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Role of glycosylation of ionotropic glutamate receptors in mammalian neurons
Danačíková, Šárka ; Horák, Martin (advisor) ; Kriška, Ján (referee)
Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. There are two distinct types of glutamate receptors, ionotropic and metabotropic, present in the mammalian excitatory synapses. My thesis is focused on the ionotropic glutamate receptors, which play critical roles in learning and memory formation. The main subtypes of ionotropic glutamate receptors are α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. All types of the ionotropic glutamate receptors, which are assembled as tetramers, contain many glycosylation sites, which can be modified by glycans or monosaccharides. The glycans and monosaccharides attached to the ionotropic glutamate receptors have been shown to regulate key processes such as folding of the subunits, transport to the cell surface as well as their functional properties. Recent literature also suggests that many neurological and psychiatric disorders such as schizophrenia exhibit abnormal glycosylation of ionotropic glutamate receptors. Thus, understanding of the molecular mechanisms, which regulate the glycosylation of the ionotropic glutamate receptors, may be important for developing new therapies for the patients with altered functioning of the glutamatergic synapses in the...
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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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Human in vitro models for epilepsy research
Hebenstreitová, Kristýna ; Danačíková, Šárka (advisor) ; Valeš, Karel (referee)
Epilepsy is the most common chronic neurological disease that affects around 1 % of the world population. It is a multifactorial disease, which is characterised by recurrent seizures. Present pharmacological treatment is symptomatic and approximately one third of the patients develop pharmacorezistant epilepsy. This bachelor thesis presents an overview of current knowledge about the modelling of epilepsy, while focusing on human in vitro models. Cell lines appear to be very promising in vitro models for modelling genetic epilepsies. For example, cell lines derived from human induced pluripotent stem cells or human neural stem cells, which both allow for introduction of potentially pathological mutations and further differentiation into many different cell types. Human in vitro models of epilepsy play important role in understanding the process of epileptogenesis, ictogenesis, mechanism of antiepileptic drugs effects and are used in the search for new active substances. Epilepsy comes with great burden of disease for the patients, that is why it is very important to research not only the molecular mechanisms of epileptogenesis, but also advancements of personalised therapy, which could give hope to many (even pharmacorezistant) patients.
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Structural determinants of regulation of surface delivery of NMDA receptors in mammalian cells
Danačíková, Šárka ; Horák, Martin (advisor) ; Bendová, Zdeňka (referee)
N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels activated by agonist glutamate and co-agonist glycine. They play a key role in mediating the fast excitatory synaptic neurotransmission in the mammalian central nervous system. To create a functional heterotetrameric receptor, the presence of two GluN1 subunits combined with GluN2 or GluN3 subunits is necessary. Previous studies confirmed the importance of M3 transmembrane helix and extracellularly localized cysteines in regulation of surface expression of functional NMDA receptors. The aim of my thesis is to elucidate an influence of clinically relevant mutations in M3 transmembrane helix and the role of all known cysteines that form disulphide bonds on surface delivery of NMDA receptor expressed in heterologous monkey kidney fibroblasts cell culture (COS-7). Using molecular biology methods, immunocytochemistry and microscopy I found that the clinically relevant mutations M641I and Y647S in GluN1 subunit and also the mutations of particular cysteines forming disulphide bonds caused substantial decrease of surface expression of NMDA receptors. Furthermore, I discovered that the effect of mutated GluN1 subunits on decrease of surface expression depends on the subunit composition. The contribution of my results lies in elucidating the...
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Role of glycosylation of ionotropic glutamate receptors in mammalian neurons
Danačíková, Šárka ; Horák, Martin (advisor) ; Kriška, Ján (referee)
Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system. There are two distinct types of glutamate receptors, ionotropic and metabotropic, present in the mammalian excitatory synapses. My thesis is focused on the ionotropic glutamate receptors, which play critical roles in learning and memory formation. The main subtypes of ionotropic glutamate receptors are α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), N-methyl-D-aspartate (NMDA) and kainate receptors. All types of the ionotropic glutamate receptors, which are assembled as tetramers, contain many glycosylation sites, which can be modified by glycans or monosaccharides. The glycans and monosaccharides attached to the ionotropic glutamate receptors have been shown to regulate key processes such as folding of the subunits, transport to the cell surface as well as their functional properties. Recent literature also suggests that many neurological and psychiatric disorders such as schizophrenia exhibit abnormal glycosylation of ionotropic glutamate receptors. Thus, understanding of the molecular mechanisms, which regulate the glycosylation of the ionotropic glutamate receptors, may be important for developing new therapies for the patients with altered functioning of the glutamatergic synapses in the...
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