Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryo

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryoSpecification of a unique cell fate during development of a multicellular organism often is a function of its position. The Drosophila central nervous system (CNS) provides an ideal system to dissect signalling events during development that lead to cell specific patterns. Different cell types in the CNS are formed from a relatively few precursor cells, the neuroblasts (NBs), which delaminate from the neurogenic region of the ectoderm. The delamination occurs in five waves, S1-S5, finally leading to a subepidermal layer consisting of about 30 NBs, each with a unique identity, arranged in a stereotyped spatial pattern in each hemisegment. This information depends on several factors such as the concentrations of various morphogens, cell-cell interactions and long range signals present at the position and time of its birth. The early NBs, delaminating during S1 and S2, form an orthogonal array of four rows (2/3,4,5,6/7) and three columns (medial, intermediate, and lateral) . However, the three column and four row-arrangement pattern is only transitory during early stages of neurogenesis which is obscured by late emerging (S3-S5) neuroblasts (Doe and Goodman, 1985; Goodman and Doe, 1993). Therefore the aim of my study has been to identify novel genes which play a role in the formation or specification of late delaminating NBs.In this study the gene anterior open or yan was picked up in a genetic screen to identity novel and yet unidentified genes in the process of late neuroblast formation and specification. I have shown that the gene yan is responsible for maintaining the cells of the neuroectoderm in an undifferentiated state by interfering with the Notch signalling mechanism. Secondly, I have studied the function and interactions of segment polarity genes within a certain neuroectodermal region, namely the engrailed (en) expressing domain, with regard to the fate specification of a set of late neuroblasts, namely NB 6-4 and NB 7-3. I have dissected the regulatory interaction of the segment polarity genes wingless (wg), hedgehog (hh) and engrailed (en) as they maintain each other’s expression to show that En is a prerequisite for neurogenesis and show that the interplay of the segmentation genes naked (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment of NB 7-3 and NB 6-4 cell fate. I have shown that in the absence of either nkd or gsb one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.

Die Funktion der Rho GTPasen in den von Toll-Rezeptoren induzierten Signaltransduktionswegen

Die Funktion von Rho GTPasen in den von Toll-Rezeptoren induzierten Signaltransduktionswegen Der Toll-ähnliche Rezeptor 2 (hTLR2) ist wie der TNFa-Rezeptor und das bei Drosophila identifizierte Imd-Protein in der Lage, über einen bisher ungeklärten Mechanismus, sowohl die Aktivierung von NF-kB als auch Apoptose zu induzieren. Im Rahmen dieser Arbeit konnte gezeigt werden, daß die aktive Form der GTPase Rho in beiden Signaltransduktionswegen eine entscheidende Kontrollfunktion übernimmt. So führt die Stimulierung von TLR2 zu einer Aktivierung von RhoA in epithelialen und monozytischen Zellinien. Die aktivierte GTPase rekrutiert die Kinase PKCz und induziert so die IkB-unabhängige Aktivierung des p65/Rel-Transkriptionskomplexes. Aktives RhoA kontrolliert darüberhinaus einen weiteren Signaltransduktionsweg, der die TLR2-abhängigen, früh-apoptptischen Membranveränderungen unter der Beteiligung der Kinasen ROCK und MLCK herbeiführt. Die Rho-abhängige Regulation dieser gegensätzlichen Signalantworten wird durch die direkte Interaktion mit spezifischen Downstreamtargets, die jeweils nur Bestandteil eines Signalweges sind, ermöglicht. Die GTPase Rho stellt somit ein Schlüsselelement in der von TLR2 induzierten primären Immunantwort dar.

Emerging contaminants in agricultural ecosystems: impact of selected pharmaceutical on water and soil ecology and pratical implications

