<p> Mass spectrometry imaging (MSI) is an advanced analytical technique that renders spatially defined images of complex label-free samples. Nanospray desorption electrospray ionization (nano-DESI) MSI is an ambient ionization direct liquid extraction technique in which analytes are extracted by means of a continuous liquid flow between two fused-silica capillaries. The droplet generated between the two capillaries is controlled by a delicate balance of solvent flow, solvent aspiration, capillary angles, and distance from the surface. This technique produces reproducible ion images with up to 10 µm resolution and can be used to identify and quantify multiple analytes on a given surface.  This thesis discusses some of the applications of this technique to biological systems, as well as the work done to develop methodology to further improve this technique’s specificity and sensitivity. Herein, applications that push the limits of the current capabilities of nano-DESI are presented, such as the high-resolution imaging of lipid species in skeletal muscle at the single-fiber level, and the quantification of low-abundance drug metabolites.  The second theme of this thesis, developing new capabilities, introduces ion mobility mass spectrometry imaging. This integrated technique increases the selectivity previously possible with MSI. To support these efforts, the work in this thesis has generated data analysis workflows that not only make these experiments possible but also further endeavor to increase sensitivity and combat instrument limitations on mobility resolution. Finally, this thesis present streamlined workflows for tandem MS experiments and modifications to a recently introduced microfluidic variant of the nano-DESI technique. In all, this thesis showcases the current capabilities of the nano-DESI technique and lays the groundwork for future improvements and capabilities.      </p>

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

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Mass spectrometry is an indispensable tool in lipidomics research. Current advances and progress in the technology of mass spectrometry have allowed for the identification, quantification and characterization of lipid molecular species to further our understanding of their biological roles. In this thesis, I assessed the influence post-mortem times have on quantitative lipidomics. Using liquid chromatography - electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) on a triple-quadrupole mass spectrometer and multiple-reaction-monitoring (MRM) mode, the glycerophosphocholine (GPC) metabolites and second messengers in the hippocampus of N3 & N4 C57BL/6 x 129/SV were profiled at various post-mortem interval (PMI). I found that disruption to the GPC metabolite and second messengers lipidome occured as early as 1 hour postmortem and fluctuate up till at least 12 hours post-mortem. Therefore, PMI is a variable in lipidomic studies that must be controlled for, and brain samples which are collected with PMI variations must be matched to avoid misinterpretation. Subsequently, I developed a working protocol to visualize the location and distribution of different classes of glycerophospholipids, ceramides, and sphingomyelin in whole mouse brain sections. This visualization technique is novel because it does not require tissue staining or immunohistochemistry; instead, it was performed using an atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) source coupled to an orbitrap mass spectrometer. As part of this lipid visualization technique, I also developed a protocol for sublimation as a simple, effective and reproducible matrix application method for brain tissue. The lipid-compatible matrix, 2,5-dihydroxybenzoic acid (DHB), was assessed and optimized for imaging lipid targets. The high mass-resolution and accuracy characteristics of the orbitrap mass spectrometer and its capability to perform tandem mass spectrometry via high-collision dissociation allowed for the identification of approximately 200 different lipid species directly from brain tissue using the visualization technique I developed. Altogether, the work in this thesis has showed that post-mortem changes in the lipidome are quantifiable and has provided a novel avenue to further assess these changes by means of imaging mass spectrometry.

No / Several lines of evidence support an autoimmune basis for alopecia areata (AA), a common putative autoimmune hair loss disorder. However, definitive support is lacking largely because the identity of hair follicle (HF) autoantigen(s) involved in its pathogenesis remains unknown. Here, we isolated AA-reactive HF-specific antigens from normal human scalp anagen HF extracts by immunoprecipitation using serum antibodies from 10 AA patients. Samples were analyzed by LC-MALDI-TOF/TOF mass spectrometry, which indicated strong reactivity to the hair growth phase-specific structural protein trichohyalin in all AA sera. Keratin 16 (K16) was also identified as another potential AA-relevant target HF antigen. Double immunofluorescence studies using AA (and control sera) together with a monoclonal antibody to trichohyalin revealed that AA sera contained immunoreactivity that colocalized with trichohyalin in the growth phase-specific inner root sheath of HF. Furthermore, a partial colocalization of AA serum reactivity with anti-K16 antibody was observed in the outer root sheath of the HF. In summary, this study supports the involvement of an immune response to anagen-specific HFs antigens in AA and specifically suggests that an immune response to trichohyalin and K16 may have a role in the pathogenesis of the enigmatic disorder.

