The excretion of extracellularpolymericsubstances (EPS) as microbial glue is an enthralling mechanism probably determining aggregate turnover and C sequestration. However the role of EPS in soils and the factors influencing the production by microorganisms are poorly understood. Moreover, interdependencies between important factors like the amount of fine-sized particles (clay content), the decomposability of organic matter and the microbial community (size and composition, as well as the excretion and composition of EPS) affecting aggregation and C sequestration are still under debate. Here, we studied the complex interactions between these factors and their role in aggregate turnover. Therefore, an incubation experiment has been conducted across a gradient of clay content (addition of montmorillonite) and substrate decomposability (starch and cellulose) as main drivers of the microbial activity. A combination of microbial parameters as well as aggregation parameters (formation and stability) have been determined. The first results showed a strong response by microorganisms to the increased clay contents, with reduced and decelerated microbial biomass production and CO 2 respiration, respectively. Interestingly, the EPS measurements indicate a change in EPS production and composition with increasing clay contents, as the ratio of EPS sugars to EPS proteins changed with increasing clay contents. The highest concentrations of EPS sugars were found in the soils without clay addition, whereas the highest EPS proteins could be found in the soils with the largest increase in clay content. Effects of these changes on aggregate turnover and changes in the microbial community are examined at the moment. This study is expected to provide insights on the role of changes in amount and composition of EPS in soils, the underlying mechanisms and there implications on aggregation. Thus, the results of this study will provide an improved base for a better understanding of C sequestration in soils.
Biological leaching is the dissolution of metal sulfides by acidophilic sulfur- and/or iron-oxidizing microorganisms. As the biological leaching activity can result in both negative and positive outcomes, both of these two contrary aspects are discussed below: I) The negative effect of bioleaching is known as acid mine drainage or acid rock drainage (AMD/ARD) and the concomitant pollution of soil and water with sulfuric acid and heavy metals. II) The positive option to use bioleaching microorganisms in biotechnological applications, for the recovery of metals from low- grade metal sulfide ores or waste materials. Attachment and biofilm formation on these ores and materials has a major impact on the leaching activity of these microorganisms. However, biofilm formation by bioleaching microorganisms on the metal-bearing sulfides is not thoroughly understood. The composition of the extracellularpolymericsubstances (EPS), which mediate adhesion to solid substrates, is fundamental for the biofilm lifestyle. The EPS consist of several different compounds such as polysaccharides, fatty acids, extracellular DNA (eDNA), proteins and others, which may vary between species, and also be dependent on the energy source and other environmental conditions prevailing during growth.
EPS represent the major constituents of microbial biofilms and determine the environmental conditions for the residing microorganisms. In a way they resemble, as Flemming et al. (2007) aptly called it, “the house of biofilm cells.” They form the extracellular matrix of biofilms, generate their porosity, density, water content and sorption properties, and afford protection for biofilm organisms (Wingender et al., 1999; Flemming and Wingender, 2002; Flemming et al., 2007; Karatan and Watnik, 2009; Flemming and Wingender, 2010). Several definitions for EPS have been given in the past. Geesey (1982) for example described EPS as “extracellularpolymericsubstances of biological origin that participate in the formation of microbial aggregates”. Characklis and Wilderer (1989) defined EPS as “organic polymers of microbial origin which in biofilm systems are frequently responsible for binding cells and other particulate materials together (cohesion) and to the substratum (adhesion)”. In general EPS are composed of a variety of macromolecules, including polysaccharides, proteins, DNA and (phospho-) lipids and can account for over 90 % of the biofilm dry weight (Wingender et al., 1999; Flemming and Wingender, 2010). In early studies, polysaccharides were the main target for analysis, as they have been assumed to be the main constituents of EPS. Hence, “EPS” were formerly considered “exopolysaccharides” or “extracellular polysaccharides”. However, further studies showed that proteins and extracellular DNA (eDNA) can be found in similar or even higher concentrations within EPS of environmental biofilms compared to polysaccharides (Platt et al., 1985; Jahn and Nielsen, 1995; Frølund et
Biofilm formation and the production of extracellularpolymericsubstances (EPS) by meso- and thermoacidophilic metal-oxidizing archaea on rele- vant substrates have been studied to a limited extent. In order to investigate glycoconjugates, a major part of the EPS, during biofilm formation/ bioleaching by archaea on pyrite, a screening with 75 commercially available lectins by fluorescence lectin-binding analysis (FLBA) has been performed. Three representative archaeal species, Ferroplasma acidiphilum DSM 28986, Sulfolobus metallicus DSM 6482 T and a novel isolate Acidianus sp. DSM 29099 were used. In addition, Acidianus sp. DSM 29099 biofilms on elemental sulfur were studied. The results of FLBA indicate (i) 22 lectins bound to archaeal biofilms on pyrite and 21 lectins were binding to Acidianus sp. DSM 29099 biofilms on elemental sulfur; (ii) major binding patterns, e.g. tightly bound EPS and loosely bound EPS, were detected on both substrates; (iii) the three archaeal species produced various EPS glycoconjugates on pyrite surfaces. Additionally, the substratum induced different EPS glycoconjugates and biofilm structures of cells of Acidianus sp. DSM 29099. Our data provide new insights into interactions between acidophilic