Pharmaceuticals are useful tools to prevent and treat human and animal diseases. Following administration, a significant fraction of pharmaceuticals is excreted unaltered into faeces and urine and may enter the aquatic ecosystem and agricultural soil through irrigation with recycled water, constituting a significant source of emerging contaminants into the environment. Understanding major factors influencing their environmental fate is consequently needed to value the risk, reduce contamination, and set up bioremediation technologies. The antiviral drug Tamiflu (oseltamivir carboxylate, OC) has received recent attention due to the potential use as a first line defence against H5N1 and H1N1 influenza viruses. Research has shown that OC is not removed during conventional wastewater treatments, thus having the potential to enter surface water bodies. A series of laboratory experiments investigated the fate and the removal of OC in surface water systems in Italy and Japan and in a municipal wastewater treatment plant. A preliminary laboratory study investigated the persistence of the active antiviral drug in water samples from an irrigation canal in northern Italy (Canale Emiliano Romagnolo). After an initial rapid decrease, OC concentration slowly decreased during the remaining incubation period. Approximately 65% of the initial OC amount remained in water at the end of the 36-day incubation period. A negligible amount of OC was lost both from sterilized water and from sterilized water/sediment samples, suggesting a significant role of microbial degradation. Stimulating microbial processes by the addition of sediments resulted in reduced OC persistence. Presence of OC (1.5 μg mL-1) did not significantly affect the metabolic potential of the water microbial population, that was estimated by glyphosate and metolachlor mineralization. In contrast, OC caused an initial transient decrease in the size of the indigenous microbial population of water samples. A second laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice Lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, the addition of 5% of sediments resulted in rapid OC degradation. The estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 °C, more than 8% of 14C-OC evolved as 14CO2 from water/sediment samples of the River Po and Venice Lagoon. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice Lagoon treated with 14C-OC, more than 30% of the 14C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that the presence of sediments would not reduce its bioavailability to microbial degradation. Another series of laboratory experiments investigated the fate and the removal of OC in two surface water ecosystems of Japan and in the municipal wastewater treatment plant of the city of Bologna, in Northern Italy. The persistence of OC in surface water ranged from non-detectable degradation to a half-life of 53 days. After 40 days, less than 3% of radiolabeled OC evolved as 14CO2. The presence of sediments (5%) led to a significant increase of OC degradation and of mineralization rates. A more intense mineralization was observed in samples of the wastewater treatment plant when applying a long incubation period (40 days). More precisely, 76% and 37% of the initial radioactivity applied as 14C-OC was recovered as 14CO2 from samples of the biological tank and effluent water, respectively. Two bacterial strains growing on OC as sole carbon source were isolated and used for its removal from synthetic medium and environmental samples, including surface water and wastewater. Inoculation of water and wastewater samples with the two OC-degrading strains showed that mineralization of OC was significantly higher in both inoculated water and wastewater, than in uninoculated controls. Denaturing gradient gel electrophoresis and quantitative PCR analysis showed that OC would not affect the microbial population of surface water and wastewater. The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in literature. In a series of laboratory experiments, the efficiency of a formulation using P. chrysosporium was evaluated for the removal of selected pharmaceuticals from wastewater samples. Addition of the fungus to samples of the wastewater treatment plant of Bologna significantly increased (P < 0.05) the removal of OC and three antibiotics, erythromycin, sulfamethoxazole, and ciprofloxacin. Similar effects were also observed in effluent water. OC was the most persistent of the four pharmaceuticals. After 30 days of incubation, approximately two times more OC was removed in bioremediated samples than in controls. The highest removal efficiency of the formulation was observed with the antibiotic ciprofloxacin. The studies included environmental aspects of soil contamination with two emerging veterinary contaminants, such as doramectin and oxibendazole, wich are common parasitic treatments in cattle farms.

Pathogenic mechanisms in mitochondrial optic neuropathies

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Hsp90 characterization and inhibition: a multi-methodological study

