Bayreuther Graduiertenschule für Mathematik und Naturwissenschaften (BayNAT)
"The New Chemistry" - Sustainable Catalysis with Alcohols
- Subject of the thesis are new iridium complexes stabilized by anionic P,N- or P,N,P-ligands. These complexes were used in homogeneous catalysis. Furthermore, mechanistic studies were performed to provide an insight into the catalytic cycles. Synthesis protocols for a multitude of different product classes have been developed.
The iridium complex 1, stabilized by a neutral P,N-ligand, reacts under basic conditions with 2-aminopyridines. By elimination of dipyridylamine the new catalyst species 2a was formed, which is more stable than catalyst 1.
Based on this finding eight new anionic P,N-ligands and the resulting iridium complexes were synthesized.
After optimization of the reaction conditions (solvent, base, temperature and catalyst loading) these catalysts were used in BH (borrowing hydrogen)/HA (hydrogen autotransfer) reactions. The selective monoalkylation of anilines with primary alcohols was investigated. In comparative experiments the superiority of the new class of catalysts versus the original catalyst 1 was clearly shown. Under mild reaction conditions (70 °C) the selectivity profile with respect to the monoalkylation has been preserved.
The catalytic protocol was subsequently extended to the alkylation of aromatic diamines. Therefore various diaminobenzenes were used as substrates. Also Dapsone®, an important drug in treatment of leprosy could be used as starting material. We succeeded in both symmetric and unsymmetric monoalkylations of diamines. Due to the selectivity profile of the catalyst regarding aromatic amines, also unprotected amino alcohols could be used as alkylating reagents.
By the use of tridentate P,N,P-ligands, a novel class of more stable catalysts compared to complexes 2a-9a, could be developed. Due to sealing the synthesis reactor with a semipermeable membrane, the retransfer of the “borrowed” hydrogen could be prevented and H2 is released. Dehydrogenation and condensation steps are now possible instead of BH/HA. By reacting secondary alcohols with β-amino alcohols, pyrroles were accessible.
After adapting the synthesis protocol to this new class of products the tolerance of functional groups was tested. Diversely functionalized alcohols were used. Under mild reaction conditions (90 °C) and very low catalyst loadings (down to 0.03 mol% iridium), a large number of novel pyrroles was accessible. Using this protocol 21 differently substituted α,α-pyrroles, 12 bicyclic pyrroles, symmetrically as well as non-symmetrically substituted oligopyrroles and three β-aminopyrroles were synthesized. The catalyst resting state was identified by NMR experiments and X-ray structure analysis to be an iridium trihydride. This trihydride is formed under catalytic conditions, by treatment of the pre-catalyst with alcohols or in hydrogen atmosphere.
In the final part of the work, a catalytic pyridine synthesis was developed. In this so far unknown heterocycle synthesis up to four different substituents could be introduced within a single reaction step. 2,6-, 2,5-, 2,4- and 2,3-substituted pyridines were synthesized selectively by using variously substituted primary or secondary alcohols and γ-amino alcohols. Furthermore, both the synthesis of bicyclic pyridines as well as the synthesis of pyridines that bear chiral substituents is possible.
Budget and fluxes of nitrogen in mountainous agroecosystems in a summer monsoonal climate under intensive land use
- A balanced nitrogen (N) cycle in intensively managed ecosystems is necessary as it underpins other ecosystem services. This study evaluated the agricultural practices in a typical mountainous catchment in South Korea in respect to N dynamics and their potential effect on water quality with the aim to develop options for a more sustainable catchment management.
In the 1st study, we used two approaches to calculate N budgets for the 5 key crops of the basin at the field scale. The gross and net N budgets for all crop types were found to be positive. Based on the small differences between the results of the two approaches we identified fertilizer N as well as soil Nmin as the dominant N input sources. As fertilizer N application was the major N input source (>50%), its reduction is the major scope of action for N savings at the field scale. A closely linked action is the synchronization of fertilizer N with soil Nmin. The large amount of fertilizer that is applied prior to planting (>60%) at the beginning of the monsoon season revealed that split applications could help reducing the fertilizer N additions and increase the low N use efficiencies (NUE). Based on the significant differences between gross and net N surplus for rice and bean fields, we identified the high amount of plant residues remaining after harvesting (>100 kg N ha-1) as a further factor for potential N savings. The 5 main crops accounted for over 80% of the total catchment N surplus (>400 Mg), even though their contribution to the area was only around 20%. A land use shift to perennial crops with lower N inputs was therefore found to be a possible but spatially limited chance to reduce N surpluses at the catchment scale. The comparison of catchment N surplus with stream N export revealed that 73-86% of the agricultural N surpluses were transported to water bodies in the catchment by either leaching or surface runoff.