The discovery of antibiotics is considered as one of the most significant scientific achievements of the 20th century – lives of millions of people and animals have been saved. Thenceforth, substantial amounts of administered antibiotics and their metabo-lites have been excreted into waste stream via urine and faeces. In this dissertation, primary focus is the qualitative balance of 14 antibiotics and one metabolite in urban water management and in urban waters, respectively. In particular, antibiotics pre-scribed to human beings are drained in the urban sewer system and finally enter the environment: (i) Continuously via the effluent of the wastewater treatment plant after a partially effective removal or degradation or (ii) Intermittent via combined sewer overflow structures due to capacity limitations of the urban drainage system. The fate and the potential effects and risks of these substances on ecosystems and hu-man health are of major concern – their direct toxic effect to all trophic levels as well as the global spread of antibiotic resistance genes are challenging. Hence, an assessment of microbial community activity due to antibiotic exposure is presented. In particular, systematic work has been carried out to study the presence and character-istics of 14 antibiotics in urban waters. In detail, investigations were conducted to gain scientific knowledge with respect to adsorption, desorption, abiotic, biotic and photolyt-ic degradation as well as activity-inhibition of microorganism communities in sewage and of natural freshwater biofilm communities, respectively, due to inevitable urban drainage overflows. In order to provide information to assist potential management strategies, which miti-gate surface water pollution and minimize the adverse impacts of antibiotics on activity of microorganism communities, the following specific topics were addressed: ⑴ The occurrence of 14 antibiotics and one metabolite were determined in sewag-es at three sampling sites in the city of Dresden, Germany. ⑵ The adsorption affinities of 14 antibiotics and one metabolite to size dependent sewer sediments were determined in experimental investigations, three sam-pling campaigns and subsequently an antibiotic-specific adsorption coefficient, normalized to organic content, was quantified. ⑶ The desorption affinity and -dynamics of 14 antibiotics and one metabolite were quantified in size dependent sewer sediments in experimental investigation and with statistical analysis. ⑷ The abiotic, biotic and photolytic degradation affinity of 14 antibiotics and one metabolite were quantified based on batch experiments with three different sewages at 7°C and 22°C, with artificial irradiation and different dilution ratios of the sewage at 30°C and subsequently a model framework decrypted ranges of abiotic, biotic and photolytic degradation coefficients. ⑸ The occurrence of three antibiotics, namely ciprofloxacin, clarithromycin and doxycycline was determined in sewage sampled during dry weather conditions in a small catchment of Dresden, which spills intermittently combined sewage (a mixture of sewage and storm water) to an adjacent brook in the case of capacity limitations of the urban drainage system during periods of intense rainfall and subsequently the three antibiotics were determined in the adjacent brook water. ⑹ Then, the activity-inhibition of microorganism community in sewage of this small catchment was quantified due to an exposition with three different antibiotics and three different antibiotic concentrations. ⑺ Last but not least, the activity-inhibition of natural freshwater biofilm communities in the adjacent brook was quantified via exposure to three antibiotics, which were individually dosed in three different concentrations, and also in mixture. ⑻ Finally, a two-dimensional hierarchical cluster analysis with dendrogram and heat map based on before mentioned activity inhibition of natural freshwater biofilm communities were conducted to identify hot spots of antibiotic tolerant and resistant bacterial subpopulations due to inevitable urban drainage system overflows.:List of Figures IV List of Tables VIII Symbols and Abbreviations XII List of Publications on the Ph.D. topic XIX 1 General Introduction 2 1.1 Background 2 1.2 Aims and Objectives 3 1.3 Innovation and Contribution to the Knowledge 4 1.4 Outline of this Thesis 4 1.5 References 6 2 Adsorption and Desorption Affinity of 14 Antibiotics and One Metabolite for particulate components in urban drainage systems 10 2.1 Introduction 11 2.2 Materials and Methods 12 2.2.1 Study area 12 2.2.2 Sewer sediment and sewage sample collection 12 2.2.3 Sediment fractionation 13 2.2.4 Antibiotic determination in sewage and sediment 13 2.3 Results and Discussion 18 2.3.1 Antibiotics in composite sewage samples 18 2.3.2 Antibiotics adsorbed to sewer sediments 19 2.3.3 Organic-bound antibiotic load as a linear function of liquid concentration 20 2.3.4 Adsorption dynamics and adsorption coefficient determined by bath experiments 20 2.3.5 Mineral composition of sewer sediment SED#1B 23 2.3.6 Initial characteristics of sediment SED#1B 23 2.3.7 Desorption dynamics and desorption coefficient of SED#1B 24 2.4 Conclusions 25 2.5 References 26 3 Abiotic, Biotic and Photolytic Degradation Coefficients of 14 Antibiotics and One Metabolite 32 3.1 Introduction 34 3.2 Materials and Methods 35 3.2.1 Study area and sample collection 35 3.2.2 Experimental set up 35 3.2.3 Modelling framework 38 3.2.4 Procedure of model calibration 40 3.3 Results and Discussion 43 3.3.1 Primary metabolic parameter 43 3.3.2 Secondary metabolic parameter 44 3.4 Conclusions 50 3.5 References 50 4 Activity-Inhibition of Microorganisms due to an Exposition with different Antibiotics and Concentrations 56 4.1 Assessing Antibiotic Resistance of Microorganisms in Sanitary Sewage 56 4.1.1 Introduction 57 4.1.2 Material and Methods 58 4.1.2.1 Sampling Site and Antibiotic Agents 58 4.1.2.2 Analyzing Antibiotics 60 4.1.2.3 Respiration Rate 60 4.1.3 Results and Discussion 60 4.1.3.1 Concentration Range of Antibiotics and Typical Sewage Parameters 60 4.1.3.2 Oxygen Uptake Rate 62 4.1.4 Summary and Conclusions 63 4.1.5 References 64 4.2 Hot Spots of Antibiotic Tolerant and Resistant Bacterial Subpopulations in Natural Freshwater Biofilm Communities due to Inevitable Urban Drainage System Overflows 66 4.2.1 Introduction 68 4.2.2 Material and Methods 69 4.2.3 Results and Discussion 72 4.2.4 Conclusions 76 4.2.5 References 76 5 Summery and General Coclusions 82 5.1 Adsorption and Desorption Affinity 82 5.2 Abiotic, Biotic and Photolytic Degradation 83 5.3 Activity-Inhibition of Microorganism Communities due to Antibiotic Exposure 84 5.4 Enhancement of the Stockholm County Council (2014) assessment of antibiotics 84 5.5 References 87 6 Proposed Directions of Future Research 90 7 Appendixes 94 7.1 Chapters 94 7.2 Figures 95 7.3 Tables 115 7.4 References 139

