpopulation of RMS cells was able to survive the co-treatment and, upon drug removal, to recover similarly to untreated cells. In this study, we identified co-chaperone BAG3 as the key mediator of this recovery: BAG3 is transcriptionally up-regulated specifically in the ST80/Bortezomib surviving cells and mediates clearance of cytotoxic protein aggregates by selective autophagy. Impairment of the autophagic pathway during the recovery phase, both by conditional knock-down of ATG7 or by inhibition of lysosomal degradation by BafylomicinA1, triggers accumulation of insoluble protein aggregates, loss of cell recovery and cell death similarly to stable short harpin RNA (shRNA) BAG3 knock-down. Our results are the first demonstration that BAG3 mediated selective autophagy is engaged to cope with proteotoxicity induced by simultaneous inhibition of constitutive PQC systems in cancer cell lines during cell recovery. Moreover, our data give new insights in the regulation of constitutive and on demand PQC mechanisms pointing to BAG3 as a promising target in RMS therapy.
Die X-chromosomal gebundene chronische Granulomatose (X-CGD) ist eine seltene Erbkrankheit, bei der die NADPH-Oxidase der Phagozyten nicht funktionell ist. Der Grund hierfür liegt meist in Mutationen in der GP91phox Untereinheit der Phagozyten-Oxidase. Hierdurch treten lebensbedrohliche Bakterien- und Pilzinfektionen bei Patienten auf, was neben einer geringen Lebensqualität zu einer erheblich verkürzten Lebenserwartung führt. Eine Stammzelltransplantation eines gesunden Spenders ist bislang der einzige heilende Therapieansatz. Für X-CGD-Patienten, die keinen passen-den Spender zur Verfügung haben, stellt die genetische Modifikation autologer hämato-poetischer Stammzellen eine alternative Form der Therapie dar. Im Jahr 2004 wurden daher in einer präklinischen Phase I/II Studie in Frankfurt zwei X-CGD-Patienten gentherapeutisch behandelt. Hierbei wurden CD34+ Stammzellen ex vivo mit einem γ-retroviralen Vektor transduziert, der eine LTR-getriebene Expressionskassette für GP91phox trägt. Nach einer nicht-myeloablativen Konditionierung wurden die genetisch modifizierten Zellen der Patienten retransplantiert. Beide behandelten Patienten zeigten schon kurz nach Therapiebeginn eine deutliche Verminderung der Infektionsanfälligkeit und somit eine stark verbesserte Lebensqualität. Auf zellulärer Ebene konnte ein gutes Engraftment der modifizierten hämatopoetischen Stammzellen im Knochenmark beobachtet werden. In funktionellen Tests konnte die Bildung superoxidproduzierender Phagozyten für die Immunabwehr gezeigt werden. Das molekulare Monitoring beider Patienten hat jedoch über die Zeit eine Verringerung der Enzymaktivität in den Phagozyten (Superoxidproduktion) gezeigt, obwohl der Anteil genetisch modifizierter Zellen nicht geringer wurde. Im Rahmen der vorliegenden Arbeit konnte durch quantitative RT-PCR-Analysen proviraler mRNA-Transkripte, eine Korrelation zwischen dem Verlust der Enzymaktivität und reduzierter Transgen-expression gezeigt werden. Durch DNA-Analysen peripherer Blutproben beider Patienten konnte eine verstärkte Methylierung an der Promotor-CpG-Insel, welche die Transgen-expression reguliert, als Ursache identifiziert werden. Weiterführende klonale Untersuchungen genmodifizierter Kolonien aus dem Knochenmark der Patienten offenbarten einen direkten Zusammenhang zwischen der Abwesenheit von Transkription bzw. Superoxidbildung und der Methylierung dieser CpG-Insel im proviralen Promotor-bereich. Somit konnte zum ersten Mal ein epigenetisches Silencing bei Patienten nach einer Behandlung mit Gentherapie nachgewiesen werden. In weiteren Versuchen konnte die vollständig ausgebildete, spezifische Methylierung des SFFV-Promotors in transduzierten Knochenmarkzellen eines Patienten durch in vitro Behandlung mit einem Methyltransferase-Inhibitor (Aza-D) in Kombination mit einem Histondeacetylase-Inhibitor (TSA) bis zu 30% reduziert werden. Dieser