Many physiological and pathological processes are mediated by the activity of proteins assembled in homo and/or hetero-oligomers. The correct recognition and association of these proteins into a functional complex is a key step determining the fate of the whole pathway. This has led to an increasing interest in selecting molecules able to modulate/inhibit these protein-protein interactions. In particular, our research was focused on Heat Shock Protein 90 (Hsp90), responsible for the activation and maturation and disposition of many client proteins [1], [2] [3]. Circular Dichroism (CD) spectroscopy, Surface Plasmon Resonance (SPR) and Affinity Capillary Electrophoresis (ACE) were used to characterize the Hsp90 target and, furthermore, its inhibition process via C-terminal domain driven by the small molecule Coumermycin A1. Circular Dichroism was used as powerful technique to characterize Hsp90 and its co-chaperone Hop in solution for secondary structure content, stability to different pHs, temperatures and solvents. Furthermore, CD was used to characterize ATP but, unfortunately, we were not able to monitor an interaction between ATP and Hsp90. The utility of SPR technology, on the other hand, arises from the possibility of immobilizing the protein on a chip through its N-terminal domain to later study the interaction with small molecules able to disrupt the Hsp90 dimerization on the C-terminal domain. The protein was attached on SPR chip using the “amine coupling” chemistry so that the C-terminal domain was free to interact with Coumermycin A1. The goal of the experiment was achieved by testing a range of concentrations of the small molecule Coumermycin A1. Despite to the large difference in the molecular weight of the protein (90KDa) and the drug (1110.08 Da), we were able to calculate the affinity constant of the interaction that was found to be 11.2 µm. In order to confirm the binding constant calculated for the Hsp90 on the chip, we decided to use Capillary Electrophoresis to test the Coumermycin binding to Hsp90. First, this technique was conveniently used to characterize the Hsp90 sample in terms of composition and purity. The experimental conditions were settled on two different systems, the bared fused silica and the PVA-coated capillary. We were able to characterize the Hsp90 sample in both systems. Furthermore, we employed an application of capillary electrophoresis, the Affinity Capillary Electrophoresis (ACE), to measure and confirm the binding constant calculated for Coumermycin on Optical Biosensor. We found a KD = 19.45 µM. This result compares favorably with the KD previously obtained on biosensor. This is a promising result for the use of our novel approach to screen new potential inhibitors of Hsp90 C-terminal domain.

Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.

Carbon fluxes and allocation pattern in an apple orchard

Carbon fluxes and allocation pattern, and their relationship with the main environmental and physiological parameters, were studied in an apple orchard for one year (2010). I combined three widely used methods: eddy covariance, soil respiration and biometric measurements, and I applied a measurement protocol allowing a cross-check between C fluxes estimated using different methods. I attributed NPP components to standing biomass increment, detritus cycle and lateral export. The influence of environmental and physiological parameters on NEE, GPP and Reco was analyzed with a multiple regression model approach. I found that both NEP and GPP of the apple orchard were of similar magnitude to those of forests growing in similar climate conditions, while large differences occurred in the allocation pattern and in the fate of produced biomass. Apple production accounted for 49% of annual NPP, organic material (leaves, fine root litter, pruned wood and early fruit drop) contributing to detritus cycle was 46%, and only 5% went to standing biomass increment. The carbon use efficiency (CUE), with an annual average of 0.68 ± 0.10, was higher than the previously suggested constant values of 0.47-0.50. Light and leaf area index had the strongest influence on both NEE and GPP. On a diurnal basis, NEE and GPP reached their peak approximately at noon, while they appeared to be limited by high values of VPD and air temperature in the afternoon. The proposed models can be used to explain and simulate current relations between carbon fluxes and environmental parameters at daily and yearly time scale. On average, the annual NEP balanced the carbon annually exported with the harvested apples. These data support the hypothesis of a minimal or null impact of the apple orchard ecosystem on net C emission to the atmosphere.

Bedeutung von Rho-GTPasen bei der Regulation zellulärer Antworten auf genotoxischen Stress