Antalet monoklonala antikroppar som används i läkemedel ökar kraftigt. Dessa läkemedel är dyra och risken för förfalskning är stor. Behovet att utveckla en metod för snabb och precis identifiering av monoklonala antikroppar är därför brådskande. För identifiering utfördes analyser med Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) på nio monoklonala antikroppar. Fokuset var att undersöka huruvida signifikanta fysiokemiska egenskaper och unika aminosyrasekvenser var närvarande och kunde urskiljas. Olika analyser med MALDI-ToF-MS användes till att både separera de monoklonala antikropparna baserat på dess fysiokemiska egenskaper, och annotera aminosyrasekvenser innehållande nyckelfragment. Med metoderna baserade på kapillärelektrofores uppnåddes också separation. CZE föredras framför CGE då mängden data som erhålls från CZE är större och provberedningen är enklare. Sammanfattningsvis utformades ett protokoll för identifieringsprocessen, vilket inleds med MALDI-ToF-MS-analyser av monoklonala antikroppar på reducerad form mot kända referenser. Därefter är en hypotes formulerad utifrån vilka antikroppar som ser mest lika ut. Slutligen analyseras dessa med CZE för fastställning av den monoklonala antikroppens identitet. / The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.

The mechanochemical polycondensation between a diamine and a dialdehyde constitutes a sustainable alternative to classical solvent-based polymerization reactions. This process not only allows for a higher conversion and a shorter reaction time as compared to standard solvent-based syntheses of this conjugated polymer, but the reaction can also be adjusted by the energy introduced via the ball mill.