Teilerfolg zeigt, dass eine klinisch relevante Reaktivierung der Transgenexpression, durch Umkehrung des Silencings am SFFV-Promotor, prinzipiell möglich ist. Das Phänomen der Abschaltung der Genexpression des γ-retroviralen Vektors in der Frankfurter Gentherapiestudie, hat ein Testsytem zur Evaluierung zukünftiger Gentherapie-Vektoren erfordert. Durch Monitoring proviraler Parameter (Kopien, Transgenexpression, Proteinexpression und Promotor-CpG-Methylierung), in der murinen embryonalen Stammzelllinie P19 konnte in dieser Arbeit ein prädiktiver Silencing-Assay erfolgreich etabliert werden. Mit Hilfe dieses Systems wurden vielversprechende Silencing-resistente Vektoren mit dem UCOE (Ubiquitous Chromatin Opening Element) identifiziert. Hierdurch wurden wichtige Grundlagen geschaffen, um zukünftige virale Vektorsysteme in Bezug auf ihre Langzeitexpression testen zu können. Zusätzlich zu der Inaktivierung der transduzierten Expressionskassette konnte in beiden Patienten ein klonales Auswachsen von Subklonen beobachtet werden, das letztendlich zu einem myelodisplastischen Syndrom bei beiden Patienten führte. Der virale Enhancer war im Gegensatz zum viralen Promotor niemals methyliert, wodurch seine transaktivierenden Eigenschaften unbeeinflusst blieben. Diese enhancervermittelte Aktivierung proliferationsfördernder Gene (Mds1-Evi1-Genlokus) konnte durch RT-PCR-Analysen zunächst in Mischpopulationen aus peripherem Blut der Patienten nach-gewiesen werden. Weiterführende klonale Analysen in Knochenmarkzellen zeigten den direkten Zusammenhang zwischen der transkriptionellen Aktivierung des Mds1-Evi1-Genlokus und den proviralen Insertionen. Somit konnte die Ursache für die therapie-assoziierte, klonale Dominanz in beiden X-CGD-Patienten aufgeklärt werden. In der Frankfurter Gentherapiestudie wurde erstmals ein klinischer Erfolg für X-CGD-Patienten erzielt. Durch intensives molekulares Monitoring konnte im Rahmen dieser Arbeit aufgedeckt werden, dass der eingesetzte γ-retrovirale Vektor über das Phänomen der Insertionsmutagenese hinaus, auch in Bezug auf die epigenetische Abschaltung der Transkription (Silencing), für zukünftige Studien modifiziert werden muss. Sicherheits-verbesserte Vektoren mit einer Resistenz gegenüber Silencing in murinen embryonalen Stammzellen konnten in dieser Arbeit charakterisiert werden. Mit diesen Genfähren könnte der angestrebte Langzeittherapieerfolg in Zukunft möglich werden.
Autophagie ist ein evolutionär stark konservierter Degradationsmechanismus für geschädigte Proteine bis hin zu ganzen Organellen eukaryotischer Zellen. Dabei umhüllt eine Doppelmembran, bisher unbekannten Ursprungs, das zu degradierende Material und bildet das Autophagosom. Dies fusioniert später mit Lysosomen, wodurch dessen Inhalt proteolytisch zersetzt und die Bestandteile der Zelle wieder zur Verfügung gestellt werden kann.
In dieser Abeit wurde der Fokus auf den mitochondrialen Abbau über Autophagie (Mitophagie) und dessen Funktion als ein mitochondrialer Qualitätsmechanismus gesetzt. Als Zellmodell wurden primäre humane Endothelzellen der Nabelschnurvene (HUVEC) verwendet. Diese zeichenen sich durch einen Übergang von einer mitotischen, jungen in eine lange postmitotische, seneszente Phase während der Kultiverungszeit aus. Dabei durchlaufen sie einer zelluläre und mitochondriale Morphologieänderung. , wodurch sich die Möglichkeit bot , die Autophagie unter verschiedenen Parametern zu betrachten.
So wird generell eine Abnahme des autophagosomalen / lysosomalen Weges mit dem Alter beschrieben und die Abhängigkeit der Mitophagie von der mitochondrialen Länge.
Mitophagie ist unter normalen Kultivierungsbedingungen ein mikroskopisch selten zu beobachtender Vorgang. Daher wurde ein mitochondriales Schädigungsystem etabliert, welches die photosensibiliesierende Wirkung des Farbstoffs MitoTracker Red Cmx Ros (MTR) nutzt, um Mitochondrien gezielt oxidativ zu schädigen und die Mitophagie zu aktivieren.
