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Effect of amyloid β on the function of endosomes and lysosomes
Tmějová, Monika ; Rudajev, Vladimír (advisor) ; Čočková, Zuzana (referee)
Alzheimer's disease is progressive neurodegenerative disease characterized by accumulation of amyloid β aggregates in brain tissue. Understanding the mechanisms of amyloid β pathogenesis and neuronal cell destruction is still not clear. The most toxic form of amyloid β are 42 aminoacids long oligomers that tends to cumulate and speed up disease progression. Membrane dynamics which affect protein degradation and recycling within the cell plays a criticale role in maintaining homeostasis. Vesicular trafficking plays fundamental role in balancing physiological level of amyloid β. Disruption of endolysosomal complex leads to cycle of disruptions within the cell which results in neuronal cell death. The main aim of this thesis was to look through different ways how amyloid β42 affects endolysosomal compartment. Results of our work confirmed toxic effect of amyloid on SH-SY5Y cell line and its ability to damage functions of lysosomes. We were not able to confirm amyloid β toxicity on endosomal function. Key words: amyloid β, Alzheimer disease, oligomers, plasma membrane, endocytosis, endosome, lysosome, neurotoxicity
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The role of RNA demethylase FTO in differentiation, regulation of energy metabolism and sensitivity to streptozotocin of neuronal and glial cell models
Čočková, Zuzana
Fat mass and obesity associated (FTO) demethylase is responsible for erasure of the most abundant epitranscriptomic mark in eukaryotic mRNA, the N6-methyladenosine (m6A) residue. Together with other m6A erasers, writers (methyltransferases) and readers it forms an m6A regulatory pathway that controls the amount, location and biological effect of m6A. The dynamic regulation of the brain's m6A methylome during neurodevelopment is essential for maintaining cerebral functions. In addition, preclinical research suggests that the m6A regulatory pathway regulates energy balance in a tissue- and cell type-specific manner. The FTO gene has been associated with lifelong risks of obesity and metabolic syndrome as well as regulation of total body energy intake and expenditure. However, little is understood about the function of the m6A pathway in control of brain energy metabolism. That is of interest in pursuit of understanding Alzheimer's disease, as this illness is characterized by profound disruptions in cerebral energy metabolism and mounting evidence suggests that disrupted brain bioenergetics may play a role in the disease's early genesis, before the appearance of clinical symptoms. In the present thesis we aimed to investigate the role of FTO in models of two brain cell types, neurons and astrocytes....
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The role of RNA demethylase FTO in differentiation, regulation of energy metabolism and sensitivity to streptozotocin of neuronal and glial cell models
Čočková, Zuzana ; Novotný, Jiří (advisor) ; Horák, Martin (referee) ; Balík, Aleš (referee)
Fat mass and obesity associated (FTO) demethylase is responsible for erasure of the most abundant epitranscriptomic mark in eukaryotic mRNA, the N6-methyladenosine (m6A) residue. Together with other m6A erasers, writers (methyltransferases) and readers it forms an m6A regulatory pathway that controls the amount, location and biological effect of m6A. The dynamic regulation of the brain's m6A methylome during neurodevelopment is essential for maintaining cerebral functions. In addition, preclinical research suggests that the m6A regulatory pathway regulates energy balance in a tissue- and cell type-specific manner. The FTO gene has been associated with lifelong risks of obesity and metabolic syndrome as well as regulation of total body energy intake and expenditure. However, little is understood about the function of the m6A pathway in control of brain energy metabolism. That is of interest in pursuit of understanding Alzheimer's disease, as this illness is characterized by profound disruptions in cerebral energy metabolism and mounting evidence suggests that disrupted brain bioenergetics may play a role in the disease's early genesis, before the appearance of clinical symptoms. In the present thesis we aimed to investigate the role of FTO in models of two brain cell types, neurons and astrocytes....