in activated sludge (Urbain et al., 1993; Froelund et al., 1996; Higgins and Novak, 1997). A probable reason why DNA stays in the exopolymeric matrix of activated sludge could be the inhibition of DNases by humic substances and the protection of DNA by metal ions (Palmgren and Nielsen, 1996). In general, functional groups of the EPS have been found to contribute to the negative charge of bioflocs (Sobeck and Higgins, 2002). Therefore, an important role is ascribed to polyvalent cations, especially Ca 2+ and Mg 2+ as they interact with the negatively charged EPS molecules and lead to a highly hydrated gel matrix which provides mechanical stability to the microorganisms and protection against environmental effects (Flemming and Wingender, 2000). Further functions of EPS include participation in cell-cell recognition and genetic transfer; the accumulation of nutrients (e.g. dissolved organic matter); the increased resistance to antibiotics and biocides through hindered diffusion and inactivation of antimicrobial agents; the immobilization of exoenzymes; a protective role against predation and digestion; and effects on the fate of organic contaminants (Wingender et
2014). FLBA allows simultaneous visualization and characterization of EPS glycoconjugates based on the lectin specificities. A requirement for FLBA is that lectins shall be tested against a specific biofilm sample (Neu et al. 2001). It is necessary due to the fact that a specific biofilm is stainable only by certain lectins. Normally, this screening allows to select suitable lectins for a particular biofilm sample. Additional EPS components can be visually characterized by combination with other stains specific for proteins, nucleic acids or lipids, among others (Neu and Lawrence 2014a; Neu and Lawrence 2014b). For instance, the Syto and Sypro series are used to detect cells via their nucleic acids and cellular proteins, respectively. FM dyes (FM1-43 and FM4-64) and Nile red are specific to stain membranes and lipophilic compounds. These stains can be used also to stain extracellular compounds in biofilms (Lawrence et al. 2007; Neu and Lawrence 2014a). DDAO (7-hydroxy-9H-1,3-dichloro-9,9- dimethylacridine-2-one) stains nucleic acids and normally does not penetrate cell membranes. Thus, it has been selected as the preferred fluorochrome for staining eDNA (Koerdt et al. 2010). Combination of several types of stains allowed us to get detailed information about biofilms of acidophiles on different energetic substrates.
Extracellular recordings provide a way of indirectly measuring the current
ow within a neu- ron. As described in the introduction, action potentials traversing the cell cause a de
ection of the extracellular electrical eld. It is a relatively uncomplicated procedure, but introduces all the problems described in this thesis. The only way to directly measure an action poten- tial, however, is to measure the voltage dierence or current
ow between the inner medium of a cell and some reference electrode. For these intracellular recordings a physical con- nection between the cell and the measurement device needs to be established. This can be achieved by touching the cell with a pipet, the intracellular electrode. The bilipid layers that constitute the cell membrane will open between the pipet and the cell, allowing to directly inject current into the cell body - thus stimulating the cell - or to measure the current
ow or voltage between the inner cell and the reference. This oers the great advantage of having - from the signal processing point of view - a simple procedure to control and monitor the ring behavior of a cell. The big disadvantage being a fairly complex experimental procedure and only a very limited number (normally one or two) of cells can be measured simultaneously. Furthermore, to allow visual guidance of the pipet movement and targeting of a cell body, mainly slices of brain tissue can be analyzed with this technique.
Approximately half of the patients with ALI/ARDS show the presence of histones in their bronchial alveolar fluid (BALF). Ward PA group showed that injection of calf thymus histones directly into the lungs via the intratracheal route activates the complement system leading to increased histone levels in ALI. They further reported that histones outside of cells were highly cytotoxic for alveolar epithelial cells promoting tissue damage and inflammation; such effects were reversed by the blockade of histones in-vivo 118 . A recent study has linked circulating histones with trauma-associated ALI/ARDS and mortality 113 . These findings were consistent with mouse trauma models that displayed pulmonary oedema, haemorrhage, microvascular thrombosis and neutrophil congestion 113 . The dying airway cells also release extracellular histones, which further contributes to the development of chronic obstructive pulmonary disease. Several nuclear proteins that are known to affect gene expression are elevated in the lungs of subjects with COPD, the most notable being those that belong to the core histones, especially H3.3. Relative to control subjects, the lung samples from subjects with COPD showed increased H3.3 in the extracellular spaces, cellular debris, airway lumen mucous, BALF and plasma 119 .