Mitotische HUVEC zeigten 2 h – 8 h nach oxidativer Schädigung eine mitochondriale Fragmentierung größtenteils begleitet von einem Verlust des Membranpotentials. Über einen Zeitraum von 72h-120h kam es zur Regeneration des mitochondrialen Netzwerks durch Neusynthese mitochondrialer Biomoleküle. Entgegen der rescue Hypothese konnten oxidativ geschädigte Mitochondrien nicht durch eine Fusion mit funktional intakten Mitochondrien gerettet werden und wurden über den autophagosomalen / lysosomalen Weg abgebaut, gekennzeichnet durch die Ubiquitin-Ligase Parkin vermittelte Markierung und finaler Kolokalisation mit den autophagosomalen und lysosomalen Markerproteinen LC3B und LAMP-2A. Auf mRNA- und Proteinebene zeigte sich in diesem Zeitraum eine erhöhte Expression autophagie-relevanter Gene (ATGs) ATG5, ATG12 und LC3B.
Der Vergleich von mitotischen mit postmitotischen HUVEC nach oxidativer Schädigung wies zwei grundlegende Unterschiede auf.
Zum einem behielten, in Gegensatz zu jungen Zellen, die Mitochondrien alter HUVEC ihre Morphologie und ihr Membranpotential bei. Diese erhöhte Widerstandfähigkeit gegenüber oxidativem Stress konnte auf die erhöhte Expression der mitochondrial lokalisierten Serin / Threonin Kinase PINK1 zurückgeführt werden, ein Schlüsselgen in Parkinson.
Die PINK1-Transkription stand invers zu der Expression der mitochondrialen Teilungsfaktoren Fis1- und Drp1, welche in postmitotischen HUVEC stark vermindert war.
Andererseits wiesen alte Zellen eine verminderte Degradationsfähigkeit geschädigter Mitochondrien auf. Dieser Umstand war durch eine verminderte lysosomale Azidität bedingt. Eine externe ATP-Zugabe förderte die Azidität der Lysosomen alter Zellen und die Fusion mit Autophagosomen, wodurch Mitochondrien und ihre geringere ATP-Produktion im Alter als ein Faktor der Autophagie ermittelt weden konnte.
Die Autophagierate steht in Verbindung mit der Lebensspanne von Zellen bis hin zu ganzen Organismen. Durch die Überexpression autophagie-relevanter GFP-Fusions-Proteine ATG5, ATG12 und LC3B, welche nach oxidativer Schädigung in ihrer Expression verstärkt wurden, förderten die Mitophagie und wurden stabil in junge HUVEC exprimiert. Diese Überexpressionen bewirkten eine verbesserte mitochondriale Qualität, veranschaulicht durch ein erhöhtes Membranpotential und die ATP-Bereitstellung, einer besseren mtDNA Integrität und sie verlängerten die Lebensspanne signifikant, wobei die Produktion von reaktiven Sauerstoffspezien (ROS), entgegen der von Harman aufgestellten Alterungstheorie, keine Verminderung zeigte. Dennoch wiesen sie einen erhöhten Gehalt oxidativ modifizierter Proteine auf, welche letztendlich auf die erhöhten Autophagosomenanzahl zurückgeführt werden konnte, in denen höchstwahrscheinlich das oxidativ geschädigte Material gelagert wird.
In dieser Arbeit kann gezeigt werden, dass Mitochondrien nach oxidativer Schädigung eine Teilung vollziehen und geschädigte Mitochondrien selektiv über Autophagie abgebaut werden. Dabei fungiert Mitophagie als ein mitochondrialer Qualitätmechanismus und steht unmittelbar mit der Lebensspanne in Verbindung.
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.
The dependence of the Escherichia coli Na+H+ antiporter A (EcNhaA) pH sensor mutant E241C on H+ and Na+ concentrations was tested using a solid supported membrane (SSM) based electrophysiological approach. Proteoliposome preparations with right side out (RSO) oriented carriers were used to investigate the passive downhill uptake mode (physiologically the reverse transport mode) at zero membrane potential. Na+ concentration gradients established with a rapid solution exchange acted as the driving force. When a Na+ concentration gradient was established at symmetrical pH, the transport activity of the E241C EcNhaA variant was similar to that of the wildtype EcNhaA, with no shift of the bell-shaped pH dependence, an increase of the KmNa at acidic pH and a decrease of the KmNa at alkaline pH, supporting the model of a competitive binding of Na+ and H+ to a common binding site.
