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Vesicular roles of Arp2/3 nucleation-promoting factors
Dostál, Vojtěch ; Libusová, Lenka (advisor) ; Malínský, Jan (referee) ; Befekadu, Asfaw (referee)
F-actin is involved in key aspects of vesicular traffic, such as membrane deformation, tubulation and vesicle motion. Branching of F-actin is mediated by Arp2/3 but this complex must first be activated by so-called nucleation-promoting factors (NPFs). These factors play an essential role in the decision where and when branched actin should form on the membrane surface. The thesis focuses on the mechanisms which underlie localization and activation of NPFs, especially in terms of the phosphoinositide composition of the vesicle membranes. I show that one of the NPFs, the WASH complex, does not exclusively depend on the retromer complex for its membrane anchoring, as previously theorized. Rather, its understudied subunit SWIP enables the complex to independently bind to the membrane. I also present data showing that the WASH complex has essential roles in maintaining lysosomal function. Additionally, I elucidate the function of another NPF known as WHAMM in the ERGIC compartment, showing that it depends on the presence of myotubularin 9 for its ability to form membrane tubules. The thesis improves our understanding of the interface between the actin cytoskeleton and intracellular membrane system.
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Cathepsin L by parasites - occurrence, features, functions
Perháčová, Terézia ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Cathepsines L are lysosomal cysteine endopeptidases with an universal function in protein catabolism. This work discusses present knowledge about their characteristics in the context of their specific function in parasites. Features and function differences are described in detail on molecular level. The emphasis is on the biochemical properties with resultant use of these enzymes. Cathepsines L of kinetoplastida, aplikomplexa, entamoeba and helmints (focused on Fasciola spp and Schistosoma spp) are each discussed in appropriate chapters. Key words: hydrolase, protease, cysteine peptidase, cathepsin L, lysosome, parasite
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Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
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Molecular genetic and biochemical studies of selected inherited metabolic disorders, development and applications of new methods
Mušálková, Dita ; Hřebíček, Martin (advisor) ; Adam, Tomáš (referee) ; Macek, Milan (referee)
Inherited metabolic disorders (IMD) form a diverse group of several hundred different diseases with a relatively high cumulative incidence (stated up to 1:600). They are associated with accumulation of the substrates and lack of the products in specific metabolic pathways, which is caused by deficiency of the enzyme or its activator, or dysfunction of the transport protein. However, the underlying cause is at the DNA level. The grounds for different phenotype manifestation in patients with the same genotype are often not known. During my work at the Institute of Inherited Metabolic Disorders, I designed several new methods for the research of IMD and applied them in the patients and their families. I created procedures for the isolation of lysosomal membranes that are used for the research of lysosomal storage disorders and general properties of lysosomes. Next, I introduced several novel assays for determination of the X-inactivation ratio, which led to a significant increase of informative women. Nowadays, we use these methods in heterozygous women with X-linked diseases in order to study the influence of X-inactivation on the manifestation of the diseases. The cases of a girl with mucopolysaccharidosis type II, a girl with OTC deficiency and a family with the mutation in HPRT1 gene are described...
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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 ; Hřebíček, Martin (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
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...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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Influence of V-ATPase inhibitors on chemoresistant neuroblastoma lines in vitro
Honzejková, Karolína ; Eckschlager, Tomáš (advisor) ; Martínková, Markéta (referee)
Tumor diseases are one of the most common causes of death worldwide. Despite the great advances in therapy in the last fifty years, this is still a serious health problem. Therefore, great efforts are still concentrated on development of new anti-cancer drugs and therapeutic approaches. Neuroblastoma (NBL) is the most common tumor in infants and the fourth most common in children. Successful treatment is greatly complicated by its heterogeneity. Chemoresistance is an undesirable phenomenon of chemotherapy. One of the chemoresistance mechanisms is the accumulation of weakly basic anticancer drugs in lysosomes. This work deals with the measurement of lysosomal uptake of these compounds in neuroblastoma cell lines UKF-NB-4 and derived, ellipticine-resistant, line (UKF-NB- 4ELLI ) under different conditions. A method for determining the cell lysosomal capacity (volume) by measuring fluorescence intensity of lysosome-specific LTR dye was introduced and the ability of bafilomycin A, a V-ATPase inhibitor, to potentiate the effects of an anticancer agent ellipticine by inhibiting its lysosomal accumulation was investigated. Keywords: neuroblastoma, lysosome, vacuolar ATPase, multidrug resistance
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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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 ; Hřebíček, Martin (advisor) ; Macek, Milan (referee) ; Adam, Tomáš (referee)
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...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav ; Ledvinová, Jana (advisor) ; Stiborová, Marie (referee) ; Holčapek, Michal (referee)
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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