In summary, this study provides evidence that chordoma cancer cells distribute extracellular vesicles, with a preference for exosomes. The change of culture conditions from normoxic to hypoxic leads to an increase EV production and alters the EV phenotype. Additionally, investigation of EV uptake supports the notion of maintained bioactivity of EVs after 24 h. Furthermore, our findings make the case for combination of several techniques for EV characterization. To investigate their potential as drug-carriers, more research about the interaction between recipient cell and EV is necessary. Especially, the examination of the EV-surface-structure is highly relevant to evaluate the uptake behaviour. Additionally, to ensure reproducibility, the determination of specific EV sizes would be advisable for future drug-loading experiments. This research defines the initial findings of a yet unknown field of chordoma-derived EVs.
The daily concentration of carotenoids in the skin of the palm of volunteers was investigated by Raman spectroscopy for a one year period. The results obtained showed individual variations in the level of carotenoid antioxidant substances in the skin of the volunteers, which strongly correlated with the specific lifestyle conditions such as dietary supplementation rich in carotenoids and the influence of possible stress factors. Carotenoid rich diets based on large amounts of fruit and vegetables, which have a large amount of carotenoids in their structure, apparently increase the measured carotenoid antioxidant level of the skin. On the other hand, the carotenoid level in the skin of the volunteers decreased subsequent to the influence of stress factors, such as exhaustion, illness, smoking and alcohol consumption. The subsequent decrease occurred relatively quickly during 24 hours, while the corresponding increase (recovery) usually lasted up to three days . The results obtained showed that the level of carotenoids in the skin reflects the state of the health of volunteers. Thus the divergence of the carotenoid concentration from the average value corresponds to the special events in the lifestyle.
It has been show n in o u r p rev io u s p a p e rs 6 th a t porphine-like substances, specifically a ,ß ,y ,d -te tra - p h en y lp o rp h in e, can be form ed abiogenically. These p ap ers also p resen t the effects of Co-60 gam m a- ra d ia tio n an d u ltraviolet an d v isible ra d ia tio n s on the ra te of fo rm a tio n of T P P . a,/?,y,(5-tetraphenyl- p o rp h in e has been selected as a m odel fo r p o rp h in e type of stru c tu re because it is easy to isolate an d identify. T his p a p e r presents a n o th e r o b serv atio n h aving d irec t b ea rin g on the fo rm a tio n of the o r ganic m ilieu, nam ely the synthesis of p o rp h in e s from sim ple p rec u rso rs and th e effect of electrical discharges in reductive and o x idative atm ospheres on the yields of porphines.
The barn owl neurophonic potential in nucleus laminaris as an example for a dipolar field in an axonal terminal zone
To test our prediction of dipolar extracellular field potential responses due to axon bundles, we recorded EFP responses from the barn owl auditory brainstem. The barn owl has a highly developed auditory system with a strong frequency-following response in the EFP (up to 9 kHz, [ Ko¨ppl, 1997b ]), called the neurophonic, which can be recorded in the nucleus laminaris (NL). In NL, the input from the two ears is first integrated to calculate the azimuthal location of a sound source, and this information is encoded in the EFP ( Carr and Konishi, 1990 ). The EFP in this region is mainly due to the afferent activity, and the contribution of postsynaptic NL spikes is small ( Kuokkanen et al., 2010 ; Kuokkanen et al., 2013 ). Furthermore, the anatomy of the afferent axons is well known and follows a stereotypical pattern ( Carr and Konishi, 1988 ; Carr and Konishi, 1990 ): Two fiber bundles enter the nucleus, with fibers from the contralateral ear entering ventrally, and from the ipsilateral ear entering dorsally. The axon bundles reach the NL from their origin without bifurcating, then bifurcate multiple times at the border of the NL, and then terminate within NL. Axon bundles have a strong directional preference and run roughly in parallel. Most of the volume within NL consists of incoming axons. This well studied physiology and anatomy makes the system an ideal candidate to investigate the EFPs of axon bundles; see the Discussion for arguments why synaptic contributions to the EFP could also be neglected here.