In the 2nd study, we followed the fate of fertilizer N in a ridge and furrow (R/F) cultivation with polyethylene (PE) mulch by using 15N tracer. N leaching was simulated with Hydrus 2D. The comparison of 4 N fertilization levels (0, 150, 250 and 350 kg N ha-1) revealed that already 150 kg N ha-1 is sufficient to reach the maximal yield of radishes. Based on the low results of fertilizer N use efficiency (FNUE), we recommend two applications during the first 25 days of growth and a further application around day 50. These split applications adjusted to the plants’ needs increase the FNUE of the radish and decrease the fertilizer N losses during the growing season. However, split applications might be impractical in plastic covered R/F cultivations because mechanical equipment to apply fertilizer under the PE mulch is required. Based on the finding that 15N retention in soil and nitrate concentration in seepage water decreased similarly for ridges and furrows during the entire growing season, we conclude that the PE mulch had no significant effect on 15N retention in soil and on nitrate concentration in seepage water and did therefore not effectively protect the fertilizer in the ridges from percolation. Based on the simulation results, we found that the ridges and furrows contributed approximately an equal amount of leached N to the total amount. We therefore conclude that the PE mulch provided little protection for the fertilizer N in the ridges during heavy rainfall. N leaching amounts were further found to increase linearly with an increase in N addition rate as it is well known for R/F cultivations without PE mulch. The PE mulch did therefore not prevent the linear increase in leaching with an increase in fertilizer N addition. We summarize that without the use of additional measures such as split applications of fertilizer, the application of PE mulch in a summer monsoon climate with heavy rainfall events does not positively influence the N leaching rates.
In the 3rd study, we monitored soil water dynamics in the field and used this data set to simulate the influence of PE mulch on water fluxes with Hydrus 2D. We simulated soil water dynamics in 1) conventional flat tillage (CT); 2) R/F cultivation without PE mulched ridges (RT); and 3) R/F cultivation with PE mulched ridges (RTpm). The comparison of the simulated pressure heads during dry and wet periods revealed that the PE mulch induced significant soil moisture patterns only during the dry periods. During monsoon events, the effect of the PE mulch was dependent on the soil texture and the hydraulic conductivity. Summarizing the advantages and disadvantages of the R/F cultivation with PE mulch on sloped fields, the practice was observed to have the lowest amount of drainage water, the lowest evaporation rates but also the highest surface runoff rates. Hence, PE mulching might be assessed as a tool to reduce percolating water, but it concurrently increases water contribution to the river network by surface runoff.
Charge and excitation-energy transfer in time-dependent density functional theory
- Learning about and understanding the mechanisms and pathways of charge and excitation-energy transfer of natural molecular complexes is a promising approach for the tailored design of new artificial energy-converting materials. Therefore, next to extensive experimental investigations, a theoretical method that is able to reliably describe and predict these phenomena from first principles is of practical relevance. In principle, density functional theory (DFT) and time-dependent density functional theory (TDDFT) appear as natural choices to study the relevant sizable molecules on a first-principles scale at bearable computational cost. However, the application of standard local and semilocal density functional approximations suffers from well-known deficiencies, in particular, as far as the simulation of charge-transfer phenomena is concerned. The present thesis approaches charge and excitation-energy transfer with the objective of improving the predictive power and extending the range of applicability of (TD)DFT.
The deficiencies of standard density functional approximations have been related to self-interaction. Hence, one major aspect of this work is the extension of the self-interaction correction in Kohn-Sham DFT that is based on the generalized optimized effective potential to TDDFT using a real-time propagation approach. The multiplicative Kohn-Sham potential allows for a transparent analysis of the exchange-correlation potential during time evolution. It reveals frequency-dependent field-counteracting behavior and step structures that appear in dynamic charge-transfer situations. The latter are important for the proper description of charge transfer. Self-interaction correction allows to access many cases that are difficult for standard TDDFT ranging from chain-like systems over excitonic excitations in semiconductor nanoclusters to short- and long-range charge-transfer excitations. At the same time, it does not spoil the reasonable accuracy that already (semi)local functionals exhibit for local excitations. Moreover, the TDDFT perspective on self-interaction correction sheds new light also on the ground-state formalism. Complex degrees of freedom in the energy-minimizing transformation of the generalized optimized effective potential approach yield smoother orbital densities that appear more reasonable when inserted into approximate functionals in the self-interaction correction formalism. This work provides new insight into the use of different functional approximations. Last but not least, the influence of spin-symmetry breaking and step structures of the potential on the preference to transfer integer units of the elementary electric charge between largely separated donor and acceptor moieties is illustrated when static external electric fields are applied. This work has been reported in three publications and one submitted manuscript.
