Neutrophils are among the first white blood cells recruited to the site of infection once microbial pathogens enter the host organism. At site, they perform a well-orchestrated chain of processes that aims to kill the microbial invader. Most prominent, neutrophils engulf microbes to inactivate them intracellularly, a process called phagocytosis. Alternatively, neutrophils can release neutrophil extracellular traps (NETs). NETs consist of chromatin decorated with antimicrobial effector proteins – a structure that can entangle bacteria and fungi. Neutrophils are crucial during fungal infections. This is reflected in the increased risk of fungal infections resulting of neutropenia. The concept of nutritional immunity describes every infection as a battle for resources. Those are mostly metal trace elements. For a long time, neutrophils were seen as powerful, but “mindless”, killers with a limited set of actions and no transcriptional capacity, but this view is in the flux. In the presented thesis, it was my goal to gain new insights into the interplay of neutrophils and fungi – with special attention to metal-nutritional aspects. We compared human neutrophils lacking the ability to undergo NETosis, due to a non-functional NADPH complex, and neutrophils from the same person that were “cured” by gene therapy. We investigated those NETs and found that their inhibitory activity towards the mold A. nidulans depends on calprotectin, a known zinc-chelator. Considering the high influx of neutrophils, we wanted to unravel the neutrophils’ contribution to the metal milieu at the site of infection and trace element changes resulting from NETosis. By combining synchrotron radiation XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial element distribution in activated neutrophils and NETs. Most strikingly, we found neutrophils to be exceptionally high in Fe and the process of NETosis to be reducing available Zn in the surrounding and the early phagosome, possibly by the formation of Zn-rich vesicles. Using RNA-sequencing, we analyzed the interplay of the C. albicans and neutrophils face-to-face. We dissected their transcriptional profile and revealed a manifold response in neutrophils that include cytokine induction and cellular rearrangement. We further were the firsts to explore the transcriptional response of C. albicans to NETs. Our data indicates a distinct response compared to intact neutrophils or other known stress triggers. Metal homeostasis was affected in Candida in both set-ups. In summary, this thesis provides new insights into the interaction of fungal pathogens with neutrophils and emphasizes the impact of nutritional aspects on this interplay. A deeper understanding of the nutritional immunity during fungal infection might open up new strategies to tackle fungal infections – a growing threat worldwide.

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The scientific progress about the important chemotherapeutic drug substance cisplatin (CDDP) and its function has often been rendered by data difficult to interpret, and still many questions about its mode of action remains to be clarified by the scientific community. However, studies of CDDP possess a high complexity due to; i) low intracellular concentration, ii) many potential biomolecule targets, iii) poor or unknown stability of the intact drug and its biomolecule adducts and iv) complex and varying sample matrices. Metallomic studies, using advanced analytical techniques may contribute to clarify the interactions between CDDP and intracellular biomolecules. For a successful outcome sample preparation conditions as well as separation and detection techniques must be carefully selected and optimized to achieve accurate results and correct interpretation of data.         This thesis describes some new and improved analytical methodologies for characterizing the Pt metallome in CDDP-exposed malignant cells. The developed methods are based on powerful liquid chromatography (LC) methods hyphenated to sensitive detection by inductively coupled plasma- (ICP) and electrospray ionization mass spectrometry (ESIMS). Consideration has also been taken about sample preparation conditions.         By selecting “chemically inert” sample preparation (cell lysis by osmosis) and separation (using only nonreactive or no additatives) conditions we could avoid the formation of platinum artifact compounds previously described in the literature (Paper I and II). Using oxygen containing organic solvents with high boiling points (dimethylformamide; DMF, 1,4-dioxane, n-propanol and ethanol) as alternatives to acetonitrile in the LC separations, significant improvements were achieved in ICPMS sensitivity and robustness. When evaluated in combination with chromatographic performance and ESIMS detection the overall best performance was achieved with n-propanol (Paper II, III and IV). From the studies in Paper II we could show that free intact CDDP can be found in malignant cells, as supporting evidence for passive or endocytotic uptake of the drug and further estimate a half-life for intracellular CDDP to about 15 minutes. Such data has not been shown before. In Paper V, the above improved LC methods were used to demonstrate differences in the platinum and cupper metallome from sensitive and resistant T289 melanoma cells exposed to CDDP at near clinical levels.         In a wider perspective we have shown the potential of using hydrophilic liquid interaction chromatography (HILIC) hyphenated to ICPMS detection as a general approach for analysis of hydrophilic metallo-compounds (Paper II). Taking advantage of the superior ICPMS performance using n-propanol gradients for reversed phase liquid chromatography (RPLC) possess a true alternative and /or complimentary technique to size exclusion chromatography (SEC) commonly applied within metallomic studies of biomolecules (Paper V). Using n-propanol in HILIC as well as in RPLC enables parallel detection by ICP- and ESIMS using only one set of chromatographic parameters (Paper III and IV), something commonly called for by scientists in the field.