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Modulation of RNA demethylase FTO function in SH-SY5Y cells: the effect on insulin signaling and mitochondrial respiration
Čočková, Zuzana ; Novotný, Jiří (advisor) ; Hlaváčková, Markéta (referee)
Aim of this thesis was to observe changes in oxidative metabolism and expression of important neuroenergetic proteins in human neuroblastoma cell line SH-SY5Y due to inhibition of FTO. FTO is a RNA demethylase that uses N6-methyladenosine as substrate. Differences in enzyme expression are connected to broad area of effects involving energy homeostasis. Mitochondria are cellular powerhouses, a key elements in production of energy and metabolic substrates, yet a source of potentially dangerous reactive oxygen species (ROS) and analogous reactive molecules. In order to better understand FTO purpose in neuronal energetic metabolism, we examined mitochondrial respiratory chain. Using high-resolution respirometry we were capable of observing impairment in mitochondrial respiration after FTO inhibition. There was considerable decline in endogenous respiration, maximal respiration rate and reserve capacity. In order to obtain more detailed view into mitochondrial respiration, expression levels of electron-transport complexes were quantified by Western blot technique. Slight reduction was identified in subunits of complex I and IV. However, the most prominent alteration was seen in complex II subunit. There were no differences in expression of complex III and ATP synthase subunits. Beside disrupted activity...
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The m6A pathway at the molecular level and its role in neurological diseases
Švendová, Aneta ; Černá, Barbora (advisor) ; Čočková, Zuzana (referee)
N6-methyladenosine is the most abundant modification in eukaryotic messenger RNA. This modification is reversible, thanks to a complex of methyltransferases and demethylases. The biological effects of m6 A are mediated through reader proteins. This complex mechanism of proteins contributes to many molecular processes such splicing, translation and transport. It also plays a role in many serious neurological diseases, such as Alzeheimer's disease, Parkinson's disease, major depressive disorder and attention deficit hyperactivity disorder. The purpose of this thesis is to describe the m6 A pathway, its regulation at the molecular level and to put it into context with neurological diseases of today. Key words: mRNA, mRNA metabolism, N6-methyladenosine, m6 A regulation, FTO, METTL, ALKBH5, neurodegenerative disorders, Alzheimer's disease, Parkinson's disease, major depressive disorder, ADHD
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Role of post-translational modifications, O-GlcNAcylation and Phosphorylation, in neurodegenerative disorders and brain hypometabolism
Špundová, Tereza ; Růžička, Jiří (advisor) ; Čočková, Zuzana (referee)
Post-translational modifications are major mechanisms that highly increase the variability in protein function. O-GlcNAcylation and phosphorylation are among the most extensively studied post-translational modifications in research to date. In physiological conditions, O- GlcNAcylation acts as a metabolic sensor that links glucose metabolism to normal neuronal functioning. Reversible phosphorylation is one of the mechanisms that can downregulate metabolism by regulating the rates of flux through metabolic pathways. The impairments in the regulation of these modifications are linked to with neurodegenerative disorders and hypometabolism. This thesis focuses on the crosstalk and correlation between these two modifications, their reciprocal relationship and their mutual impact on their regulations in models of neurodegenerative diseases and disease non-related models. Keywords: hypometabolism, O-GlcNAcylation, phosphorylation, post- translational modifications, neurodegenerative disorders, hibernation, caloric restriction, memory, learning
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Modulation of RNA demethylase FTO function in SH-SY5Y cells: the effect on insulin signaling and mitochondrial respiration
Čočková, Zuzana ; Novotný, Jiří (advisor) ; Hlaváčková, Markéta (referee)
Aim of this thesis was to observe changes in oxidative metabolism and expression of important neuroenergetic proteins in human neuroblastoma cell line SH-SY5Y due to inhibition of FTO. FTO is a RNA demethylase that uses N6-methyladenosine as substrate. Differences in enzyme expression are connected to broad area of effects involving energy homeostasis. Mitochondria are cellular powerhouses, a key elements in production of energy and metabolic substrates, yet a source of potentially dangerous reactive oxygen species (ROS) and analogous reactive molecules. In order to better understand FTO purpose in neuronal energetic metabolism, we examined mitochondrial respiratory chain. Using high-resolution respirometry we were capable of observing impairment in mitochondrial respiration after FTO inhibition. There was considerable decline in endogenous respiration, maximal respiration rate and reserve capacity. In order to obtain more detailed view into mitochondrial respiration, expression levels of electron-transport complexes were quantified by Western blot technique. Slight reduction was identified in subunits of complex I and IV. However, the most prominent alteration was seen in complex II subunit. There were no differences in expression of complex III and ATP synthase subunits. Beside disrupted activity...
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