In the field of excitation-energy transfer, recent discoveries of quantum coherence effects shed new light on the mechanisms behind energy-transfer rates. The latter are affected by a number of different properties of the isolated molecules, but involve also effects due to the environment of the system. This thesis addresses excitation-energy transfer phenomena from two perspectives. First, I use real-time propagation TDDFT to investigate the intermolecular coupling strength and the coupling mechanism between single fragments of supermolecular setups. These investigations base on standard closed quantum system TDDFT and exploit the coherent oscillation of excitation energy between separated molecules after the initial excitation process. Second, I use open quantum system ideas in the framework of TDDFT to study the influence of the system’s environment on the energy-transfer time scales and pathways in a circular arrangement of molecules using an effective energy-dissipation mechanism. The first part of these results is published. The second part is presented in this thesis and includes work in progress.
Comparisons of N2O and CH4 fluxes as affected by land use systems and climate in small catchments in Korea
- In the course of global and climate change humankind has to face extreme weather events with increased intensity and frequency and it has to deal with feeding an increasing number of people which is accompanied by shortage of resources such as water. Since half of humankind directly depends on freshwater and other ecosystem services provided by mountainous areas, it is essential to study such complex terrains and how natural as well as agricultural systems react to climatic and other anthropogenic changes.
Emissions of greenhouse gases like Nitrous oxide (N2O) and Methane (CH4) are of global concern, too, because they are involved in global warming and therewith: climate change. Major sources of N2O are agriculturally managed soils, and very important sources of CH4 are rice paddies. Thus, it is of great importance to study intensively managed agricultural systems and the effects of the management practices on greenhouse gas emissions.
The major focus of this thesis is to quantify dry crop fields’ and forests’ N2O emissions as well as rice paddies’ N2O and CH4 emissions and to identify climatic as well as management related factors and underlying processes which are driving the N2O fluxes in a complex terrain.
A prolonged early summer drought in 2010 led to significant N2O consumption in soil of three different forest sites. The following above-average monsoon rainfall period indeed turned the N2O consumption into emission but could not turn the N2O balance of a forest on sandy-loam substrate from negative into a positive one, which means that for the first time a negative N2O balance was observed for a forest soil during the growing season. The N2O emissions of those forest sites were clearly driven by soil moisture and temperature and there appeared to be an effect of the substrate on N2O emissions as well, as it is increasingly often observed that sandy-loam soils show significant N2O consumption.
Plastic mulching – a worldwide used method in agriculture to increase crop production by enhancing soil temperature, creating more stable soil moisture conditions and restricting arable weed growth – turned out to have a mitigating effect on N2O emissions. DNDC (Denitrification and Decomposition) modeling results matched best with the measurement results when the maximum daily soil temperature and half of the daily precipitation was assumed to occur as dominating climate conditions underneath the impervious polyethylene (PE) film, suggesting that N2O production underneath the plastic cover was driven by soil moisture and temperature. N2O emissions from a non-fertilized soy bean field, which has Nitrogen fixation as an additional Nitrogen source, were similar to the N2O emissions from a radish field after application of an intermediate amount of N fertilizer of 200 kg ha-1.
Comparing N2O and CH4 emissions from rice paddies under different water management practices showed that intermittent irrigation (II) (no continuous flooding, no water logging)
had the least global warming potential (GWP) which was only 30% of the global warming potential (GWP) of a traditionally irrigated (TI) paddy (continuous flooding and water logging). Another practice of 2.5 months of continuous flooding, followed by midseason drainage and reflooding which created moist but non-water logged conditions (FDFM) lead to 66% of the traditionally irrigated paddies combined CH4 and N2O emissions. These results suggest that a trend towards less flooding has a great potential to mitigate greenhouse gas emissions from a sandy or sandy-loam substrate, respectively. Studying the three paddies’ subsoil conditions revealed that N2O production and consumption processes had mainly taken place between 25 and 50 cm soil depth judging by N2O concentrations and δ15N-N2O values along the soil profiles of all the investigated paddies as well as gene abundances of denitrifying and nitrifying bacteria of the FDFM paddy.
Apart from these important findings on N2O flux dynamics of three different land use systems, it is noticeable that the N2O emissions of the study region are in general very low which is very pleasing and implies that the area deals with global change challenges and associated intensive agriculture in a way that comparatively only small amounts of N2O degas. But this raises the question after the “why?” considering that large amounts of fertilizer are applied on the fields. This thesis does not have a final answer to that question but it discusses whether the sandy substrate may play a major role for the N dynamics of the whole area. There is evidence that NO3- - as the substrate for denitrification - leaches easily due to the soil conditions. To finally figure out why the N2O emissions are that low a more detailed investigation on the fate of NO3- would be desirable.
