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 <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 >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.

The NreABC system of Staphylococcus carnosus combines nitrate and oxygen sensing by an NreA,NreB sensor complex

Staphylococcus carnosus is a facultative anaerobic bacterium which features the cytoplasmic NreABC system. It is necessary for regulation of nitrate respiration and the nitrate reductase gene narG in response to oxygen and nitrate availability. NreB is a sensor kinase of a two-component system and represents the oxygen sensor of the system. It binds an oxygen labile [4Fe-4S]2+ cluster under anaerobic conditions. NreB autophosphorylates and phosphoryl transfer activates the response regulator NreC which induces narG expression. The third component of the Nre system is the nitrate receptor NreA. In this study the role of the nitrate receptor protein NreA in nitrate regulation and its functional and physiological effect on oxygen regulation and interaction with the NreBC two-component system were detected. In vivo, a reporter gene assay for measuring expression of the NreABC regulated nitrate reductase gene narG was used for quantitative evaluation of NreA function. Maximal narG expression in wild type S. carnosus required anaerobic conditions and the presence of nitrate. Deletion of nreA allowed expression of narG under aerobic conditions, and under anaerobic conditions nitrate was no longer required for maximal induction. This indicates that NreA is a nitrate regulated inhibitor of narG expression. Purified NreA and variant NreA(Y95A) inhibited the autophosphorylation of anaerobic NreB in part and completely, respectively. Neither NreA nor NreA(Y95A) stimulated dephosphorylation of NreB-phosphate, however. Inhibition of phosphorylation was relieved completely when NreA with bound nitrate (NreA•[NO3-]) was used. The same effects of NreA were monitored with aerobically isolated Fe-S-less NreB, which indicates that NreA does not have an influence on the iron-sulfur cluster of NreB. In summary, the data of this study show that NreA interacts with the oxygen sensor NreB and controls its phosphorylation level in a nitrate dependent manner. This modulation of NreB-function by NreA and nitrate results in nitrate/oxygen co-sensing by an NreA/NreB sensory unit. It transmits the regulatory signal from oxygen and nitrate in a joint signal to target promoters. Therefore, nitrate and oxygen regulation of nitrate dissimilation follows a new mode of regulation not present in other facultative anaerobic bacteria.

Identification of target genes of the T-box proteins in Drosophila melanogaster

In a prior bioinformatic analysis by Hüyseyin Binbas, potential Tbx targets sequences in wing-related genes have been identified. Guided by this information, enhancer trap/reporter lacZ insertions were characterized by X-gal staining first in wildtype and then in l(1)omb imaginal discs.rnIn several lines I observed an increase in reporter expression in a l(1)omb mutant background. Since Omb is assumed to function predominantly as a transcriptional repressor, this may indicate direct regulation. Repression by Omb was observed e.g. for brk and tkv. These genes are negatively regulated by Dpp, while omb is induced by Dpp. Omb which mediates the effects of Dpp on proliferation could, thus, also mediate the Dpp effect on patterning of the wing disc. However, brk and tkv were not completely derepressed in l(1)omb indicating that Dpp represses these genes also by an Omb-independent mechanism.rnMore frequently I observed loss of reporter expression in an l(1)omb mutant background. In these cases, regulation by Omb presumably is indirect. For example, STAT92E-lacZ expression in the wildtype eye was symmetrically expressed at the dorsal and ventral margins. In l(1)omb, ventral expression was selectively lost. Loss of omb is known to cause ventral overproliferation of the eye by activation of the Jak/STAT pathway. STAT92E expression is negatively regulated by Jak/STAT signaling suggesting that loss of omb activates Jak/STAT further upstream in the pathway.rnRegional overproliferation of eye and wing in the l(1)omb mutant background proved a complicating issue in the search for Omb targets. This effect made it difficult to decide whether an expanded reporter expression pattern was due to tissue expansion or reporter gene derepression. For instance hth-lacZ appeared to expand along the ventral eye disc margin in l(1)omb. Without addtional experiments it cannot be concluded whether this is due to de-repression or to activation in association with the proliferative state. Parallel to my experiments, evidence accumulated in our laboratory that loss of omb may attenuate Wg and Hegehog signaling. Since these diffusible proteins are the main patterning molecules in the wing imaginal disc, with dpp being downstream of Hh, many of the observed effects could be secondary to reduced Wg and Hh activity. Examples are ab-lacZ, Dll-lacZ and vgBE-lacZ (reduced expression on the dorso-ventral boundary) and inv-lacZ (late larval expression in the anterior wing disc compartment is lost) or sal-lacZ. Epistasis experiment will be required to clarifiy these issues.rnFurthermore, loss of omb appeared to induce cell fate changes. It was reported previously that in an omb null mutant, the dorsal determinant apterous (ap) is ectopically expressed in the ventral compartment (an effect I did not observe with the strongly hypomorphic l(1)omb15, indicating strong dose dependence). Ventral repression of ap is maintained by epigenetic mechanisms. The patchy and variable nature of ectopic expression of ap or grn-1.1-lacZ points to an effect of omb on epigenetic stability.rnIn the second part of my thesis, an analysis of Omb expression in the Drosophila embryonic ventral nervous system was performed. Omb was found co-expressed with Eve in the medial aCC and RP2 motorneurons as well as the fpCC interneuron and the mediolateral CQ neurons. Additionally, Omb was detected in the Eg positive NB7-3 GW serotonergic motoneuron and the N2-4 neurons. Omb was not found in Repo positive glial cells. During embryonic stage 14, Omb showed some coepression with Dpn or Pros. At the embryonic stage 16, Omb was expressed in minor subset of Mid and Wg positive cells.