Interfacial polymerization (see fig.12) is conventionally used for encapsulation in polymeric shells. The reaction of polymerization happens on the boundary between two immiscible (usually) liquid phases. One reactive monomer is dissolved in one phase, another reactive monomer - in another. When monomers meet on the interface the polymerization reaction takes place. The whole process is divided into two steps. On the first step an emulsion is formed. The first reactive monomer is dissolved in the dispersed phase, while the continuous phase usually contains emulsion stabilizer. On the second step the second reactive monomer is added to the continuous phase, leading to the polymeric shell formation on the surface of the emulsion droplets. The liquid content of the dispersed phase turns to be entrapped inside the polymer after the polymerization reaction is finished. The size and the shape of the final capsules are expected 94 to be equal to the ones of the original emulsion droplets, which serve as liquid templates for the capsules. Different kinds of emulsions can be used for interfacial polymerization. The most common way is to use conventional oil-in-water emulsions 95-125 , while inverse water-in-oil 103,126-128 and oil-in-oil 129-134 emulsions also have been used.
The next part of the work deals with description of anisotropic self-healing compos- ites. Here, a transversely isotropic material behavior is considered in order to simu- late polymeric composites reinforced by an uniaxial oriented fiber family. Therefore, an anisotropic part (taken from literature) is added to the Helmholtz free energy function. This anisotropic part depends on the preferred direction of the reinforcement and is not affected by damage, i.e., the damage formulation is only connected to the isotropic part of the Helmholtz free energy function due to the fact that it is assumed that only the matrix material becomes damaged during the loading. The mechanical behavior of the reinforced composite is shown by a numerical example.
PTX was present in its free form. At higher PTX concentrations (> 0.1 μg/mL), Taxol showed higher cytotoxicity than PTX-loaded polymeric micelles formulations due to high toxicity of Cremophor EL/ ethanol mixture. Fig. 6 D shows that at PTX concentration > 1 μg/mL, the cytotoxicity did not show significant difference between PTX- loaded micelles with or without biotinylation in HEK293 cells lacking the biotin receptor (p > 0.05). However, at PTX concentrations of 10 and 100 μg/mL, the biotinylated micelles were significantly more cy- totoxic than PTX-loaded micelles without biotin modification in A549 cells which overexpress the biotin receptor (p < 0.05) ( Fig. 6 C). At PTX concentrations of 1–100 μg/mL, the polymer concentration was above CMC and therefore the significant killing enhancement of the formulation is likely caused by the released PTX after internalization of the micelles. IC 50 values as determined using the MTS assay are sum-
The detection of the humic substances is rather delicate as the UV-vis light path through the capillary is relatively short (50–75 µm). Using a series of adjustments at the DAD detection cell, it is possible to detect Aldrich humic acid with a concentration as low as 20 mg/L. The detection limit of the ICP-MS for the plutonium species is as low as 20 ppb. As a first step, in order to verify the applicability of the method, the timely synchronization of the two detectors was investigated using a highly concentrated solution of iodine and iodide, as they can be easily detected by both detectors. Two separate peaks were obtained with the DAD, as shown in Fig. 4. The separation was monitored at the same time by ICP-MS set on mass 127. Two different peaks corresponding to iodine carrying species were obtained with this method, too. The electropherogram obtained with the DAD using the optimal wavelength compared to the one obtained with the ICP-MS in order to evaluate their time correlation are shown in Fig. 4.
For classification of unknown samples we build a PLS model based on eight spectra of each substance. The remaining two spectra were used as test samples for the PLS model prediction. PLS utilizes sample dependent information to create the model. We used the class membership in binary form (0, 1) for non-members and members for the pure substances, and ratios for the mixtures. The sample identification works well for the pure substances, which means that all tested samples could be assigned to the correct class. Even more ratios of pure substances within mixtures can be classified with good accuracy. This demonstrates the applicability of FTIR spectroscopy for the identification of various substances.
In nanotechnology micron-sized and nano-sized hydrogels represent a versatile class of "smart" respon- sive materials. Hydrogels consist of an intramolecular cross-linked polymeric network which can absorb large amounts of solvent. Hydrogels have dimensions ranging from 10 to 1000 nm with gels in the lower range being termed nanogels and gels in the upper range being termed microgels. However, there is no clear definition and sometimes hydrogels having sizes in the micron regime are also referred as micro- gels. In this thesis the term microgel will be used for hydrogels with dimensions up to 600 nm. Due to their size and high surface area microgels are able to interact with cellular and subcellular domains. In addition, microgels show very distinct properties compared to their macroscopic counterparts. It is commonly accepted that microgels will react faster to chemically and /or physically changes caused by external stimuli. The characteristic time of swelling is proportional to the square of the linear dimension of the gel. 60