Describing Charge Transfer in Extended Donor-Acceptor Systems with Density Functional Theory
- It is a long-standing problem of (time-dependent) density functional theory ((TD)DFT) that traditional functionals severely underestimate charge transfer (CT) excitations. In particular, the theoretical description of donor-acceptor (DA) systems is plagued by this shortcoming. DA systems are frequently used as light absorbing components in organic photovoltaic devices. The lowest electronic excitation in these molecules is usually influenced by CT.
In order to support the systematic development of new DA systems that are needed to improve the efficiency of organic solar cells it is a prerequisite for theory to reliably predict the electronic properties of this system class.
We demonstrate that the tuned range-separated hybrid (RSH) approach predicts these excitations in accordance with experiment. The approach can be regarded as an implicitly defined density functional within the generalized Kohn-Sham (GKS) scheme of DFT. Its main ingredient is the range-separation parameter that determines the splitting between long- and short-range exchange. It is obtained from first principles by enforcing the ionization potential theorem of GKS theory.
We consider DA systems of various sizes that are composed of thiophene as donor and benzothiadiazole or naphthalene diimide as acceptor. We show how the optical and electronic properties can be tailored by changing the conjugation length and the arrangement of the donor and acceptor components. We also address the downsides that accompany the use of tuned RSH functionals. Due to the way the approach is implicitly defined anew for each system it is not size consistent. By calculating ground state properties of atoms and diatomic molecules we report size consistency errors and demonstrate consequences of the size consistency violation, e.g., the incorrect prediction of binding energies.
In order to reliably predict CT excitations within the Kohn Sham scheme of DFT the exchange correlation potential approximation has to incorporate particle number discontinuities. A candidate potential with the necessary features is the Becke-Johnson potential that is based on semi-local ingredients and is therefore computationally attractive for the treatment of very large systems. We show, however, that the potential cannot be applied in TDDFT because it is not a functional derivative and violates the zero-force theorem. We discuss a procedure on the basis of density path integrals that transforms the BJ potential into a functional derivative of a corresponding energy expression.
Design, Synthesis and Application of Cylindrical Polymer Brushes: From Nanostructures to Advanced Materials
- This thesis focuses on the design, synthesis and application of cylindrical polymer brushes (CPBs). Herein, we investigated the scission behavior of polyelectrolyte CPBs on different surfaces, developed novel synthetic pathways for well-defined CPBs via reversible addition-fragmentation chain transfer (RAFT) polymerization, designed and prepared complex functional CPBs for light-harvesting and energy transfer, and utilized CPBs as templates for the synthesis of novel one-dimensional (1D) organic/inorganic hybrid nanostructures.
The ‘grafting-from’ approach was chosen as the general method to synthesize well-defined CPBs with various chemical and structural compositions. The linear polymer backbones (polyinitiators) were obtained by anionic polymerization or RAFT polymerization, whereas the side chains were grafted by atom transfer radical polymerization (ATRP) or RAFT polymerization. The obtained CPBs possess a narrow molecular weight distribution in both the backbone and the side chains.
The polymer backbone of core-shell CPBs consisting of a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) core block and a poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) shell block was ruptured upon drying on solid surfaces, when suf-ficient Coulombic interactions between the shell block and the surface were formed. We controlled this scission behavior by tuning the surface interactions through switching the surface nature, shell quaternization, varying pH, or adding multivalent counterions. This study demonstrates that core-shell CPBs serve as a tool to directly compare the weak intermolecular forces with the strong carbon-carbon covalent bonds.
A novel ‘grafting-from’ approach was developed to overcome the challenges of synthesizing well-defined CPBs from a linear polymer backbone with a high density of RAFT functionalities. In this so-called “CTA-shuttled” R-group approach, a certain amount of low-molecular-weight chain transfer agent (CTA) was added to the polymerization system, serving as shuttles to transfer active radicals among the individual growing CPBs. Well-defined CPBs with polystyrene or poly(tert-butyl acrylate) branches and core-shell CPBs with polystyrene-block-poly(N-isopropylacrylamide) branches were synthesized, with the molecular weight distribution much narrower than that from the conventional R-group approach. Monte Carlo simulations confirmed that the advantage of the “CTA-shuttled” R-group approach consists in the release of the active radicals from the trapping CPB systems.
