Doctoral Thesis
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Gram-negative bacteria secrete lipopolysaccharides (LPS), leading to a host immune
response of proinflammatory cytokine secretion. Those proinflammatory cytokines are
TNF-α and IFN-γ, which induce the production of indoleamine 2,3-dioxygenase (IDO). IDO production is increased during severe sepsis, and septic shock. High IDO
levels are associated with increased mortality. This enzyme catalyzes the degradation of tryptophan (TRP) to kynurenine (KYN) along the kynurenine pathway (KP).
KYN is further degraded to kynurenic acid (KYNA). Increased IDO levels accompany
with increased levels of KYNA, which is associated with immunoparalysis.
Due to its central role, the KP is a potential target of therapeutic intervention.
The degradation of TRP to KYN by IDO was intervened by 1-Methyltryptophan (1-
MT), which is assumed to inhibit IDO. By administering 1-MT, the survival of
1-MT-treated mice suffering from sepsis increased compared to mice not treated with
1-MT. The levels of downstream metabolites such as KYN and KYNA were
expected to be decreased. Surprisingly, in healthy mice and pigs, an increase in KYNA
after 1-MT administration was reported. Those unexpected metabolite alterations after 1-MT administration, and the mode of action, were not the focus of recent
research. Hence, there is no explanation for KYNA increase, while KYN did not change.
This thesis aims to postulate a possible degradation pathway of 1-MT along the KP
with the help of ordinary differential equation (ODE) systems.
Moreover, the developed ODE models were used to determine the ability of 1-MT to
inhibit IDO in vivo. Therefore, a multiplicity of ODE models were developed, including
a model of the KP, an extension by lipopolysaccharide (LPS) administration, and 1-MT
administration.
Moreover, seven ODE models were developed, all considering possible degradation pathways of 1-MT. The most likely degradation pathway was combined with the ODE model
of LPS administration, including the inhibitory effects of 1-MT.
Those models consist of several dependent equations describing the dynamics of the KP.
For each component of the KP, one equation describes the alterations over time. Equations for TRP, KYN, KYNA, and quinolinic acid (QUIN) were developed.
Moreover, the alterations of serotonin (SER) were also included. All together belong
to the TRP metabolism. They include the degradation of TRP to SER and to KYN,
which is further degraded to KYNA and QUIN. Every degradation is catalyzed by an enzyme. Therefore, Michaelis-Menten (MM) equations were used employing the substrate
constant Km and the maximal degradation velocity Vmax. To reduce the complexity of
parameter calculation, Km values of the different enzymes were fixed to literature values.
The remaining parameters of the equations were determined so that the trajectories of
the calculated metabolite levels correspond to data. The parameters of different models were determined. To propose a degradation pathway of 1-MT leading to increased
KYNA levels, seven models were developed and compared. The most likely model was
extended to test whether the inhibitory effects of 1-MT on IDO can be determined.
Three different approaches determined the ODE model parameters of the different hypothesis of 1-MT degradation. In the first approach, ODE model parameters were fixed
to values fitted to an independent data set. In the second approach, parameters were
fitted to a subset of the data set, which was used for simulations of the different hypotheses. The third approach calculated ODE model parameters 100 times without
fixed parameters. The parameter set ending up in trajectories of the TRP metabolites,
which have the smallest distance to the data, was assumed to be the most likely. The
ODE model parameters were fitted to data measured in pigs. Two different
experimental models delivered data used in this thesis. The first experimental model
activates IDO by LPS administration in pigs. The second one combines the IDO
activation by LPS with the administration of 1-MT in pigs.
The most likely hypothesis, according to approach 1 was the degradation of 1-MT to
KYNA and TRP. For the second data set the most likely one was the direct degradation of 1-MT to KYNA. With approach 2 the most likely degradation pathways were
the combination of all degradation pathways and the degradation of 1-MT to TRP and
TRP to KYNA. With approach 3 the most likely way of KYNA increase was given by
the direct degradation of 1-MT to KYNA. In summary, the three approaches revealed
hypothesis 2, the direct degradation of 1-MT to KYNA most frequently. A cell-free
assay validated this result. This experiment combined 1-MT or TRP with or without
the enzyme kynurenine aminotransferase (KAT). KAT was already shown to degrade
TRP directly to KYNA. The levels of TRP, KYN and KYNA were measured. The
highest KYNA levels were yielded with an assay adding KAT to 1-MT, corresponding
to hypothesis 2. The models describing the inhibitory effects of 1-MT revealed that
the model without inhibitory effects of 1-MT on IDO was more likely for all three approaches.
