Global ETD Search

21	Příprava a charakterizace buněčných modelů lysosomálních dědičných onemocnění - mukopolysacharidos / Preparation and characterization of cell models of lysosomal hereditary diseases - Mucopolysaccharidoses Presová, Gabriela January 2020 (has links) Mucopolysaccharidoses are a group of diseases that belong to lysosomal storage disorders. A common sign of these monogenic multisystem diseases is a gene mutation leading to a deficiency of the lysosomal enzyme participating in glycosaminoglycan degradation. It results to their accumulation in the tissues and organs, where they cause a progressive damage. There is no efficient treatment available for most mucopolysaccharidoses. Moreover, the research is complicated because of the low prevalence and type of affected tissues. Animal models of these human diseases are used for an evaluation of newly developed therapeutic approaches. However, they also have many limitations due to the different pathogenesis and catabolic pathways of the accumulated substrates between humans and animals. Therefore, animal models are replaced by human cell models. In this thesis, the development of four mucopolysaccharidoses human cell models is reported (MPS IIID, MPS IVA, MPS IVB, MPS VI). Corresponding genes (GNS, GALNS, GLB1, ARSB) were inactivated using CRISPR/Cas9 technology, where plasmids containing specific inserts are delivered to the target human induced pluripotent stem cells (iPSC), using electroporation. Isolated clones, which represent iPSC disease models, were characterized by Sanger sequencing, enzyme...
22	Charakterizace promotorových oblastí genů HGSNAT a GBA, a příspěvek ke studiu patogeneze MPS IIIC a Gaucherovy choroby / Characterization of promoter regions of HGSNAT and GBA genes, and a contribution to the study of pathogenesis of MPS IIIC and Gaucher disease Richtrová, Eva January 2014 (has links) Pathogenesis of mucopolysaccharidosis type IIIC (MPS IIIC) and Gaucher disease has not been yet fully clarified, and the causes of phenotypical variability between the patients with the same genotype in Gaucher disease remain obscure. Because the variants in the regulatory regions of genes can cause phenotypical differences mentioned above, I have studied promoter regions of HGSNAT and GBA genes mutated in these lysosomal disorders. I have shown that there is an alternative promoter of GBA (P2). Additional studies were aimed to elucidate possible physiological functions of P2, and its possible role in the pathogenesis of Gaucher disease. I have found that P2 is not tissue specific, and that its variants do not influence the variability of phenotype in Gaucher patients with the same genotype. P2 is used differentially neither during the differentiation of monocytes to macrophages nor in macrophages from controls and Gaucher patients, in whom there is a prominent storage only in cells of macrophage origin. We have thus not found any changes that would suggest a role for P2 in the pathogenesis of Gaucher disease. I have characterized the promoter region of HGSNAT and shown that the binding of Sp1 transcription factor is important for its expression. Sequence variants found in HGSNAT promoter in...
23	Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism Kamani, Mustafa 08 August 2013 (has links) Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways. Sphingolipids Metabolism Lysosomal storage disorders adamantane Glucosylceramide Lactosylceramide P-glycoprotein MDR1 Gaucher disease Fabry disease siRNA Gb2 galabiosylceramide Glycolipids Inhibitors Knockout mouse Crossbreeding Glucosylceramide synthase Lactosylceramide synthase Ganglioside Globo-series Gb3 synthase pharmacological chaperone Glucocerebrosidase Substrate reduction therapy ATP8B1 Cholesterol ABC Transporters P-type ATPase Flippase FAPP2 GLTP Glycosyltransferase Glycolipid analogues ERAD ABCB1 knockdown GM3 and EGFR GM3 and insulin receptor Glycolipid synthesis Glycolipid metabolism enzyme enhancement therapy 0487 0307 0491 0379 0306
24	Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism Kamani, Mustafa 08 August 2013 (has links) Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water-soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation. Two predominant theories have been developed to explain how GlcCer accesses the Golgi lumen: one involving direct translocation from the cytosolic-to-lumenal leaflet of the Golgi by the ABC transporter P-glycoprotein (P-gp, ABCB1, MDR1), and the other involving retrograde transport of GlcCer by FAPP2 to the ER, followed by entry into the vesicular transport system for Golgi lumenal access. To examine the in vivo involvement of P-gp in GSL metabolism, we generated a knockout model by crossbreeding the Fabry disease mouse with the P-gp knockout mouse. HPLC analyses of tissue Gb3 levels revealed a tissue-specific reduction in MDR1/Fabry mice. TLC analyses, however, did not show such reduction. In addition, we performed a gene knockdown study using siRNA against P-gp and FAPP2. Results show these siRNA to have distinct effects on GSL levels that are cell-type specific. These results give rise to the prospect of unique therapeutic approaches by targeting P-gp or FAPP2 for synthesis inhibition of particular GSL pathways. Sphingolipids Metabolism Lysosomal storage disorders adamantane Glucosylceramide Lactosylceramide P-glycoprotein MDR1 Gaucher disease Fabry disease siRNA Gb2 galabiosylceramide Glycolipids Inhibitors Knockout mouse Crossbreeding Glucosylceramide synthase Lactosylceramide synthase Ganglioside Globo-series Gb3 synthase pharmacological chaperone Glucocerebrosidase Substrate reduction therapy ATP8B1 Cholesterol ABC Transporters P-type ATPase Flippase FAPP2 GLTP Glycosyltransferase Glycolipid analogues ERAD ABCB1 knockdown GM3 and EGFR GM3 and insulin receptor Glycolipid synthesis Glycolipid metabolism enzyme enhancement therapy 0487 0307 0491 0379 0306

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