Leibfritz, Dieter
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Leibfritz, Dieter
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Leibfritz, Dieter
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Item-typ:Veröffentlichung, Collagen 1 fibers and hypoxic tumor microenvironments in breast cancer and their effect on transport of molecules within the tumor matrix(2013-10-23); ; ; The work presented in this thesis represents a culmination of four years of research work focused on understanding the role of collagen I (Col1) fibers, a major component of the tumor extracellular matrix in breast cancer. We have investigated the relationship of Col1 fibers with metastasis, hypoxia, macromolecular transport, water diffusion and fractional anisotropy using clinical breast cancer specimens and human breast cancer xenografts genetically engineered to fluoresce under hypoxia. The thesis describes in the detail of the rationale, study design, results and discussion for our studies, which have been either published or submitted as four separate papers. The work here also presents multimodal in vivo imaging techniques which could be used clinically for non-invasive diagnoses of breast cancer progression. These techniques will help provide better patient management.Dissertation291 99 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Lipidanalyse von Bioflüssigkeiten durch kombinierte SPE/HPLC-, MS- und NMR-Techniken(2013-05-07); ; ; Body fluids like blood plasma are easy to gain and reflect the metabolic state of an organism. One of the challenging tasks is the analysis of intact and underivatized lipids, which have various important functions in organism (energy storage, membrane building block, cellular signalling, precursor of relevant biomolecules etc.). The lipid composition consists of different, structural diverse lipid classes with several subclasses including numerous lipid species. These species vary in chain length, degree of saturation and/or kind of bonding of the attached fatty acid(s). Anomalous lipid compositions were correlated to different diseases like diabetes and many others. In the present work a hyphenated approach consisting of different separation (SPE, HPLC) and analytical techniques (MS, NMR) was applied for the investigation of lipids in body fluids. Such an approach combines the advantages of the different techniques resulting in increased selectivity, sensitivity and structure elucidation power. The thesis shows the way from finding a suitable lipid extraction method to the identification of individual lipid species in blood plasma and serum samples with a main focus on method development and optimization. Beside several SPE and LC-MS methods for the separation of various lipid groups, classes or species, a new isocratic HILIC-ESI-MS method for the class separation of phospholipids is mentionable. Furthermore, the analytic procedure was used for lipid analysis of diabetic mouse samples (diabetes mellitus type 2) to demonstrate the application for samples with pathological background. Detailed experimental features and results obtained on different lipids are part of this work.Dissertation449 296 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, NMR Spektroskopische Untersuchungen von Körperflüssigkeiten (Kinderurin)(2007-10-17); ; ; Urine is an information-rich biofluid that can provide insights into the metabolic state of an organism. While urine is easy to gain, the investigation is difficult since every metabolite needs a single analysis. NMR offers several advantages for metabolomic investigations. It requires minimal or no sample preparation and small sample volume. It is non-destructive and allows the observation of a large number of metabolites simultaneously and without preselection. A goal of this study was the assignment of resonances of recent metabolites in the one- and two-dimensional NMR spectra of human urine since the inherent complexity of urine NMR spectra is a common barrier to apply targeted investigations. The identification of recent metabolites was assisted by using SPE and HPLC.Changes in metabolic profiles resulting from diet may be difficult to differentiate from normal or pathological physiologic variation. Thus another aim was the definition of dietary influence on the composition of urine. We examined the effect of eating banana, pecans and oranges on the composition of urine. The metabolites were quantified on the basis of peak heights and were expressed in terms of Creatinine, which can provide an internal standard.Dissertation279 232 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Evaluation of the Effects of Tyrosine Kinase Inhibitors on the Metabolism of Human Tumor Cells using an NMR-based Biochemical Profiling Strategy(2006-05-17); ; ; Imatinib (Gleevec) is one of the first molecularly targeted cancer therapy drugs, successfully approved for the treatment of chronic myelogenous leukemia. The goal of this project was to assess the metabolic profiles and to gain mechanistic insights into the effects of tyrosine kinase inhibitors on cell metabolism. Most studies were