Imitating the natural “energy cascade” architecture, we developed single-molecular, rod-like nano-light harvesters (NLHs) on the basis of CPBs. Herein, a number of block copolymer side chains carrying light absorbing antennae groups (9,9-diethylfluorene, energy donors) were tethered to a linear polymer backbone containing emitting groups (anthracene, energy acceptors). These NLHs provide very efficient energy absorption and energy transfer from antennae to energy acceptors. Furthermore, we were able to manipulate the efficiency of energy transfer by tuning the distance between energy donors and energy acceptors in physical and/or chemical ways. This CPB-based NLH architecture presents a novel concept to design light harvesting materials and can readily be transplanted to any other applications in photoelectronic devices.
Core-shell CPBs with a poly(acrylic acid) (PAA) core block and a PDMAEMA shell block were employed as templates for the preparation of various rare-earth metal cations (Ln3+) incorporated silica hybrid nanoparticles (NPs). A tight chelation of Ln3+ ions in the PAA core and a crosslinked silica layer deposited on the shell provide a very stable encapsulation of Ln3+ ions within the hybrid NPs and thus a high biocompatibility. The silica hybrid NPs obtain unique and diverse properties from the incorporated Ln3+ ions, such as visible photoluminescence, paramagnetic behavior, and a longitudinal relaxation time (T1) shortening effect. This novel template-directed approach succeeds in combining different functional centers via loading in-situ mixed Ln3+ ions into individual CPBs resulting in multicomponent hybrid NPs, which possess both visible photoluminescence and T1 contrast enhancement and can thus be applied as multimodal bioimaging probes.
Development of an artificial silk protein on the basis of a lacewing egg stalk protein
- Silks are widely used in textile industry as clothing and furnishings due to their tensile strength, smoothness, soft texture, lustre, and drape. Most commonly silk of the mulberry silkworm Bombyx mori (B. mori) is used in such applications, however, silks evolved independently in many different arthropods for various purposes.1 During evolution the different silks were optimised for their task-specific uses over millions of years, e.g. adopting different mechanical properties. The mechanical properties mainly derive from the protein secondary structure and its higher order arrangement in silk fibres. Spider silk, for example, is known for its tensile properties surpassing nylon, Kevlar®, silkworm silk, and high-tensile steel.2-5 Beyond their mechanical properties, some silks are also reported to be biocompatible and non-immunogenic.6 One beneficial feature of silk proteins is the possibility to process them into various morphologies.7, 8
Several of these silk features make them interesting for material scientists, intending to produce silks with tuneable properties depending on the desired application, ranging from technical ones such as high performance fibres to medical ones such as drug delivery.
This thesis deals with the characterisation and reproduction of a less explored silk, the lacewing egg stalk silk. Mechanical testing revealed a strong dependence on the relative humidity. In the dry state at 30% relative humidity, the stalks are quite rigid and break at an elongation of 2% whereas at 70% and 100% relative humidity they elongate up to 434%. This extension is accompanied by a secondary structure change from cross-ß to parallel-ß. The cross-ß structure in unstretched stalks provides bending stiffness and rigidity to the stalk, and this bending stiffness gets lost when the stalks are stretched. In this thesis a model is proposed which explains these differences at various relative humidity on the molecular level, wherein changes in the strength of hydrogen bonds upon exposure to water (a hydrogen bond donor/acceptor) in combination with multiple disulphide cross-links (which are not affected by water) act together and are responsible for this behaviour.
Based on consensus sequences of published sequence data (derived from MalXB2 an egg stalk protein of Mallada signata (M. signata)),9 an engineered egg stalk protein named N[AS]8C was recombinantly produced.
To produce an artificial stalk, a droplet of a solution of purified N[AS]8C was placed on a substrate, and tweezers were used to pull out a fibre. After drying, and post treatment, the properties of the artificial stalks were investigated in comparison to the natural ones. Mechanical testing revealed similar behaviour at 30% relative humidity, but at 70% and 100% relative humidity the artificial stalks were not as extensible as the natural ones. This corresponds to the fact, that no cross-ß structure was formed, and, therefore, no rearrangement into parallel-ß structure was possible.
Subsequently, N[AS]8C was processed into non-fibrous morphologies. It was possible to produce capsules, hydrogels, foams, and films. The foams show an interesting micro and nano structure which differs from that of recombinant spider silk. The cavities are filled with a mesh of nano fibres building a 3D scaffold.
Films are a morphology with potential for application in cell culture. Fibroblast attachment on N[AS]8C films is quite poor. Therefore, we tried to induce guided fibroblast growth on patterned protein films. A first layer of the films was cast from ntagCysC16-c(RGDfK), an engineered spider silk protein coupled with the integrin recognition motif RGD to provide a protein layer to which fibroblasts attached well. The second protein layer was produced using a PDMS (polydimethylsiloxane) template and N[AS]8C. Fibroblasts grown on these films adhere only to the RGD modified spider silk and not to the N[AS]8C areas. A second feature of such films is to orient the fibroblasts on films with alternating lines of the two proteins. Such films might be useful for tissue engineering to control cell adhesion and get a structured cell pattern. This is essential for many tissues such as bones, muscles, and epithelia tissue. The low cell adhesion properties of N[AS]8C films might be interesting for coatings for applications where cell adhesion is not desired such as stents or catheters.