The phylogeny of the genus Gazella and the phylogeography and population genetics of arabian species
(2014)
Biodiversity is caused by a fundamental evolutionary process: speciation. When species can spread into new habitats and are allowed to colonize new ecological niches, speciation can become accelerated and is then called radiation. This can happen, e.g., when formerly separated land masses become connected. A prime example of such a scenario is the Arabian Peninsula that connects Africa and Asia since the Oligocene (approx. 30 Ma ago). Since then, the peninsula promoted several faunal exchanges between both continents. The mammalian genus Gazella is an excellent candidate for investigating this faunal exchange. Species are distributed on both, the African and Asian continent as well as on the Arabian Peninsula that is located in between. The aim of my thesis was to cast new light on the evolution and speciation of the genus and, furthermore, to evaluate the currently problematic taxonomy to infer suggestions for improved conservation actions for threatened gazelle species. Therefore, I investigated the taxon Gazella genetically and identified factors that promoted the speciation of this diverse genus. I assessed intraspecific genetic variability for species that inhabited the Arabian Peninsula to infer the past demography of those species and to estimate the history of species divergence and past population parameters.
In the first part of my thesis I inferred a mitochondrial phylogeny based on cytochrome b gene sequences using samples of all nine extant species of Gazella and also of closely related taxa (chapter 2). Besides the monophyly of the genus Gazella two reciprocally monophyletic clades were detected that evolved in allopatry: one predominantly African and one predominantly Asian clade. Within both clades species pairs could be inferred with species being ecologically adapted to different habitats: one species is a desert-dweller (probably the ancestral character state combination), while the other one is adapted to rather mountainous and humid habitats. These adaptations also correlate with the behavior of the species with the mountainous forms being sedentary, territorial and living in small groups and the desert forms being migratory, non-territorial and living in larger herds.
The second part of my thesis focuses on the Arabian gazelle species. In a study about G. subgutturosa I could show that the Arabian form G. marica (sand gazelle)—previously recognized as a subspecies of G. subgutturosa—is genetically distinct from the nominate form (chapter 3). Moreover, a phylogenetic tree based on cytochrome b gene sequences revealed a polyphyly of G. subgutturosa and G. marica with sand gazelles being more closely related to G. leptoceros and G. cuvieri of North Africa. Consequently, I suggested the restoration to full species level for G. marica corroborating earlier conservation practices of breeding both taxa separately in captivity.
In case of G. dorcas such a genetic differentiation could not be detected (chapter 4). Despite the large distribution range from Mali in the west to Saudi Arabia in the east only low genetic variation was detectable in mitochondrial sequence data. Statistically parsimony network analyses revealed pronounced haplotype sharing across regions. Using a coalescence approach I observed a steep population decline that started about 25,000 years ago and which is still ongoing. The decline could be correlated with human hunting activities in the Sahara. Hence, hunting of G. dorcas (already in ancient times) had a much larger impact on gazelle populations than previously thought and even led to the extinction of the Arabian form of G. dorcas.
In chapter 5 of my thesis I provided a rigorous test to genetically distinguish between the potential species G. gazella and G. arabica. Previously recognized as a single species mitochondrial sequence analyses provided first hints for the separation of both taxa. But without the investigation of nuclear loci the observed pattern could also be the result of male biased dispersal combined with female philopatry. Therefore, I amplified mitochondrial sequence markers and nuclear microsatellite loci for both taxa and found support for the earlier view of two separate species. No signs of recurrent gene flow could be detected between neighboring populations of G. arabica and G. gazella. The split of both species could be estimated one million years ago and the recommendation of breeding both taxa separately in captivity for conservation purposes is fully justified.
Several populations of G. arabica suffer from a severe decline. In chapter 6 I asked whether the population occurring on the Farasan archipelago—being at stable individual numbers for decades—may serve as potential source for future reintroduction on the Arabian mainland, although the gazelles show a reduced body size. Analyzing the genetic differentiation of Farasan gazelles, a genetic cluster could be inferred being endemic to the archipelago. However, only approx. 70% of Farasan individuals were assigned to this specific cluster, while the others showed at least intermediate or even complete assignment to the mainland cluster. This indicates ongoing introgression that is probably mediated by human translocations of gazelles from and onto the islands. Considering the uniform dwarfism of Farasan gazelles, reasons for the smaller body size might be direct consequences of resource limitations, i.e., phenotypic plasticity. If the population decline on the mainland will hold on Farasan gazelles could serve as stocks for future reintroductions.