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Carbohydrate dimers in tumor immunotherapy
Krupová, Monika ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Carbohydrate dimers in tumor immunotherapy Monika Krupová (Department of Biochemistry, Faculty of Science, Charles University in Prague) One possible approach to tumor immunotherapy is an activation of killer lymphocytes through specific ligands for their surface receptors. CD69 is a molecule greatly widespread among various cells of haematopoietic origin. Since the physiological ligand for this receptor is still unknown, ligand mimetics are used for modulation of its activity. The mimetics tested in this work are based on monomeric or oligomeric carbohydrated attached through two different chemical groups to the central linker of varying length, giving rise to thiourea and triazole series. The ability to precipitate soluble NKR-P1 and CD69 receptors was evaluated in precipitation assays and the optimal length of the linker for NKR-P1 receptor was found to be decyl. On the other hand, cross-linking of CD69 is not so dependent on the length of the linker. The aim of this work was to describe in vitro effect of the tested compounds on cellular signalization, natural killing of leukemic cell lines and activation-induced apoptosis. Compounds of triazole series containing two disaccharides (GalNAc β1→4 GlcNAc) linked by a linker were found to have the strongest effect on the production of...
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Úloha Mst1 / FoxO dráhy při indukci apoptosy
Lettlová, Sandra ; Bezouška, Karel (advisor) ; Liberda, Jiří (referee)
Vitamin E analogue -tocopheryl succinate (-TOS) from the group of mitocans, the drugs targeting mitochondria, is a selective inducer of apoptosis in various cancer cell types, which involves the accumulation of reactive oxygen species (ROS). It was found that ROS generation causes p53-independent upregulation of the pro-apoptotic protein Noxa that induces apoptosis by displacement of the BH3-only protein Bak from its inactive complex with the anti-apoptotic protein Mcl-1 to form a pore in outer mitochondrial membrane. Current research has demonstrated that generation of ROS causes activation of Mst1, a component of the Hippo pathway that presents a universal size-control mechanism in all metazoans, whose deregulation is linked to tumorigenesis. Treatment of Jurkat cells with - TOS revealed that activated Mst1 kinase phosphorylates the Forkhead box O1 (FoxO1) transcription factor that then translocates to the nucleus and activates transcription of genes important for apoptosis induction, including NOXA. This explains the p53 independent apoptosis induction and presents Mst1-FoxO1-Noxa as a new pathway involved in the process. Current research has also documented that activated Mst1 kinase controls the expression of the c-MYC oncogene and its target genes, whose products are involved in glucose...
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Effect of iron overload on the induction of apoptosis in mammalian cells
Kabíčková, Tereza ; Balušíková, Kamila (advisor) ; Klíma, Martin (referee)
Iron cations are an important metal ions required to number of essential cell functions. On the other hand, ferrous iron can be very toxic as well. When surplus iron is present in cells, it can catalyze the formation of reactive oxygen species (especially hydroxyl radicals) by Fenton reaction. Iron homeostasis is predominantly regulated by very strict mechanisms on the level of iron uptake into the body. Moreover, iron absorption, transport and storage within the body can be also regulated using complex mechanisms which differ on the level of individual cells and on the level of whole organism. Deregulation of iron homeostasis causing an iron overload and generation of reactive oxygen radicals can evoke serious cell damage leading up to apoptotic cell death. Excess iron storage and subsequent development of oxidative stress can affect lot of different tissues in the body. The organ damages such as fibrosis, cirrhosis, hepatocellular carcinoma, heart failure, loss of β cells and glucose intolerance or diabetes mellitus in patients with iron overload are very often seen. Nevertheless, the apoptosis induced by iron overload has not been well elucidated yet. There are no complex informations about the precise mechanism by which oxidative stress affects different cell types or whether there are other...
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Interference of selected DNA viruses with apoptotic processes
Sauerová, Pavla ; Forstová, Jitka (advisor) ; Štěpánek, Luděk (referee)
This work is focused on selected DNA viruses and some of their mechanisms used for inhibition or induction of the apoptotic processes. The selected DNA viruses are Hepatitis B virus, polyomaviruses, papillomaviruses and herpesviruses. Viruses developed different strategies for fighting the host defense mechanism during their evolution. One of the host defense mechanisms that reacts against virus infection is apoptosis. In case of viruses we can observe the phenomenon of inhibition or induction of apoptosis (which both depend on the life cycle phase of the virus). The purpose of these "fighting" strategies is to ensure successful replication, virus releasing from the cell and finally to let it spread in an organism or among them. Some "fighting" strategies are similar e.g. targeting and manipulation on p53 oncosupresor level or production of Bcl-2 homologs; other strategies are very specific. Certain viruses have mechanisms which allow them to survive in a host organism for a long time.