Dual-Responsive Polymer and Hybrid Systems: Applications for Gene Delivery and Hydrogels
- This thesis focused on the synthesis of functional materials based on water-soluble and responsive polymers, in particular poly((2-dimethylamino)ethyl methacrylate) (PDMAEMA). The dual-responsive behavior and polycationic character at physiological pH of PDMAEMA lead to outstanding properties and thus, to a versatile component for water-based applications. The main concept of the thesis was to combine the ability for gene delivery of PDMAEMA with the magnetic properties of iron oxide nanoparticles to enable an activity of the gene vector in an applied external magnetic field. Another point was to apply the dual-responsive behavior of PDMAEMA (temperature and pH) for physically cross-linked hydrogels.
Initial studies on magnetic dual-responsive gene vectors revealed a facile synthesis of PDMAEMA-grafted iron oxide nanoparticles utilizing dopamine as physically binding anchor group for the polymer chains. Here, a dopamine-based ATRP initiator was applied for the surface modification of the nanoparticles, which enabled a controlled polymerization technique via the “grafting-from” approach. Gene transfection experiments with CHO-K1 cells show that the transfection efficiency is significantly higher than for poly(ethyleneimine) (PEI), which is regarded as the “gold standard” among the polycationic gene vectors. Although the hybrid particles show a considerably high molecular weight (4.3 MDa), which should lead to a significant increase of the cytotoxicity as observed for linear PDMAEMA their cytotoxicity is remarkably low, lower than that of PEI. Thus, the excellent performance in gene delivery experiments can be attributed to the star-like architecture of the PDMAEMA. Moreover, the uptake of our superparamagnetic gene vector into the cells enables a magnetic cell separation by applying an external magnetic field.
However, due to the non-covalent bonds of dopamine to the iron oxide nanoparticles, the PDMAEMA chains undergo a detachment with time from the nanoparticle surface. This led to the synthesis of PDMAEMA-based magnetic core-shell-corona nanoparticles. Here, the iron oxide nanoparticles were covered with a thin silica shell in order to link the PDMAEMA chains covalently to the inorganic core via silane chemistry. This approach revealed stable dual-responsive hybrid nanoparticles with irreversible binding of the polymer chains and a high long-term stability in aqueous media. These hybrid star-like particles also show excellent gene delivery. The inter-polyelectrolyte complex formation between the PDMAEMA corona of the core-shell-corona particles and pDNA showed that the pDNA molecules are individually complexed with single nanoparticles at N/P ratios (polymer nitrogen / pDNA phosphorous) where the best transfection results are obtained. The magnetic cell separation was further improved by using a Magnetic Activated Cell Sorting system (MACSTM). The magnetically separated cells maintain a high transfection efficiency as well as viability and could even be further cultivated.
Another aspect of this thesis was to include PDMAEMA as stimuli-responsive block in a double switchable block copolymer-based hydrogel. For this purpose, we chose a physically cross-linked ABCBA pentablock terpolymer system, which was polymerized via sequential ATRP and consist of a water-soluble PEO middle block, two dual-responsive (temperature/pH) PDMAEMA B-blocks as well as two thermo-responsive poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) A-blocks (PDEGMA-b-PDMAEMA-b-PEO-b-PDMAEMA-b-PDEGMA). The aggregation behavior in dilute solution was investigated via temperature-dependent Dynamic Light Scattering (DLS) revealing that both stimuli-responsive blocks can be triggered separately and the coil-to-globule transition temperatures of the stimuli-responsive blocks were found to be strongly dependent on the block lengths for low molecular weights. In concentrated solutions, however, rheology studies did not show a further change in the mechanical properties after gelation for the investigated ABCBA pentablock terpolymer compositions. As a result, the principle of our complex system points towards a successful synthesis of a dual-responsive ABCBA pentablock terpolymer hydrogel system, which may show two distinct phase transition even for the gel state, if longer block lengths of the outer A- and B-blocks would be applied.
Flow and transport processes as affected by tillage management under monsoonal conditions in South Korea
- A sustainable agriculture, which provides enough yields to satisfy the food demand and minimizes the impacts on ecosystem services such as provision of high water quality, is challenging especially in monsoon regions. In this thesis, plastic mulched ridge cultivation (RTpm) under monsoonal conditions and its impact on flow processes and nitrate transport was investigated.