SAPK/JNK regulieren nach genotoxischem Stress eine Vielzahl von Zielsubstraten, die bedeutsam für Reparatur und Überleben der Zelle sind, somit nehmen sie Einfluss auf das zelluläre Schicksal der Zelle. Ob DNA-Schäden eine Phosphorylierung von Stress-Kinasen nach sich ziehen ist bisher noch wenig untersucht. Mit reparaturdefizienten Zellen wurde der Einfluss von DNA-Schäden, durch Cisplatin/Transplatin/UV-C, auf die SAPK/JNK Aktivierung untersucht. Die Aktivierung der Stress-Kinasen erfolgte agenzspezifisch und abhängig von verschieden Reparaturfaktoren. Die Aktivierung korrelierte in reparaturdefizienten Zellen teilweise mit dem späten Auftreten von DNA-Strangbrüchen, war jedoch unabhängig von erhöhten initialen DNA-Schäden. Diese Befunde zeigten, dass die späte Aktivierung der SAPK/JNK DNA-schadensabhängig verläuft und das Cisplatin und Transplatin bei Verwendung von äquitoxischen Dosen zu einer vergleichbaren Aktivierung von SAPK/JNK führten. Die Hemmung der Rho-GTPasen sowohl durch Statine als auch mittels Clostridium difficile Toxin B zeigte weiterhin, dass Rho-GTPasen möglicherweise die späte DNA-schadensabhängige Aktivierung der Stress-Kinasen vermitteln. Die Hemmung von Rho-GTPasen durch physiologisch relevante Konzentrationen von Statinen führte in primären humanen Endothelzellen (HUVECs) zu einer Protektion vor IR-Strahlung und Doxorubicin. In beiden Fällen konnte eine Hemmung des pro-apoptotischen Transkriptionsfaktors p53 sowie der Chk1, welche einen Zellzyklusarrest reguliert, mit der Statin-Behandlung erreicht werden. Effektor-Caspasen wurden dabei durch den HMG-CoA-Reduktase Hemmer nicht beeinflusst. Ausschließlich bei dem Statin-vermittelten Schutz vor Doxorubicin kam es zu einer Reduktion von initialen DNA-Schäden, in Form von DNA-Strangbrüchen. Die IR-induzierten Strangbrüche in der DNA blieben von der Statin-Inkubation hingegen unbeeinflusst. Aufgrund ihrer protektiven Eigenschaften gegenüber IR- und Doxorubicin-induzierter Zytotoxizität in Endothelzellen und ihrer pro-apoptotischen Wirkung auf Tumorzellen könnten Statine möglicherweise die unerwünschten Nebenwirkungen von Zytostatika und einer Strahlentherapie günstig beeinflussen

Nanotechnology control of self organized biomolecules and biomaterials for medical research

Nanotechnology entails the manufacturing and manipulation of matter at length scales ranging from single atoms to micron-sized objects. The ability to address properties on the biologically-relevant nanometer scale has made nanotechnology attractive for Nanomedicine. This is perceived as a great opportunity in healthcare especially in diagnostics, therapeutics and more in general to develop personalized medicine. Nanomedicine has the potential to enable early detection and prevention, and to improve diagnosis, mass screening, treatment and follow-up of many diseases. From the biological standpoint, nanomaterials match the typical size of naturally occurring functional units or components of living organisms and, for this reason, enable more effective interaction with biological systems. Nanomaterials have the potential to influence the functionality and cell fate in the regeneration of organs and tissues. To this aim, nanotechnology provides an arsenal of techniques for intervening, fabricate, and modulate the environment where cells live and function. Unconventional micro- and nano-fabrication techniques allow patterning biomolecules and biocompatible materials down to the level of a few nanometer feature size. Patterning is not simply a deterministic placement of a material; in a more extended acception it allows a controlled fabrication of structures and gradients of different nature. Gradients are emerging as one of the key factors guiding cell adhesion, proliferation, migration and even differentiation in the case of stem cells. The main goal of this thesis has been to devise a nanotechnology-based strategy and tools to spatially and temporally control biologically-relevant phenomena in-vitro which are important in some fields of medical research.