based on nuclear magnetic resonance and mass spectrometry metabolic profiling. Treatment with imatinib induced apoptosis in human leukemia cells. Different stages of apoptotic cell death have been distinguished showing specific metabolic patterns. Imatinib resistant cell lines have shown characteristic profiles in regards to their glycolysis and lipid metabolism. After imatinib withdrawal, resistant cells underwent apoptosis and were regaining some of the properties of their parental clones. These findings may be important in the evaluation, identification, prediction of patients developing resistance and treatment of cancer. Based on these results, metabolic profiling of blood from imatinib-treated patients has the potential to be developed into a diagnostic tool to detect the imatinib resistance at an early stage.Dissertation247 112 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Methodological developments towards quantitative short TE in vivo :sup:1:/sup:H NMR spectroscopy without water suppression(2002-08-15); ; ; Water suppression (WS) has been used exclusively as a routine for almost two decades in the in vivo 1H NMR spectroscopy to avoid the experimental and postprocessing difficulties caused by the dominant water signal, which is 3 to 5 orders magnitude larger than the signals of metabolites of interest. However, WS has some disadvantages as compared to the spectroscopy without WS. This thesis is devoted to methodological developments towards short echo time (TE) in vivo proton NMR spectroscopy without WS. The methodology developed in this thesis integrates experimental and software approaches for the optimization of the spectroscopic techniques, which concerns the two major parts, namely, the spectral measurements and spectral quantification. An experimental method was developed to eliminate frequency modulation sidebands induced by gradient pulses, a major obstacle of in vivo 1H NMR spectroscopy without WS. First order phase errors were eliminated by radio frequency pulse sequence optimization. The dominant water signal was modeled and extracted with a scheme developed on the basis of the Matrix Pencil Method, The extracted water signal was used as a reference for the lineshape correction. With these approaches the imperfections of the spectra were removed. Spectral quantification scheme was developed by combining the baseline characterization by a wavelet transform based technique and the time domain spectral fitting using full prior knowledge of the metabolite model spectra. The model spectra were obtained by spectral simulation instead of in vitro measurements. The performance of the methodology was tested and verified by Monte Carlo studies, phantom measurements and in vivo measurements on rat brain, and compared with existing methods. More than 10 metabolites can be quantified from spectra measured on a 4.7 T NMR imaging system. The estimated concentrations of major metabolites are in good agreement with literature values.Dissertation227 265 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, The Development and Use of New Methods and Strategies for the Monitoring of Nephrotoxicity and the Study of Renal Physiology: Proteomics- and Metabonomics-based Studies(2006-06-20); ; ; A Key for avoiding the negative effects of immunosuppressant caused nephropathy after transplantation is early detection. This study shows a set of biomarkers, which could serve for the early detection of nephrotoxicity. Systematic development of a new model enables for the evaluation of drug induced nephrotoxicity and could be used for the development of new drugs in an early phase. We found indicators for the drug induced formation of reactive oxygen species, which could be causing the toxicity. The NMR-based model correlated with the 8-isoprostaglandin-F2aplha levels and the glomerular filtration rates showing the trend in toxicity:control<sirolimus<=tacrolimus<Sirl. Tac.<cyclosporine<Sirl. cylosporine. The combination of calcineurin inhibitors with sirolimus shows synergistic toxicity and that the combination of tacrolimus with sirolimus is favorable compared to cyclosporine with sirolimus.Investigations on changes caused by hyperosmotic stress in inner medullary collecting duct cells showed the importance of sorbitol and the polyol pathway as the most important mechanism in an environment of 900 mOsm/KgH2O media tonicityDissertation247 151 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Synthese von Gd(II)-Komplxen für die molekulare MRI(2009-02-04); ; ; The synthesis of functionalized Gd(III)-complexes (DTPA, TTHA, DOTA, DO3A) is described in this work. Several receptor mediated aproaches for molecular MRI were studied here. A novel opiate DTPA Gd(III)-complex was checked and showed on incubated C6-Glioma Cells in a MRI agarose gel phantom a concentration depended T1-contrast.Dissertation237 313 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Metabolism in Hepatocytes and Adipocytes under inhibition of carbonic anhydrases studied with multinuclear NMR-spectroscopy(2009-06-17); ; ; Obesity is defined as an intense overweight