On hillslopes, we monitored surface and subsurface flow processes in four plastic mulched potato fields using a network of tensiometers, FDR sensors, runoff collectors and flow dividers as well as Brilliant Blue FCF tracer experiments. The obtained datasets were used to calibrate the process-based models HYDRUS 2/3D and EROSION 3D in order to quantify drainage water fluxes, surface runoff and erosion rates of RTpm compared to ridge tillage without coverage (RT) and conventional flat tillage (CT). In a flat terrain, N fate under fertilizer rates at 50, 150, 250 and 350 kg NO3− ha−1 was investigated in a plastic mulched radish field using suction lysimeters, tensiometers and a 15N tracer experiment. We used datasets of nitrate concentrations and matric potentials to calibrate a water flow and solute transport model using the numerical code HydroGeoSphere.
RTpm affects soil water dynamics dominantly during dry periods, when ridge soil was drier compared to furrow soil caused by the protective plastic coverage and root water uptake in ridges. Hence, pressure head gradients induced lateral flow from furrows to ridges. Under monsoonal conditions, soil was fully saturated and down slope lateral flow occurred in the coarse textured topsoil. The dye tracer experiments showed that matrix flow dominated in the sandy topsoil. Lateral funnel flow above the tillage pan was the prominent preferential flow path. Unexpectedly, macropore flow in deeper soil horizons was not detected. The field and modeling studies revealed that surface runoff was substantially increased under RTpm compared to RT and CT. However, the field topography primarily controlled surface runoff and erosion rates. The concavity of the field led to flow accumulation and high erosion losses in the center of the field, while a convex shape resulted in less soil erosion.
NO3− leaching was found to be the prominent pathway especially during the early season. Furthermore, the biomass production did not significantly differ between NO3− fertilizer rates of 150 to 350 kg ha−1. Hence, we recommend NO3− fertilizer application of 150 kg ha−1, a better fertilizer placement and split applications. We simulated whether the given recommendations on fertilizer best management practices (FBMPs) decreased NO3− leaching amounts. Compared to RT under conventional fertilization in ridges and furrows, the simulations showed that NO3− leaching can be considerably reduced up to 82% by combining RTpm, fertilizer placement only in ridges and split applications with a total fertilizer NO3− amount of 150 kg ha−1.
Based on these findings, the impact of RTpm on flow and transport processes has to be evaluated differently depending on terrain complexity. In a flat terrain, where surface runoff processes are absent or minimal, RTpm has several advantages. Beside functions such as weed control, and earlier plant emergence due to higher temperatures, plastic mulching decreases drainage water and NO3− leaching. Thus, RTpm enhances nutrient retention below the plastic mulch and reduces NO3− groundwater contamination risk. On slopes, where precipitation contributes substantially to surface runoff, RTpm even increases runoff, soil erosion and surface leaching of agrochemicals into aquatic systems.
This thesis provides several recommendations, aiming to minimize environmental impacts and to decrease costs of fertilizer and herbicide inputs. To reduce surface runoff and soil erosion at sloped fields, we suggest applying perforated plastic mulch and a ridge configuration following contours of the field. Furthermore, we recommend omitting application of herbicides in furrows to allow weed growth, which slows down runoff processes. These suggestions would increase infiltration and subsurface flow processes automatically become more important. However, absent preferential flow to deeper soil layers indicated a low groundwater contamination risk. Since funnel flow above the tillage pan was found to be the most important preferential flow path, we propose the establishment of riparian buffer zones. This would also help to reduce the discharge of sediments and fertilizers via surface runoff into the streams. Finally, FBMPs such as fertilizer placement only in ridges and split applications were found to decrease nitrate leaching considerably. Hence, we suggest applying FBMPs with impermeable plastic mulch in flat terrain, while on hill slopes FBMPs should be applied with perforated plastic mulch. To reduce the leaching and erosion risk after harvest when the plastic mulched ridges are removed, we recommend cultivating cover crops.