Perfluorinated compounds in environmental matrices of Lake Victoria Gulf

ABSTRACT One of the major ecological challenges on Lake Victoria resources is the existence of “hot spots”, caused by human waste, urban runoff, and industrial effluents. The lake is tending towards eutrophication which is attributed to the increasing human population in its watershed. A report of the levels of perfluorooctane sulfonate and perfluorooctanoic acid in environmental matrices of Lake Victoria is presented, and the management implication of perfluorinated compounds and similar potential organic pollutants examined. Two widely consumed and economically important fish species namely Lates niloticus (Nile perch) and Oreochromis niloticus (Nile tilapia) were obtained from Winam gulf of Lake Victoria, Kenya, and analysed for perfluorooctane sulfonate and perfluorooctanoic acid in muscles and liver using liquid chromatography coupled with mass spectroscopy. Variability in the concentrations of perfluorooctanoic acid or perfluorooctane sulfonate in river waters (range perfluorooctanoic acid 0.4 – 96.4 ng/L and perfluorooctane sulfonate < 0.4 – 13.2 ng/L) was higher than for Lake waters (range perfluorooctanoic acid 0.4 – 11.7 ng/L and perfluorooctane sulfonate < 0.4 – 2.5 ng/L respectively). Significant correlations were tested between perfluorinated compounds levels in sediments, fish and water. Wastewater treatment plants and other anthropogenic sources have been identified as significant sources or pathways for the introduction of perfluoroalkyl compounds into Lake Victoria ecosystem. In this study, elevated concentrations of perfluorooctanoic acid and perfluorooctane sulfonate was found in two wastewater treatment plants (WWTPs) in Kisumu, City of Kenya. An alternative analytical method to liquid chromatography/ mass spectroscopy for analysis of perfluorocarboxylic acids in abiotic and biotic matrices where high concentrations are expected is also presented. Derivatisation of the acid group to form a suitable alkyl ester provided a suitable compound for mass spectroscopy detection coupled to gas chromatography instrumental analysis. The acid is esterified by an alkyl halide i.e benzyl bromide as the alkylating agent for Perfluorocarboxylic acids quantification. The study also involved degradability measurements of emerging perfluorinated surfactants substitutes. The stability of the substitutes of perfluorinated surfactants was tested by employing advanced oxidation processes, followed by conventional tests, among them an automated method based on the manometric respirometry test and standardized fix bed bioreactor [FBBR] on perfluorobutane sulfonate (PFBS), a fluoroethylene polymer, fluorosurfactant (Zonyl), two fluoraliphaticesters (NOVEC ™ FC4430 and NOVEC ™ FC4432) and 10-(trifluoromethoxy) decane-sulfonate. Most of these emmerging surfactants are well-established in the market and have been used in several applications as alternatives to PFOS and PFOA based surfactants. The results of this study can be used as pioneer information for further studies on the sources, behaviour and fate of PFOA and PFOS and other related compounds in both abiotic and biota compartments of Lake Victoria and other lakes. Further an overview in degradation of emerging perfluorinated compounds substitutes is presented. Contribution in method development especially for acid group based fluorosurfactants is presented. The data obtained in this study can particularly be considered when formulating policies and management measures for preservation and sustainability of Lake Victoria resources.

Early steps in ventral nerve cord development in chelicerates and myriapods and formation of brain compartments in spiders

The central point of this work is the investigation of neurogenesis in chelicerates and myriapods. By comparing decisive mechanisms in neurogenesis in the four arthropod groups (Chelicerata, Crustacea, Insecta, Myriapoda) I was able to show which of these mechanisms are conserved and which developmental modules have diverged. Thereby two processes of embryonic development of the central nervous system were brought into focus. On the one hand I studied early neurogenesis in the ventral nerve cord of the spiders Cupiennius salei and Achaearanea tepidariorum and the millipede Glomeris marginata and on the other hand the development of the brain in Cupiennius salei.rnWhile the nervous system of insects and crustaceans is formed by the progeny of single neural stem cells (neuroblasts), in chelicerates and myriapods whole groups of cells adopt the neural cell fate and give rise to the ventral nerve cord after their invagination. The detailed comparison of the positions and the number of the neural precursor groups within the neuromeres in chelicerates and myriapods showed that the pattern is almost identical which suggests that the neural precursors groups in these arthropod groups are homologous. This pattern is also very similar to the neuroblast pattern in insects. This raises the question if the mechanisms that confer regional identity to the neural precursors is conserved in arthropods although the mode of neural precursor formation is different. The analysis of the functions and expression patterns of genes which are known to be involved in this mechanism in Drosophila melanogaster showed that neural patterning is highly conserved in arthropods. But I also discovered differences in early neurogenesis which reflect modifications and adaptations in the development of the nervous systems in the different arthropod groups.rnThe embryonic development of the brain in chelicerates which was investigated for the first time in this work shows similarities but also some modifications to insects. In vertebrates and arthropods the adult brain is composed of distinct centres with different functions. Investigating how these centres, which are organised in smaller compartments, develop during embryogenesis was part of this work. By tracing the morphogenetic movements and analysing marker gene expressions I could show the formation of the visual brain centres from the single-layered precheliceral neuroectoderm. The optic ganglia, the mushroom bodies and the arcuate body (central body) are formed by large invaginations in the peripheral precheliceral neuroectoderm. This epithelium itself contains neural precursor groups which are assigned to the respective centres and thereby build the three-dimensional optical centres. The single neural precursor groups are distinguishable during this process leading to the assumption that they carry positional information which might subdivide the individual brain centres into smaller functional compartments.rn

