Doctoral Thesis
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The aim of this thesis is to concentrate on the investigation of these ROS&RNS composition distribution and their production pathways in the gas phase produced by a plasma jet. By understanding the physical mechanisms behind the generation of the ROS&RNS a precise tuning and design of the composition distribution in the gas phase can be achieved. One crucial physical parameter is the dissipated power inside the plasma. Only if this parameter is known a meaningful comparison of different feed gas settings is possible. Therefore, a concept for measuring the dissipated power inside the plasma for the modified micro-scaled atmospheric pressure plasma jet( µAPPJ) is designed. Additionally, due to achievements within this thesis it is now possible to ignite a homogeneous discharge in argon and helium within the geometry of the µAPPJ. The used feed gas is a determining factor concerning the electron energy distribution function and consequently influencing the production mechanism of the ROS&RNS. First of all, the electrical characterisation of the modified µAPPJ was performed including the alpha-to-gamma transition. It is shown that the alpha-to-gamma transition power is increasing with increasing frequency. For the first time it is now feasible to investigate the influence of the dissipated power on the neutral gas temperature, the metastable atom densities and the ROS&RNS production for the modified µAPPJ with argon and helium as feed gas. Due to the possibility of changing the feed gas and controlling the dissipated power a fundamental insight into the production mechanism of the ROS&RNS generated by the plasma jet is achieved. With rising dissipated power the temperature and the metastable densities as well as the ozone and nitrogen dioxide concentrations are increasing. By adding molecular oxygen and nitrogen to the feed gas of a plasma jet the ROS&RNS composition can be tuned. However, also the dissipated power is changed by the small amount of admixtures. Due to the developed dissipated power measurements within this thesis it was possible to disentangle the influence of the admixture on the power and on the ROS&RNS production. If the dissipated power is fixed for the µAPPJ with argon and helium feed gas, respectively, the highest amount of ozone was measured with oxygen admixture in an argon discharge, the highest amount of dinitrogen pentoxide with nitrogen admixture in an argon discharge and the highest amount of nitrogen dioxide with nitrogen admixture in a helium discharge. Beyond the influence of the dissipated power and the molecular admixture on the ROS&RNS production the feed gas temperature is a crucial parameter for the corresponding chemical reactions. By changing this parameter the distribution of ozone and nitrogen dioxide can be tuned precisely in such a way that with increasing temperature the ozone density goes down and the nitrogen dioxide density rises. Another determinant for the ROS&RNS composition produced by an atmospheric pressure plasma jet is the influence of ambient air. If the ambient air is changing from pure nitrogen to pure oxygen atmosphere the ozone density produced by the plasma jet is increasing. For the same conditions the nitrogen dioxide has a maximum at an oxygen-to-nitrogen ratio of 1:1. To avoid the influence of the ambient air on the reactive species production the afterglow of the µAPPJ was prolonged with a glass tube. By increasing the amount of molecular admixtures to the feed gas with each in equal quantities a totally different ROS&RNS composition can be obtained compared without the glass tube. It figures out that for small molecular admixtures the reactive species composition is nitrogen dominated and for higher admixtures it is oxygen dominated. Consequently, by shielding the ambient air from the active effluent and by admixing molecular oxygen and nitrogen the ROS&RNS composition can be designed.
Non-thermal atmospheric pressure plasma has drawn more and more attention to the field of wound healing research during the last two decades. It is characterized by a unique composition, which includes amongst others free radicals, ions and electrons. Furthermore, non-thermal plasma exhibits temperatures that are below those inducing thermal cell damage. Next to its well-established anti-bacterial properties, plasma can have lethal as well as stimulating effects on mammalian cells. Therefore, the medical application of non-thermal plasma on chronic wounds seems to be a promising tool to enable healing processes. However, less is known about the plasma-mediated induction of intracellular signaling pathways in human immune cells, which play a leading part in the process of wound recovery and removal of pathogens. Therefore, this thesis examined the cellular effects of a non-thermal atmospheric pressure plasma treatment on human immune cells using the argon plasma jet kinpen 09. Here, the CD4+ T helper cell line Jurkat, the monocyte cell line THP-1 as well as the corresponding primary cells were investigated. First, cell survival and apoptosis induction was assessed in response to non-thermal plasma treatment by growth curves and flow cytometric assays. On the one hand it could be shown that primary cells are more susceptible to plasma treatment than the respective cell lines. On the other hand, monocytes responded less sensitive to plasma exposure than lymphocytes. Furthermore, this thesis outlined the impact of non-thermal plasma treatment on the gene expression level of immune cells. Therefore, DNA microarray analysis was performed with the cell lines Jurkat and THP-1. It became obvious that plasma exposure modulated the expression of several genes in both cell types. Differential expression of distinct target genes was further validated by quantitative PCR in the immune cell lines. Here, elevated gene expression levels of JUN and FOS in Jurkat cells and increased transcription of JUND in THP-1 cells in response to plasma treatment were made visible. JUN, FOS and JUND are components of the transcription factor AP-1, which is involved amongst others in gene expression of IL-8 and HMOX-1. Consequently, transcriptional induction of the inflammatory cytokine IL-8 as well as the enzymes HMOX-1 and GSR was detected in plasma-treated THP-1 cells. In addition, alterations in the protein activation levels were analyzed in plasma-treated Jurkat, THP-1 cells and primary monocytes. Since some of the identified target genes are known to be associated with the MAPK pathways, the regulation of these cascades was further investigated by western blot analysis. In all investigated cell types the pro-proliferative signaling molecules ERK 1/2 and MEK 1/2 as well as the pro-apoptotic signaling proteins p38 MAPK and JNK 1/2 were activated in a plasma treatment time dependent manner. In contrast to Jurkat and primary monocytes, the anti-apoptotic HSP27 was only induced in THP-1 cells in response to plasma exposure. Moreover, modulation of cytokine production and secretion was examined in the different immune cell types and co-cultured THP-1 and HaCaT keratinocytes by ELISA or flow cytometry. While Jurkat cells showed no plasma-mediated regulation of cytokine expression, THP-1 cells revealed an increased IL-8 secretion after long plasma time duration (360 s). Additionally, the intracellular expression levels of IL-6 and IL-8 were modulated in primary monocytes by plasma exposure. While short plasma treatment caused no alteration of the number of cells expressing IL-8 an up-regulation of the intracellular IL-6 level occurred after 30 s of plasma treatment. Long plasma treatment times resulted in a significant decrease of the intracellular IL-8 and IL-6 production levels. Furthermore, co-cultured THP-1 and HaCaT cells as well as mono-cultured THP-1 and HaCaT cells were examined regarding their cytokine secretion profile. Here, cells treated with plasma (180 s) as well as LPS and plasma (180 s and LPS) were compared with untreated cells. IL-6, IL-8 and GM-CSF secretion was induced by both plasma and plasma combined with LPS treatment in mono-cultivated HaCaT cells and co-cultured cells. Though, the highest cytokine secretion levels were reached in the plasma and LPS exposed co-culture. In contrast, mono-cultivated THP-1 cells only showed an increased secretion of IL-6, IL-8 and TNFa after incubation with plasma together with LPS exposed medium. In conclusion, this study revealed for the first time the non-thermal plasma-modulated expression of numerous genes and cytokines and the activation state of various signaling cascades in human immune cells. Thus, it contributes to gain a better understanding of the immune-modulatory impacts of plasma that might promote the wound healing process.