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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Regulation of NMDA receptor trafficking in mammalian cells
Hemelíková, Katarína ; Horák, Martin (advisor) ; Novotný, Jiří (referee) ; Valeš, Karel (referee)
N-methyl-D-aspartate (NMDA) receptors are a subclass of glutamate receptors that play an essential role in mediating excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). The activation of NMDA receptors plays a key role in brain development and memory formation. Abnormal regulation of NMDA receptors plays a critical role in the etiology of many neuropsychiatric disorders. NMDA receptors form a heterotetrameric complex composed of GluN1, GluN2(A-D) and GluN3(A, B) subunits. The NMDA receptors surface expression is regulated at multiple levels including early processing (synthesis, subunit assembly, endoplasmic reticulum (ER) processing, intracellular trafficking to the cell surface), internalization, recycling and degradation. NMDA receptors are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications including phosphorylation and palmitoylation. However, the role of N-glycosylation in regulating of NMDA receptor processing has not been studied in detail. The aim of this study was to clarify the mechanisms of regulation of surface expression and functional properties of NMDA receptors. We used a combination of molecular biology, microscopy, biochemistry and...
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Endoplasmic reticulum-associated degradation and its role in virus infection
Svobodová, Terezie ; Forstová, Jitka (advisor) ; Mašek, Tomáš (referee)
The endoplasmic reticulum-associated degradation pathway, ERAD, is an important mechanism for maintaining cellular homeostasis. The function of ERAD is degradation of accumulated unfolded proteins in the endoplasmic reticulum. ERAD is carefully regulated by pathway called "Unfold protein response" and by "ERAD tuning" mechanism. Some viruses have adopted the ways how to exploit this system or its factors for their own benefit. These utilizations include targeting of specific host proteins for degradation, transfer of viral products or virions from the endoplasmic reticulum into the cytoplasm, or the use of a membrane platform arising from the cooperation with "ERAD tuning" for viral replication. Role of ERAD in viral infection can manifest itself in different ways, it can contribute to degradation not only of host proteins but also of viral products. In this work I summarize mechanisms of ERAD pathway and their regulatory pathways. Meanwhile, in the specific examples, I present roles of ERAD pathway and associated systems in viral infections.
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Induction and course of programmed cell death in cancer cells after taxane application."
Kábelová, Adéla ; Jelínek, Michael (advisor) ; Gemperle, Jakub (referee)
The taxanes are a class of commonly used anticancer agents, which are very effective in treatment of breast, ovarian, prostate or lung cancer. Taxanes bind to the β-tubulin subunit of microtubules and lead to their stabilization and inhibition of depolymerization. Such microtubules lose their function to form mitotic spindle, thus arresting cells in G2/M phase and resulting in apoptosis. Unfortunately some cells are able to escape from taxanes-induced apoptosis by developing various mechanisms of resistance including alteration in taxanes target microtubules or upregulation of specific transporters that pump the drug out of cells. Other types of resistance are connected with process of programmed cell death (PCD), especially with proteins that after taxane application participate in its successful progress. Taxanes can directly or indirectly modify the activity of Bcl-2-family proteins that control mitochondrial and endoplasmic reticulum integrity, thus regulating the initiation of PCD. Caspases are executioners of PCD and caspase-2 activated by cytoskeletal disruption seems to be especially important in taxanes- induced apoptosis. In some cases can taxane treatment also result in caspase-independent cell death. Special role has protein p53 that seems to be involved only in apoptosis caused by low taxanes...
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Endoplasmic reticulum stress
Červenka, Jakub ; Schierová, Michaela (advisor) ; Horníková, Lenka (referee)
The accumulation of unfolded or misfolded proteins in endoplasmic reticulum (ER) leads to ER stress and the activation of unfolded protein response (UPR). Recent studies show that ER stress or UPR are associated with many diseases such as diabetes, hepatitis type C, prion disease, different kinds of tumors or Alzheimer's, Parkinson's and Huntington's disease and also with physiological processes like cell differentiation. When UPR is activated in yeast Saccharomyces cerevisiae, Ire1 protein oligomerizes, transautophosphorylates and activates itself. After this, Ire1 cleaves HAC1 mRNA to remove an intron. The spliced form of HAC1 mRNA is translated into the Hac1 transcription factor, which induces transcription of genes for chaperones of lumen ER, proteins involved in ERAD, synthesis of lipids etc. The cell uses this to reestablish homeostasis in ER. In mammals, the UPR is more complex and except Ire1 dependent pathway, it comprises Perk and Atf6 pathways, which are missing in yeast. Nevertheless, Perk is activated and regulated by the similar mechanism as Ire1 in S. cerevisiae. In consideration of broad spectrum of methods for genetic manipulation, rapid growth and well annotated genome, the yeast S. cerevisiae is a useful model for study of general mechanisms of UPR in mammals.
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Calcium homeostasis and modulation of nociceptive synaptic transmission
Sojka, David
This study was designed to improve our knowledge regarding mechanisms of nociceptive signaling at spinal cord level. One of the forms of spinal cord synaptic transmission modulation is central sensitization, a manifestation of synaptic plasticity at spinal cord level, which was found to be present at many chronic pain syndromes. This study deals mainly with a development of calcium imaging technique with a final goal to study mechanisms of central sensitization in vitro on population of dorsal horn neurons. We have analyzed synaptically evoked intracellular Ca changes as a result of dorsal root stimulation in a superficial dorsal horn area in spinal cord slices and found two types of Ca responses: one synchronized with electrical stimulation and a second one, delayed response due to Ca release from internal stores. The delayed Ca release was not previously shown to be present in these neurons and it was not dependent on activation of ionotropic glutamatergic receptors, suggesting involvement of metabotropic receptor pathway. The presence of this delayed type of Ca response could have a significant role in the induction of some types of chronic pain syndromes, since intracellular calcium increase is thought to be a key trigger point in spinal cord neurons sensitization. An important role in neuronal calcium...
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