Oxygen-containing polycyclic aromatic hydrocarbons (OPAHs) and their parent-PAHs in soil

In spite of the higher toxicity of oxygen-containing polycyclic aromatic hydrocarbons (OPAHs) than of their parent-PAHs, there are only a few studies of the concentrations, composition pattern, sources and fate of OPAHs in soil, the presumably major environmental sink of OPAHs. This is related to the fact that there are only few available methods to measure OPAHs together with PAHs in soil. rnThe objectives of my thesis were to (i) develop a GC/MS-based method to measure OPAHs and their parent-PAHs in soils of different properties and pollution levels, (ii) apply the method to soils from Uzbekistan and Slovakia and (iii) investigate into the fate of OPAHs, particularly their vertical transport in soilrnI optimized and fully evaluated an analytical method based on pressurized liquid extraction, silica gel column chromatographic fractionation of extracted compounds into alkyl-/parent-PAH and OPAH fractions, silylation of hydroxyl-/carboxyl-OPAHs with N,O-bis(trimethylsilyl)trifluoracetamide and GC/MS quantification of the target compounds. The method was targeted at 34 alkyl-/parent-PAHs, 7 carbonyl-OPAHs and 19 hydroxyl-/carboxyl-OPAHs. I applied the method to 11 soils from each of the Angren industrial region (which hosts a coal mine, power plant, rubber factory and gold refinery) in Uzbekistan and in the city of Bratislava, the densely populated capital of Slovakia.rnRecoveries of five carbonyl-OPAHs in spike experiments ranged between 78-97% (relative standard deviation, RSD, 5-12%), while 1,2-acenaphthenequinone and 1,4-naphtho-quinone had recoveries between 34-44%% (RSD, 19-28%). Five spiked hydroxyl-/carboxyl-OPAHs showed recoveries between 36-70% (RSD, 13-46%), while others showed recoveries &amp;amp;lt;10% or were completely lost. With the optimized method, I determined, on average, 103% of the alkyl-/parent-PAH concentrations in a certified reference material.rnThe ∑OPAHs concentrations in surface soil ranged 62-2692 ng g-1 and those of ∑alkyl-/parent-PAHs was 842-244870 ng g-1. The carbonyl-OPAHs had higher concentrations than the hydroxyl-/carboxyl-OPAHs. The most abundant carbonyl-OPAHs were consistently 9-fluorenone (9-FLO), 9,10-anthraquinone (9,10-ANQ), 1-indanone (1-INDA) and benzo[a]anthracene-7,12-dione (7,12-B(A)A) and the most abundant hydroxyl-/carboxyl-OPAH was 2-hydroxybenzaldehyde. The concentrations of carbonyl-OPAHs were frequently higher than those of their parent-PAHs (e.g., 9-FLO/fluorene &amp;amp;gt;100 near a rubber factory in Angren). The concentrations of OPAHs like those of their alkyl-/parent-PAHs were higher at locations closer to point sources and the OPAH and PAH concentrations were correlated suggesting that both compound classes originated from the same sources. Only for 1-INDA and 2-biphenylcarboxaldehyde sources other than combustion seemed to dominate. Like those of the alkyl-/parent-PAHs, OPAH concentrations were higher in topsoils than subsoils. Evidence of higher mobility of OPAHs than their parent-PAHs was provided by greater subsoil:topsoil concentration ratios of carbonyl-OPAHs (0.41-0.82) than their parent-PAHs (0.41-0.63) in Uzbekistan. This was further backed by the consistently higher contribution of more soluble 9-FLO and 1-INDA to the ∑carbonyl-OPAHs in subsoil than topsoil at the expense of 9,10-ANQ, 7,12-B(A)A and higher OPAH/parent-PAH concentration ratios in subsoil than topsoil in Bratislava.rnWith this thesis, I contribute a suitable method to determine a large number of OPAHs and PAHs in soil. My results demonstrate that carbonyl-OPAHs are more abundant than hydroxyl-/carboxyl-OPAHs and OPAH concentrations are frequently higher than parent-PAH concentrations. Furthermore, there are indications that OPAHs are more mobile in soil than PAHs. This calls for appropriate legal regulation of OPAH concentrations in soil.