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The influence of acyclic nucleotide phosphonates PMEG and PMEDAP on p38 kinase signaling in human leukemic cells
Nejedlá, Michaela ; Entlicher, Gustav (advisor) ; Slaninová, Jiřina (referee)
PMEG [9-(2-phosphonomethoxyethyl)guanine] and PMEDAP [9-phosphonomethoxy- ethyl)-2,6-diaminopurine] are acyclic nucleoside phosphonates possessing cytotoxic properties. Antiproliferative effect of PMEG was demonstrated in various tumor cell lines in vitro. PMEG also represents an active component of some experimental prodrugs with enhanced selectivity and efficacy (such as GS-9219). PMEDAP seems to have weaker effect in vitro compared to PMEG, however it exhibited pronounced antitumor effect in SD-rats with spontaneous lymphoma. Therefore it was included in the present study as well. The aim of this study was to describe the interactions of PMEG and PMEDAP with p38 MAP kinase signaling and its relationship to the apoptosis. We investigated the influence of these compounds on the expression of four genes encoding p38 MAPK isoforms and whether this change is translated into the protein. It was found that PMEG up-regulates p38β and γ mRNA in CCRF-CEM cells and p38 β and δ in HL-60 cells. The effect of PMEDAP was less pronounced than that of PMEG. However, total p38 protein level remained unaffected by PMEG and PMEDAP. Activation of p38 MAPK cascade was also measured in the cells exposed to these agents using phospho-specific antibodies. We found that neither PMEG nor PMEDAP activated p38 kinase...
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IGFBP3 expressing rekombinant vaccinia virus used for tumor therapy
Musil, Jan ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...
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Role of DD- and DED-containing adaptor proteins in apoptotic signaling
Čaja, Fabián ; Janštová, Vanda (referee) ; Anděra, Ladislav (advisor)
Proteins containing a bundle of six anti-paralel α-helices in so-called "death domain" (DD) and similar structures (DED, CARD) represent important players in apoptotic signaling. To DD/DED/CARD domains-containing proteins belong pro- apoptotic membrane receptors from the TNFR superfamily, then adaptor proteins and enzymes as proteases or kinases. These pro-apoptotic "death receptors" interact with adaptor proteins and initiator caspases containing DDs or DEDs and activate apoptotic signaling cascade. DEDs and DDs are in addition found in many proteins participating in activation of caspases or other non-apoptotic signaling. Many experimental models document that defects in and deregulations of proteins containing DDs and DEDs can have severe if not lethal consequences for an organism. Abberations in these proteins in many cases could lead to cancerogenesis, immunodeficiencies or developmental defects.
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The role of fatty acids in apoptosis induction in pancreatic beta cells
Žigová, Ivana ; Libusová, Lenka (referee) ; Němcová, Vlasta (advisor)
Type 2 diabetes belongs to lifestyle diseases. To its development contributes also the decrease in functional cell mass caused particularly by long-term action of increased blood levels of fatty acids and glucose. Experiments from recent years demonstrated that apoptosis of pancreatic cells is induced especially by saturated fatty acids, in contrast to unsaturated fatty acids which do not exert significant cytotoxic effect and they are even able to inhibit the proapoptotic effect of saturated fatty acids. Despite intensive research, the mechanisms of fatty acid-induced apoptosis of pancreatic cells are not convincingly explained. Understanding of the processes that lead to destruction of functional cells could enable development of new therapies for type 2 diabetes treatment that would be targeted directly at one of the causes of this disease. The aim of this work is to summarize the recent knowledge about mechanisms by which fatty acids induce and regulate apoptosis of pancreatic cells.
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The role of caspase 2 in apoptosis induction in tumor cells.
Schmiedlová, Martina ; Horníková, Lenka (referee) ; Kovář, Jan (advisor)
Within the cell, caspase-2 probably fulfills several functions. Caspase-2 can be involved in apoptosis induction, DNA repair as well as cell cycle regulation. Caspase-2 has the character of initiator and also executioner caspase. A stimulus for caspase 2 activation can be oxidative stress or DNA damage. Caspase-2 is activated by cleavadge during an interaction with protein complexes. One of protein complexes,i.e. PIDDosome, is made of protein PIDD, RAIDD and pro-caspase-2. Withine the PIDDosome, caspase-2 is activated. Activated caspase-2 occures in a short S form and in long L form. L form of caspase-2 has proapoptotic effects and S form of caspase-2 has antiapoptotic effects. Caspase-2S has been only detected on mRNA level but not on protein level. The main role of caspase-2L is apoptosis induction in normal and tumor cells. Caspase-2 in tumour cells is activated by extrinstic as well as intristic apoptotic pathway. Apoptosis induction by caspase-2 is for example studied in connection with breast cancer treatment with taxanes. Caspase-2 ability of apoptosis induction in cancer cells independently of p53 protein is employed in cancer treatment including overcoming the resistance to apoptosis induction which is based on loosing p53 activity. Caspase-2 is involved in apoptosis induction by different...
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Immunological testing of combined LELTE / saccharide dendrimers
Kádek, Alan ; Man, Petr (referee) ; Bezouška, Karel (advisor)
Immunological testing of combined LELTE / saccharide dendrimers. (in Czech) Alan Kádek (Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic) CD69 is a widespread receptor of the immune system cells. Although a physiological ligand for this receptor is still unknown, in Laboratory of protein architecture its affinity towards a range of compounds including calcium, carbohydrates and charged compounds such as carboxylated calixarenes, has been proved. Moreover, a pentapeptide sequence LELTE derived from a mycobacterial chaperonine protein hsp65 has been recently identified as a ligand for CD69 representing a "danger" signal for the immune system. However, this peptide is not immunoactive per se, but only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using bi- or polyfunctional reagents. In this thesis I evaluate for four newly synthesized compounds their in vitro immunological effects on cellular signalization, proliferation, natural killing of tumors and on induction of apoptotic cell death in immunocytes. The tested compounds present LELTE peptide through attachment to a cyclopeptidic RAFT scaffold (K-K-K-P-G)2 through the ε-amino groups of lysine residues, alone or in combination with a...
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