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Qualitative analysis of selected glycosphingolipids subclasses isolated from human blood plasma using HILIC-ESI-MS/MS
ŠIKLOVÁ, Michaela
This bachelor thesis deals with the qualitative analysis of selected subclasses of glycosphingolipids isolated from human blood plasma using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HILIC-ESI-MS/MS). Glycosphingolipids are ubiquitous and structurally very diverse components of cell membranes involved in many cellular functions. It has been shown that changes in the structures of glycosphingolipids and their distinct expression can pose a key factor in the development of numerous diseases and different types of cancer.The theoretical part of this thesis is dedicated to a summary of fundamental knowledge about glycosphingolipids, their biosynthesis, classification, biological functions and their association with various types of diseases and cancers. Furthermore, methods of isolation, purification and analytical possibilities for the determination of glycosphingolipids in biological samples are described here.In the experimental part, blood samples were taken and processed together with the preparation of the necessary solutions, plasma samples, and standards. Moreover, a test analysis, optimization of mobile phase gradient and comparison of the most frequently used extraction methods according to Folch, Bligh and Dyer, Matyash, and Alshehry alongside fractionation of lipid extract is described in this part. An integral part of the experimental section was the identification and structural characterization of glycosphingolipids within selected subclasses, including the generation of GSL profiles in human blood plasma. In conclusion, the results of the work and answers to the stated research questions are summarized, including the evaluation of possible improvements in this field.
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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Pathobiochemistry of lysosomal storage disorders: Study of Fabry disease and generation of cellular models of X-linked disorders.
Rybová, Jitka ; Ledvinová, Jana (advisor) ; Entlicher, Gustav (referee) ; Živný, Jan (referee)
Human autopsy or biopsy tissue samples, mouse models and cell cultures of various types represent the most common materials in the investigation of cell pathogenesis of inherited diseases. This dissertation is devoted to all these approaches in the study of two X-linked lysosomal storage diseases, Fabry disease (FD,α-galactosidase A (AGAL) deficiency) and mucopolysaccharidosis type II (MPSII, idunorate-2- sulfatase (IDS) deficiency). The primary goal of the work was analysis of lipid blood group B antigens with terminal α-galactose (B-GSL) in the pancreas of FD patients with blood group B (FD-B).,In addition to the main glycosphingolipid (GSL) substrate, globotriaosylceramide (Gb3Cer), B-GSLs represent another minor substrate of AGAL. The deposition of undegraded B-GSL has been demonstrated in FD-B pancreas where it was significantly higher than in other organs such as the kidneys and lungs which accumulate mainly Gb3Cer. High concentration of lipid and non-lipid B-antigens was primarily confirmed in exocrine acinar epithelial cells of FD-B, accompanied by massive accumulation of ceroid (secondary sign of lysosomal storage). Unlike acini, the endocrine portion of the pancreas remained unaffected by accumulation of AGAL substrates. This interesting phenomenon of cell biology shows how a specific...
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