condition and is an increasingly serious problem for the health of population, particularly in the modern Western countries. The excessive body weight is associated with various diseases, especially cardiovascular diseases, diabetes mellitus type 2, certain types of cancer, sleep apnea, and osteoarthritis.There are therefore already a number of therapeutic procedures aimed at the treatment or prophylaxis of obesity. A novel therapeutic procedure for the treatment and/or prophylaxis of obesity, which can complement the previously known form of therapy, intends the inhibition of de novo lipogenesis (DNL). DNL means the synthesis of endogenous fatty acids from carbohydrate in the mammalian organism. There are various conceivable possibilities to inhibite DNL in mammalian cells, all of which aim to reduce the turnover of the TCA cycle. The inhibition of carbonic anhydrases (CAs) isozymes involving in several steps of the de novo lipogenesis can inhibit the DNL. The CAs catalyze a very simple physiological reaction, the interconversion of carbon dioxide and bicarbonate. It is comprehensible that inhibition of mitochondrial CA (i.e. CA V) and/or cytosolic CA (i.e. CA II) subsequently affect pyruvate carboxylation. The carbohydrates are not only used to synthesis lipids, but also TCA cycle intermediates and TCA cycle related amino acids such as glutamate. Sulphonamide/ Sulphamate are carbonic anhydrase inhibitors (CAIs) and good candidates to inhibit DNL on the step of pyruvate carboxylation in mitochondria and/or in cytosol. Inhibition of CA will reduce the influx into the anaplerotic activity of pyruvate carboxylase (pc) and subsequently into the citric acid (TCA) cycle, which will modify the intermediates of the TCA cycle and its effluxes (in particular glutamate) and lipid synthesis. The inhibition of carboanhydrase affects also the synthesis of alanine and lactate. To study the effect of CAIs upon the synthesis of lipids and other metabolites in HEP-G2 and 3T3-L1 cells multinuclear NMR spectroscopy was used. To investigate the effect of CAIs on the cellular metabolism, the HEP-G2 and 3T3-L1 cells were incubated for 6, 12, or 24h in the presence or absence of CAIs, and metabolic alterations of labeled substrates were followed up using NMR spectroscopy. The data of the present study clearly show that topiramate (TPM) and acetazolamide (ACT) are nonspecific inhibitors of CA isozymes (IC50 ≥1000 à �à µM), but DNL in the presence of lower concentrations of ethoxyzolamide (ETZ) is more effectively inhibited. The results also show that compound 1 (a newly synthesised sulfonamide) is a very efficient candidate to inhibit CA II and CA V and possibly also other CA isozymes. The data of this study show also that compound 1 has no effect on DNL, if labelled acetate is used as tracer. Therefore, the effect of compound 1 on DNL operates at the level of CA V and/or CA II and not at the level of acetyl-CoA carboxylase (ACC).HEP-G2 cells were also incubated for 6h with compound 5 (another newly synthesised sulfonamide) and three follower compounds of compound 1 (i.e. compound 2, compound 3, compound 4) or drug vehicle (0.1% DMSO) to study the CA inhibitory effect on bicarbonate fixation in cultured HEP-G2 cells. The results of this study clearly show that compound 5 is the most efficient CAI whereas the followers of compound 1 are mediocer CAIs for DNL. DNL is inhibited to 100% in the presence of 50 à �à µM compound 5 or 100 à �à µM compound 1, thus the effects of 50 à �à µM compound 5 on bicarbonate fixation in cultured HEP-G2 cells is comparable with the inhibitory effect of 100 à �à µM compound 1. The HEP-G2 cells were incubated in the presence of CAIs or drug vehicle (0.1% DMSO) for control with [U-13C]glucose for 6 or 24h. The results of this study show that after 6h the effect of 50 à �à µM compound 5 and 100 à �à µM compound 1 on the glucose influx into the TCA cycle via pc and on pdh are similar. However after 24h in the presence of 50 à �à µM compound 5, the influx of labelled glucose into the TCA cycle via pc is decreased by only 20% and pdh activity is not affected, whereas after 24h the pc and pdh activities are reduced in the presence of 100 μM compound 1 to 100% and 80% of control respectively. The results of experiments with [2-13C]acetate in the presence of compound 5 show that compound 5 has possibly an indirect inhibitory effect on pdh. The results of this study also show that, similar to compound 1, the effect of compound 5 on DNL is at the level of CA and not at the level of acetyl-CoA carboxylase.Dissertation266 123 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Automatische Quantifizierung von Metabolitenkonzentrationen in :i:in vivo:/i: Spektren(2004-03-10); ; ; :p:A number of dedicated quantification tools have been developed for the interpretation of :i:in vivo:/i: magnetic resonance spectroscopy (MRS) data. To be useful in a clinical setting these tools have to meet the requirements of both a high level of automation and a general applicability in a wide range of clinical examinations. Existing tools put their main emphasis on