The correctness of hypothesis 2 has to be confirmed by further in vitro experiments. It
also has to be investigated which reactions promote the degradation of 1-MT to KYNA.
The missing inhibitory properties of 1-MT on IDO, determined by the in silico ODE
models, align with previous research. It was shown that the saturation of 1-MT was too
low, e.g. in pigs, to inhibit IDO efficiently.
In this study, the first possible degradation pathway of 1-MT along the KP is proposed.
The reliability of the results depends on the quality of the experimental data, and the
season, when data were measured. Moreover, the results vary between the different
approaches of parameter fitting. Different approaches of parameter fitting have to be
included in the analysis to get more evidence for the correctness of the results.
Underground hard coal mining operations irreversibly disrupt the pre-existing mechanical equilibrium of the geological media. The employment of high-recovery methods modifies the stress field of the sedimentary sequence, generating movement and faulting of the rock layers above and below mined seams. These new fracture zones do affect the original conditions of the hydrogeological system by modifying flow pathways and increasing the permeability of the rock sequence. Moreover, the surface area of rock exposed to air and water is increased, conditioning the water-rock interaction. Despite this rather clear conceptualization, flow and reactive transport processes in fractured overburdens are rarely modeled simultaneously. Discrete setups that consider fractures and porous matrix require extensive characterization of both media, which is impractical for regional case studies. As a result, most post-mining models explicitly ignore fracture structures by employing the equivalent porous approach or even both media with lumped parameter models. However, omitting either medium represents a delicate simplification, considering that mining-related fractures control the rate and direction of water flow within moderately permeable but relatively highly porous rock sequences.
In this dissertation, the specific contribution of fractured and matrix continua to the transient discharge and water quality of a post-mining coal zone is quantified and evaluated. For this purpose, dual and multiple interacting continua models are employed to simulate fluid flow and reactive mass transport in fractured and variable water-saturated rock sequences. The effectiveness of the models is evaluated by simulating the origin, generation and transport of acid mine drainage (i.e., water with elevated concentrations of hydrogen, iron, sulfate and chloride) within the shallow overburden of the Ibbenbüren Westfield. Compared to other coal districts in Germany, this area is strongly delimited by the local geology and topography, resulting in a well-defined hydrogeological system to test the models. Petrographic and chemical analyses performed on core samples from the area show the strong influence of mining-derived fractures on the water-rock interaction within the Carboniferous sequence. The presence of oxidized pyrite along with amorphous iron hydroxide phases in weathering fronts on both sides of the fractures demonstrates the exchange of solutes and gases between the fractured and the porous matrix media.
Based on the previous evidence, the TOUGHREACT software is employed to characterize flow and reactive transport processes in the Westfield. However, each of the two processes is simulated at separate stages to have more control in the adjustment of sensitive parameters for which little information is available. For the flow component, a dual continuum model, with Richard’s equations is used to characterize the unsaturated water flow in both fractured and matrix media. Under this approach, the model adequately reproduces the bimodal flow behavior of the discharges measured in the mine drainage for the years 2008 and 2017. Simulation results show how the fractured continuum generates intense discharge events during the winter months while the rock matrix controls smooth discharge limbs in summer, when water is slowly released back to the fractures. With the flow component calibrated, the second part of the study incorporates the geochemical processes into the model based on actual data from the rock samples. Their simulation requires extending the two-continuum setup to a multiple continua model with five nested block strings: one for the fractures and four for the rock matrix. This further subdivision prevents under-representations of kinetic reactions with short equilibrium length scales and numerical instabilities due to lack of chemical and flow gradients. As a result, the new multiple continua model provides good agreement with respect to long- and short-term concentrations and discharge trends measured in the mine drainage. The flow of oxygen and meteoric water through the fractured continuum leads to a high and steady release of hydrogen, iron and sulfate ions derived from pyrite oxidation in the matrix continua closest to the fractures. Moreover, high chloride concentrations result from the mixing and gradual release of relatively immobile solutes in the matrix as they interact with percolating water in the fracture. Both findings are equally congruent with the reactive pyrite oxidation and iron hydroxide precipitation fronts identified in the fractured core samples.
In the end, the multiple continua models, the simulation procedure and the results of the benchmark and sensitivity analysis scenarios developed for the Westfield pave the way for the application of the approach in other mining zones. The first candidate emerges in the Ibbenbüren Eastfield, where a coupled elemental-isotopic approach included in this thesis has confirmed that water-conducting fracture zones are primary elements for solute generation and transport in the first 300 meters of the overburden. In the latter case, calibration and verification of the models can be complemented with measurements of δ34S in sulfates and δ18O, δ2H, and Tritium in water.