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L-type voltage gated calcium channels are membrane bound ion channels that regulate the intracellular concentration of calcium ions as a response to changes in the membrane potential. They exist in four isoforms and recent studies have brought extra attention to the Cav1.2 isoform as it has been shown to be critical for the maturation of oligodendrocyte progenitor cells in the brain. These cells are involved in neural myelination and if they do not mature properly, it can lead to abnormal neural myelination. This is a condition that is strongly correlated with neurodegenerative diseases such as Multiple Sclerosis and has been associated with psychiatric diseases such as schizophrenia. The number of tools for studying these proteins are few, and therefore this thesis aimed to expand the toolbox by developing an analogue (FluoBar2) to an existing probe (FluoBar1). The analogous probe could resolve some of the issues currently associated with labeling using FluoBar1 as well as open up the possibility of conducting pulse-chase experiments when used with FluoBar1. The short absorption and emission wavelengths of FluoBar1 causes large amounts of autofluorescence in cellular imaging, making the images hard to interpret. In FluoBar2, the Pacific Blue fluorophore used in FluoBar1 is exchanged for 5-carboxyfluorescein, causing the absorption and emission to be redshifted which theoretically should decrease the cellular autofluoresence. The project begun with the synthesis of FluoBar1 which was isolated in a 0.19% overall yield. During this, the synthetic route towards the linker used in FluoBar1 and FluoBar2 was developed as well, increasing the yield with respect to the most expensive reagent from 41.5% to 61%. An attempt at synthesizing FluoBar2 was made and its presence was supported by 1H NMR as well as UPLC-MS, but it was not fully isolated and characterized.

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Analytical surface sampling tools enable direct chemical studies of solid biological samples, including morphologically diverse tissue, and provide both fast chemical analysis with minimum sample preparation and information about molecular spatial distribution. Spatially resolved investigations of tissue sections could help to reveal pathological mechanisms at specific tissue locations. Mass spectrometry imaging (MSI) is a valuable method for direct tissue analysis that can give a full molecular profile of heterogeneous biological in a short period of time. However, MSI is not always capable of identifying isomeric and isobaric species, especially for low-weight metabolites. Moreover, the complex chemical matrix of biological samples significantly influences the performance of MSI, including introducing artefacts such as ion suppression. Therefore, separation by liquid chromatography or capillary electrophoresis prior mass spectrometric detection is beneficial to mitigate such artefacts and increase sensitivity. Recently, the new setup for surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS) was presented that enables direct sampling from a tissue location followed by separation and mass spectrometric detection. This thesis is aimed to show the developments and improvements of SS-CE-MS along with applications of quantitative spatial metabolomics. Paper I demonstrates SS-CE-MS using a sheath liquid interface to the MS and its robustness and reproducibility both for the injection process and the connection to mass spectrometer. Analysis of small polar molecules, lipids, and proteins in both tissue and blood samples is described in addition to an increased throughput using multisegmented electrokinetic injection. In Paper II five quantitative approaches for SS-CE-MS were developed and evaluated. Off-line one-point calibration was found to be optimal for tissue analysis and further used for mapping metabolites, including isomeric species, in four regions of rat brain. Specifically, aromatic amino acids were found decreased in cortex and the isomers valine and leucine were more abundant than their isomers betaine and isoleucine, respectively. Overall, this thesis shows SS-CE-MS as promising new quantitative method for future applications to healthy and pathological tissue investigation.

I det svenska djupförvaret för kärnbränsle ska kompakterad bentonit användas som barriär mellan kopparkapslar med utbränt kärnbränsle och berget. I kontakt med vattenförande sprickor kan bentonitbarriären under vissa omständigheter avge montmorillonitkolloider. Förutom att barriärens egenskaper urholkas pga förlusten av material kan kolloiderna, om de är stabila, underlätta transporten av sorberade radionuklider ut mot biosfären. Den här studien fokuserar på att undersöka vilka effekter grundvattenkemin har på montmorillonitkolloiders stabilitet. Grundvattnets sammansättning, pH och jonstyrka, kommer sannolikt att förändras under djupförvarets livslängd, delvis pga inträngandet av glactialt smältvatten. Initialt kommer omgivande berg att värmas upp pga värmealstring från det radioaktiva sönderfallet i det utbrända kärnbränslet. Effekterna av pH, jonstyrka och temperatur på montmorillonitkolloiders stabilitet har analyserats genom att följa hur kolloiderna aggregerar med tiden. Minskningen av partikelkoncentration med tiden mättes med Photon Correlation Spectroscopy (PCS). Aggregationsexperimenten visar att, vid ett givet pH och en given temperatur, ökar hastighetskonstanten för aggregation med ökande jonstyrka. Kritiska koaguleringskoncentrationen (CCC) för NaCl och CaCl2 för Na-montmorillonit och Ca-montmorillonit beräknas utifrån ett samband mellan hastighetskonstanterna och jonstyrkan. Hastighetskonstanten för aggregation minskar med ökande pH eftersom ytpotentialen ökar. Effekten blir tydligare vid högre jonstyrkor och högre temperaturer, men kan däremot inte observeras vid låga temperaturer. Temperatureffekten på bentonitkolloidernas stabilitet är pH-beroende. Vid pH≤4 ökar hastighetskonstanten för kolloidaggregation med ökande temperatur, oavsett jonstyrka.Vid pH≥10 minskar hastighetskonstanten med ökande temperatur. I mellanliggande pH-område minskar hastighetskonstanten för aggregation med ökande temperatur, förutom vid den högsta jonstyrkan, där den ökade. Beräkningar baserade på DLVO-teori matchar de experimentella resultaten. / In deep geological repositories in Sweden, encapsulated nuclear waste will be surrounded by compacted bentonite in the host rock. In future contact with water-bearing fractures, this bentonite barrier can release montmorillonite colloids under certain conditions. This process can lead to loss of buffer material. Furthermore, these colloids, if stable, may facilitate the transport of associated radionuclides towards the biosphere. Colloid stability is determined by groundwater chemistry. This study addresses the effects of groundwater chemistry on the stability of montmorillonite colloids. During the lifetime of the repository, the pH and ionic strength of the groundwater are expected to vary, partly due to intrusion of glacial melt water. Initially, the temperature will be higher in the surrounding host rock due to heat released from radioactive decay in the spent nuclear fuel. The effects of these parameters on the stability of montmorillonite suspensions were evaluated by studying the aggregation kinetics. The change in particle concentration with time was monitored by Photon Correlation Spectroscopy (PCS). Aggregation kinetics experiments showed that for a given pH and temperature, the rate constant for colloid aggregation increased with increasing ionic strength. The relationship between the rate constant and the ionic strength allowed the NaCl and CaCl2 critical coagulation concentration (CCC) for Na- and Ca-montmorillonite to be determined. The aggregation rate constant decreased with increasing pH as the surface potential increased. This effect became more pronounced at higher ionic strengths and higher temperatures but could not be observed at low temperature. The effect of temperature on the stability of the suspensions is pH-dependent. At pH≤4, the rate constant for colloid aggregation increased with increasing temperature, regardless of ionic strength. At pH≥10, the aggregation rate constant decreased with increasing temperature. In the intermediate pH interval, the aggregation rate constant decreased with increasing temperature except at the highest ionic strength, where it increased. The experimental results were in agreement with DLVO calculations. / QC 20101105

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