The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D 1H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man- (1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.

Many important biological processes occur near or in membranes. The role of membranes is not merely confined to compartmentalization, they also form the matrix for membrane associated proteins and are of functional importance. Membrane associated proteins on the other hand require specific membrane properties for proper function. The interactions between membranes and proteins are thus of paramount importance and are at the focus of this work. To draw valid conclusions about the nature of such interactions the membrane mimetics required in biophysical methods must faithfully mimic crucial properties of biological membranes. To this end, new types of small isotropic bicelles which mimic plant and bacterial membranes were characterized by their size and lipid dynamics using solution-state NMR. Small isotropic bicelles are specifically well suited for solution-state NMR studies since they maintain a bilayer while being sufficiently small to conduct interpretable experiments at the same time. Monogalactosyl diacylglycerol and digalactosyl diacylglycerol, which are highly abundant in thylakoid membranes, were successfully incorporated into bicelles. Also, it was possible to make bicelles containing a lipid mixture extracted from Escherichia coli cells. A fundamental physical property of lipids in bilayers is their phase behaviour and thus the dynamics that lipids undergo in a membrane. Here, the dynamics of 13C-1H bonds in lipids were studied by nuclear spin relaxation. From such studies it was found that the glycerol backbone of lipids in bicelles is rigid while the flexibility of the acyl chain increases towards its end. Bulky head groups are rigid, while smaller head groups are more dynamic than the glycerol backbone. Acyl chain modifications, like unsaturations or cyclopropane moities, that are typically found in E. coli lipids, locally increase the rigidity of the acyl chain. Membrane interactions of a putative membrane anchor of the glycosyltransferase WaaG, MIR-WaaG, were studied by fluorescence methods, circular dichroism and solution-state NMR. It was found that MIR-WaaG binds to vesicles that mimic the anionic charge of E. coli inner membranes and that α-helical structure is induced upon interaction. The NMR-structure of MIR-WaaG agrees well with the crystal structure and from paramagnetic relaxation enhancement studies it could be concluded that a central part of MIR-WaaG is immersed in the membrane mimetic. Based on these results a model of the membrane interaction of WaaG is proposed where MIR-WaaG anchors WaaG to the cytosolic leaflet of the E. coli inner membrane via electrostatic interactions. These are potentially enhanced by membrane interactions of Tyr residues at the membrane interface and of hydrophobic residues inside the membrane.

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Linker histones (H1) are a family of lysine-rich proteins that bind to or near the point at which DNA enters and exits the nucleosomal core. A number of studies have shown that H1 expression levels are altered in cancer and that variant-specific changes can be observed in different tumor cells. Although several crystal structures are published for core-histone/DNA complex (nucleosome), the location of linker histone and its interactions are poorly understood. This study attempts to answer several questions regarding the interactions of histone H1 with double stranded partner DNA. Preliminary NMR assignments of this protein have been determined. We also investigated the structural changes in histone H1.0 globular domain induced by DNA binding. During the course of this project it was observed that subtle changes in pH could affect NMR spectral quality. We investigated the pH dependence of the protein stability by performing Circular Dichroism (CD) experiments.

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BACKGROUND. Prostate cancer (PCa) remains a major health concern in men of the Western World. However, we still lack effective diagnostic tools a) for an effective screening with both high sensitivity and specificity, b) to guide biopsies and avoid histology sampling errors and c) to predict tumor aggressiveness in order to avoid overtreatment. Therefore, a more reliable, highly cancer-specific and ideally in vivo approach is needed. The present study has been designed in order to further develop and test the method of "metabolomic imaging" using magnetic resonance spectroscopy (MRS) at 7T to address those challenges. METHODS. Thirty whole prostates with biopsy-proven PCa were in vitro analyzed with a 7T human MR scanner. A voxel grid containing the spectral information was overlaid with the MR image of the middle transverse cross-sectional plane of each case. Subsequent histopathological evaluation of the prostate specimen followed. After the spectral output was processed, all voxels were compared with a metabolomic PCa profile, which had been established within a preliminary study, in order to create a metabolomic map indicating MRS cancer-suspicious regions. Those regions were compared with the histologically identified tumor lesions regarding location. RESULTS. Sixty-one percent of the histological cancer lesions were detected by metabolomic imaging. Among the cases with PCa on the examined slice, 75% were identified as cancerous. None of the tested features significantly differed between detected and undetected cancer lesions. A defined "Malignancy Index" (MI) significantly differentiated between MRS-suspicious lesions corresponding with a histological cancer lesion and benign lesions (p = 0.006) with an overall accuracy of 70%. The MI furthermore showed a positive correlation with the Gleason grade (p = 0.021). CONCLUSION. A new approach within PCa diagnostics was developed with spectral analysis including the whole measureable metabolome - referred to as "metabolomics" - rather than focusing on single metabolites. The MI facilitates precise tumor detection and may additionally serve as a marker for tumor aggressiveness. Metabolomic imaging might contribute to a highly cancer-specific in vivo diagnostic protocol for PCa. / Diese Studie befasst sich mit der Diagnostik von Prostatakarzinomen mittels metabolomischer Bildgebung auf Grundlage der Magnetresonanzspektroskopie. An dreißig Organen mit gesichertem Prostatakarzinom wurde nach Prostatektomie in vitro ein Magnetic-Resonance-Spectroscopy-Imaging (MRSI)-Scan mit 7T durchgeführt. Die Auswertung der prostatischen Spektren erfolgte mittels eines in einer Vorstudie definierten Prostatakrebs-Profils, das die metabolomischen Daten des gesamten messbaren Metaboloms einbezog. Die Ergebnisse der metabolomischen Bildgebung wurden anschließend mit denen der histologischen Untersuchung nach dem MRSI-Scan verglichen. 61% der histologisch nachgewiesenen Krebsläsionen konnten mittels der metabolomischen Bildgebung detektiert werden. Ein "Malignancy Index" unterschied signifikant zwischen malignen MRSI-Läsionen sowie den falsch Positiven. Er dient nachweislich als möglicher Marker für Tumoraggressivität. Mit der metabolomischen Bildgebung wurde ein Verfahren vorgestellt, das hochspezifisch für das Prostatakarzinom ist, eine Tumorlokalisation vor Biopsieentnahme ermöglicht sowie eine Abschätzung der Tumoraggressivität und möglichen Progression erlaubt. Dennoch sind weitere Studien erforderlich, um das Verfahren sowie insbesondere die Sensitivität zu verbessern.

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