one or other of these two aspects, but not on both simultaneously. The quantification tool of this work, PRISMA, was therefore developed to meet both of these requirements and to deliver reproducible and consistent results with good reliability. :p:The design of PRISMA allows on the one hand an easy adaptation to different applications due to a very flexible model parameterization. On the other hand it guaranties a robust and automatic result generation by an assumption-free assignment of the metabolites. For the baseline a new model is introduced which is based on a finite time signal. :p:The robust behavior of the PRISMA quantification is demonstrated on diverse :sup:1:/sup:H MRS data acquired from different anatomical regions. Besides synthetic data signals from brain and prostate measurements of healthy and pathological tissue were analyzed. The measurements were done at varying field strength, echo times and signal to noise ratios using single voxel and multi voxel techniques (SVS, SI). In addition, typical characteristics of PRISMA such as reproducibility and reliability were investigated and found to compare well with the performance of other quantification tools.Dissertation364 183 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Isolation und Identifikation von Lipiden in Körperflüssigkeiten.(2007-12-19); ; ; The analysis of intact and underivatised lipids is one of the challenging tasks of body fluids as well as cell and tissue extracts. The lipid composition consists of different classes such as neutral and positively or negatively charged lipids with manifold subclasses of structural diversity. Phospholipids consist of a polar head group ( i.e. phosphorylcholine, etc.) attached to the sn-3 position of glycerol and varying saturated and unsaturated fatty acids esterified to the sn-1 and sn-2 position whereby fatty acids in position sn-1 were mostly saturated. Structural diversity will have a distinct effect on membrane properties (i.e. fluidity, permeability, oxygen scavenger, etc.). Anomalous membrane compositions were correlated to neoplastic and neurodegenerative diseases, diabetes and many others. Further more they are involved in cell death (apoptosis, necrosis), cellular signaling and are precursers for lysophosphatidylcholines, diacylglycerols, phosphatic and arachidonic acid. Many different analytical techniques have been proposed to characterize pathophysiological deviations of the native lipid composition, but most of them are destructive. Further more most of these studies recorded only the total amount of the individual lipid classes without acquiring additional information on the composition of structural subclasses. With NMR spectroscopy it is possible to measure intact biomaterials nondestructively without any preceding derivatisation, but its low sensitivity limits a wide application. To overcome the limitations of a single analytical tool the combined approach in lipid analysis should improve the reliability of the results and eventually will reveal yet unknown biomarkers or species. Therefore, an efficient analytical tool is needed to identify and quantify in situ low concentrated known, but also still unidentified naturally occurring species to get a quick insight into biochemical processes. The combination of HPLC separation power, MS sensitivity with accurate mass measurement of molecular and fragment ions and NMR structure elucidation power will meet most suitably the challenge and will overcome the limits any single technique, but also demonstrate the potential of their combination ultimately to analyze native lipid mixtures. By means of the HPLC separation with novel isocratic reversed phase HPLC-methods (separation according to the fatty acid composition) it was possible to get semi quantitative information of the individual species in the phosphatidylcholin, phosphatidylethanolamin, phosphatidylserine, phosphatidylinositol and sphingomyeline extracts. MS peak detection offers a high sensitivity to detect low concentrated compounds but has only a low specificity. MS/MS experiments overcome this problem and allow the assignment of individual fatty acids to the sn-1 or sn-2 position within the glycerol moiety. By high resolution spectra it is additional possible to generate the compound's molecular formula to detect abnormalities like oxidation of the double bond etc. The separated phospholipids were pooled and sampled in a 600 MHz NMR-spectrometer for recording 1D and 2D spectra to assess the potential, power and universality of this method. Based on the MR-data (1H, 13C or 31P) the lipid class, degree of unsaturation and anomalies were easily identified. The hyphenated approach benefits most by the NMR spectroscopy structure elucidation power in complex mixtures. In routine analysis data base identification by retention time, precision mass and 1H NMR helps to save money and time. Only new, abnormal or unclear sample compounds should be selected for more time consuming 2D NMR techniques. Detailed experimental features and results obtained on these lipids are given in the document.Dissertation253 113