Assessing the role of sCYLD and Bcl-3 in immune regulation

CYLD is a deubiquitinating enzyme, which negatively regulates NF-κB signaling by removing Lys63-linked polyubiquitin chains from its substrates. In mice, there are two variants of CYLD: full-length CYLD (FL-CYLD) and its short splice variant sCYLD. sCYLD lacks the NEMO and TRAF2 binding sites and CYLDex7/8 mice, which have been generated in our laboratory, overexpress sCYLD in the absence of the full length transcript. In this thesis, we show that bone marrow-derived macrophages (BMDCs) overexpressing sCYLD display a hyperactive phenotype. They have increased levels of the inflammatory cytokines IL-6 and TNFα, have exaggerated stimulatory capacity and fail to induce tolerance in in vivo experiments. CYLDex7/8 BMDCs have increased levels of nuclear Bcl-3, which we could show to be directly induced by sCYLD expression. NF-κB signaling was markedly upregulated in CYLDex7/8 BMDCs.rnBcl-3 overexpressing BMDCs with normal CYLD expression, however, were not hyperactive, suggesting that Bcl-3 overexpression is not sufficient for causing the observed phenotype. Taken together we propose a model in which the exclusive overexpression of sCYLD with high nuclear levels of Bcl-3 in BMDCs is accompanied by an increased NF-κB activation, resulting in a hyperactive phenotype.rnWe further analyzed macrophages overexpressing sCYLD using the LysMcre CyldFL/FL strain, but could not detect differences in activation marker expression, cytokine secretion or iNOS production. LysMcre CyldFL/FL mice immunized with MOG35-55 peptide showed a more severe course of experimental autoimmune encephalomyelitis (EAE), which could not be explained by enhanced levels of MHC class II on CNS-resident macrophages and microglia or increased T cell infiltration.rnMice overexpressing Bcl-3 in T cells develop spontaneous colitis. They have less peripheral memory/effector T cells and less Th1 cells, whereas Th17 numbers are normal. Naïve T cells overexpressing Bcl-3 show defects in in vitro differentiation to the Th1 or Th17 fate. CD4+ T cells overexpressing Bcl-3 show enhanced survival capacity in in vitro culture, but have a defect in proliferative capacity when stimulated in vitro or when adoptively transferred into lymphopenic hosts.

Origin of cold gas and dust in massive elliptical galaxies

The recent availability of multi-wavelength data revealed the presence of large reservoirs of warm and cold gas and dust in the innermost regions of the majority of massive elliptical galaxies. To prove an internal origin of cold and warm gas, the investigation of the spatially distributed cooling process which occurs because of non-linear density perturbations and subsequent thermal instabilities is of crucial importance. The first goal of this work of thesis is to investigate the internal origin of warm and cold phases. Numerical simulations are the powerful tool of analysis. The way in which a spatially distributed cooling process originates has been examined and the off-centre amount of gas mass which cools when different and differently characterized AGN feedback mechanisms operate has been quantified. This thesis demonstrates that the aforementioned non-linear density perturbations originate and develop from AGN feedback mechanisms in a natural fashion. An internal origin of the warm phase from the once hot gas is shown to be possible. Computed velocity dispersions of ionized and hot gas are similar. The cold gas as well can originate from the cooling process: indeed, it has been estimated that the surrounding stellar radiation, which is one of the most feasible sources of ionization of the warm gas, does not manage to keep ionized all the gas at 10^4 K. Therefore, cooled gas does undergo a further cooling which can lead the warm phase to lower temperatures. However, the gas which has cooled from the hot phase is expected to be dustless; nonetheless, a large fraction of early type galaxies has detectable dust in their cores, both concentrated in filamentary and disky structures and spread over larger regions. Therefore a regularly rotating disk of cold and dusty gas has been included in the simulations. A new quantitative investigation of the spatially distributed cooling process has therefore been essential: the contribution of the included amount of dust which is embedded in the cold gas does have a role in promoting and enhancing the cooling. The fate of dust which was at first embedded in cold gas has been investigated. The role of AGN feedback mechanisms in dragging (if able) cold and dusty gas from the core of massive ellipticals up to large radii has been studied.