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Annulate lamellae and their role in viral infection
Doležal, Vojtěch ; Bruštíková, Kateřina (advisor) ; Vopálenská, Irena (referee)
Annulate lamellae are subdomain of endoplasmatic reticulum made of lipid membrane. This membrane has inserted pore complexes structurally and spatially similar to the nuclear pores. Annulate lamellae naturally occur in germ cells, but their formation can be induced even in cell types in which they naturally do not occur. They significantly participate in nuclear assembly after cell division. They also participate in regulation of Ca2+ release from endoplasmatic reticulum into the cytoplasm. Annulate lamellae formation is also induced by infection by some viruses. The first part of this work summarizes the history of annulate lamellae, their occurence in different cell types, localization within cells, their structure and fuction. Second part of this work summarizes the occurence of annulate lamellae in virus infected or transformed cells and their function in viral infection if it is known. Keywords: Annulate lamellae, pore complex, nucleoporin, oocyte, virus, endoplasmatic reticulum
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Phosphoinositides in membranes associated with RAB1
Růžička, Václav ; Doubravská, Lenka (advisor) ; Bouřa, Evžen (referee)
Small GTPases of the RAB protein family are key regulators of numerous cellular pathways, including membrane trafficking and autophagy. With the control of membrane processes are also associated phosphorylated phosphatidylinositol derivatives, known as phosphatidylinositol phosphates (PIPs). Our focus lies on the RAB1A and RAB1B GTPases and the processes of early protein secretion they regulate. The enzymes that control specific phosphorylated forms of phosphatidylinositol seem to be functionally related to this pathway, indicating that these lipids also regulate at least some aspects of protein secretion. However, the mechanism by which they do so remains unclear. This bachelor thesis therefore summarises the current knowledge of phosphatidylinositol phosphates on RAB1A and RAB1B associated membranes, including their role on the endoplasmic reticulum (ER) and its export sites (ERES), on the intermediate compartment between the ER and the Golgi apparatus (ERGIC), and on the Golgi apparatus itself. This thesis also specifically examines direct interactions between RAB1 GTPases and phosphatidylinositol phosphates or their related enzymes. In summary, it can be concluded that phosphatidylinositol phosphates play a significant role in the early stages of protein secretion.
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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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Proximity proteome of intramembrane serine protease RHBDL4
Boháčová, Šárka ; Stříšovský, Kvido (advisor) ; Brábek, Jan (referee)
Regulated intramembrane proteolysis is an interesting process involved in a multitude of cellular pathways. Enzymes which catalyse this are termed intramembrane proteases (IMPRs), cleaving proteins passing through the membrane within their transmembrane domain. Rhomboid proteases are serine IMPRs. They are widely distributed among organisms and evolutionarily conserved, but despite many efforts, their physiological roles are largely unexplored. RHBDL4 is a mammalian rhomboid protease localised to the endoplasmic reticulum. It is involved in the development of colorectal cancer, which makes it an important focus of research, but its physiological function is not well understood. In order to explore it, I established and employed a proximity proteomics approach, termed APEX2. It is based on biotinylation of proteins in the spatial proximity of the target in the physiological environment of intact living cells. Labelled proteins are subsequently purified, identified and quantified by mass spectrometry. Exploring the physiological vicinity of RHBDL4, its interaction partners and substrates can be revealed and the detailed subcellular compartment, where RHBDL4 resides, can thus be inferred. During three independent experiments in HCT116 cell line, three proteins emerged repeatedly in the RHBDL4...
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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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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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Binding proteins of MTMR9
Holšteinová, Aneta ; Doubravská, Lenka (advisor) ; Cebecauer, Marek (referee)
Myotubularins are lipid phosphatases that dephosphorylate phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate the position three of the inositol ring. This allows them to regulate the structure of the lipid layer of the membrane compartment. The first member of the family was described in association with a severe hereditary myopathy. From that point on, another thirteen members have been added to the family. The catalytically inactive MTMR9 carrying the conserved mutation in the phosphatase domain regulates the localization of the marker of the early secretory pathway, RAB1A, the cis-Golgi structure and the secretion. MTMR9 interacts with the catalytically active MTMR6 and MTMR8 that specifically localizes and increases their phophatase activity. The aim of this diploma thesis was to find out whether the phenotype observed in cells with altered MTMR9 levels is dependent on the catalytically active phosphatases MTMR6 and MTMR8. We proved the influence of MTMR6 and MTMR8 on the distribution of tranfected RAB1A between the intermediate compartment and the Golgi apparatus. MTMR6 and MTMR8 also take part in regulating the cis-Golgi structure. By the use of two different approaches we did not manage to clarify the influence of MTMR6 and MTMR8 on secretion. Changes in the catalytic...
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Connection between Unfolded Protein Response (UPR) and plant immunity
Kapr, Jan ; Burketová, Lenka (advisor) ; Vosolsobě, Stanislav (referee)
This bachelor thesis is concerned with the specific pathway in a response to endoplasmic reticulum stress in plant cells - the Unfolded Protein Response (UPR) and its role in plant immunity signalling. The work summarizes the main recent knowledges of molecular components of plant immunity and response to plant pathogens, focusing on important molecules that are also connected to UPR. The role of salicylic acid as a molecule on a crossroad between UPR signalling pathways and local and systemic resistence, is highlighted in this work. Recently, the phospholipids have also been shown to be important component of signaling pathways in response to biotic stress in plants and their role is also mentioned.
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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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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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