Foamy Virus RNase H - Aktivität, Struktur und Funktion
Das für die Replikation des RNA-Genoms von Foamy Viren (FV) notwendige Enzym, die Protease-Reverse Transkriptase (PR-RT), beinhaltet die Protease-, die Polymerase- und RNase H-Domäne. Letztere ist für den Abbau der RNA im entstehenden RNA/DNA Hybrid verantwortlich. Während die FV PR-RT als Monomer vorliegt, besteht die HIV-1 RT aus einem p66/p51-Heterodimer. Erstaunlicherweise ist die isolierte HIV-1 RNase H im Vergleich zur z.B. E. coli oder separaten MoMLV RNase H nicht aktiv. Aus den Sequenzvergleichen verschiedener RNase H-Domänen ergibt sich, dass die Prototyp FV (PFV) RNase H im Gegensatz zur HIV-1 RNase H einen Sekundärstrukturbereich aufweist, bei dem es sich um die sogenannte C-Helix mit einer sich anschließenden basischen Schleife (basic protrusion) handelt. Da zu Beginn der Arbeit keine 3D-Struktur einer retroviralen RNase H mit basic protrusion bekannt war, sollte in dieser Arbeit die Struktur der PFV RNase H und die Funktion der basic protrusion bei der Substratbindung geklärt werden.
Die Tertiärstruktur der PFV RNase H-Domäne konnte mit NMR-Spektroskopie gelöst werden. Somit war es möglich, die basic protrusion einschließlich der C-Helix zu identifizieren. Die isolierte RNase H-Domäne zeigte in fluoreszenzbasierten Tests sowie in qualitativen RNase H-Versuchen mit radioaktiv markiertem Substrat Aktivität. Um die Funktion der C-Helix und der sich anschließenden basischen Schleife bei der Substratbindung zu analysieren, wurden NMR-Titrationsexperimente durchgeführt. Dafür wurde die PFV RNase H-Domäne zunächst durch den Austausch der zwei katalytisch wichtigen Reste Aspartat 599 und Histidin 724 zu Asparagin inaktiviert (RNase H-(D599N-H724N)), um den Abbau des Substrates während der Messungen zu vermeiden. Die Auswertung von [15N, 1H]-HSQC- und [1H, 15N, 1H]-NOESY-HSQC-Spektren erbrachte eine Übereinstimmung der Tertiärstrukturen der RNase H-(D599N-H724N) mit der wt RNase H.
Die NMR-Titrationsexperimente zeigten, dass die C-Helix in der PFV RNase H wie ein Lineal agiert, das die sich anschließende basische Schleife zum Substrat orientiert. Darüber hinaus besitzt die basic protrusion zusätzlich eine Reihe an positiv geladenen Resten, die gut lösungsmittelzugänglich sind und dadurch erste Kontakte mit dem Substrat ermöglichen. Damit bietet die basic protrusion eine Art Plattform für die Substratbindung. Der HIV-1 RNase H fehlt nicht nur die C-Helix, zusätzlich ist die sich anschließende Schleife vermutlich zu kurz, um das Substrat zu binden. Da diese Schleife außerdem nur über einen basischen Rest verfügt, ist wahrscheinlich auch die Gesamtaffinität dieses Bereichs für die Substratbindung zu gering. Strukturvergleiche der PFV RNase H mit der HIV-1 RT zeigen, dass die fehlende basic protrusion der HIV-1 RNase H durch eine Schleife aus der Verbindungs-Subdomäne der p66-Untereinheit kompensiert wird. Dieser Bereich könnte somit ein neuer Angriffspunkt für Inhibitoren in der antiretroviralen Therapie bei HIV 1 sein.
Um zukünftig weitere Strukturanalysen mit der PR-RT bzw. mit einzelnen Domänen durchführen zu können, wurden in einem weiteren Projekt verschiedene N- und C-terminale Deletionsvarianten der PR-RT des Simian Foamy Virus hergestellt. Durch Aktivitätstests mit diesen Deletionsvarianten konnten die Abgrenzungen für die PR-, die Polymerase-, die RNase H-Domäne und die Verbindungs-Subdomäne in der PR-RT identifiziert werden. Dabei zeigte sich, dass die Region H107-N143 C-terminal von der PR wichtig für die Funktion der Polymerase ist. Die Deletion der RNase H-Domäne und Verbindungs-Subdomäne führte zu einer drastischen Abnahme der Substrataffinität, Integrität und Polymerisationsfähigkeit des Enzyms. Trotzdem konnte eine minimale Polymerase-Domäne bestimmt werden (RT(107 454)), die ohne PR- und RNase H-Domäne sowie ohne die Verbindungs-Subdomäne in der Lage ist, zu polymerisieren. Für die Dimerisierung und damit Aktivierung der PR, die durch Bindung von zwei PR-RTs an das sog. PARM-Element (engl. protease activating RNA motif) auf der genomischen RNA geschieht, sind jedoch die RNase H-Domäne und die Verbindungs-Subdomäne unverzichtbar. Dadurch ist die RNase H nicht nur für die reverse Transkription essenziell; in FV stellt sie auch ein Regulationselement für die PR-Aktivierung dar und ist somit indirekt an der Prozessierung von Gag (Strukturproteine) und Pol (virale Enzyme) beteiligt.