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Bacterial toxins translocating across the membrane of eucaryotic cells
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
The bacterial protein toxins endowed with the ability to translocate accross the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eucaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transfering other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with others artificially prepared agens on synthetic membranes allows detailed understanding of mode of action of individual pore- forming toxins. Some of the toxins were actually described in such a detail, that can serve as investigation tools for characterization of new translocated molecules. One of such example is the transfer of nucleotides or whole nucleic acid molecules accross the membrane pore of α- hemolysine of S. aureus. This applications is in recent days commercially used for DNA sequencing.
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Metabolism of methionine and its role in cellular processes in Saccharomyces cerevisiae.
Pavelková, Eliška ; Zikánová, Blanka (advisor) ; Žáčková Suchanová, Jiřina (referee)
Methionine is a proteinogenic amino acid which can be in case of its lack synthesize by yeast, in contrast to mammals. Methionine is also indispensable for cells because it is a precursor molecule for S-adenosylmethionine (AdoMet). AdoMet participates in a biosynthesis of other molecules such as polyamines or biotin. AdoMet is a donor of a methyl group in transmethylation reactions of proteins and lipids. Due to this fact, AdoMet is involved in regulation of a variety of cellular processes. Although yeast can synthesize methionine, from energy point of view, it is more advantageous to take accept methionine from the extracellular environment. Extracellular methionine concentration also affects the expression of permeases involved in its transport into the cell and methionine biosynthesis. Intracellular availability of methionine is monitored by tRNA thiolation. Cell growth and aging is positively influenced based on the amount of thiolated tRNA. Methionine biosynthesis, translation rate and carbohydrate metabolism are negatively influenced. Recently, it has been found out that under certain conditions, lack of methionine induces non nitrogen starvation autophagy, rapidly decreases growth rate and extend life-span of cells in Saccharomyces cerevisiae. Study of the role of methionine in the cellular...
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Gene PALB2 and its role in breast cancer
Zdařilová, Klára ; Janatová, Markéta (advisor) ; Žáčková Suchanová, Jiřina (referee)
Breast cancer is the most common cancer among women in the Czech Republic. Mutations in two major predisposition genes, BRCA1 and BRCA2, account only for 16 % of familial risk of breast cancer. Gene PALB2 was discovered in 2006 as a tumor suppressor. Protein product of PALB2 plays a major role in pathway of DNA repair of double-strand breaks through the homologous recombination mechanism. PALB2 links BRCA1, BRCA2 and RAD51 and is required for their recruitment to DNA damage foci and initiate homologous recombination. In a response of DNA damage PALB2 participates on regulation of the cell cycle. Protein function of PALB2 is necessary to maintain the integrity of the genome and in case of loss this function, because of the gene inactivation, it leads to genomic instability, which may be the basis for the development of tumorogenesis. Heterozygous mutations in PALB2 increase the risk of breast cancer predisposition, these mutations has been demonstrated even in pancreatic cancer and less often in ovarian cancer. Therefore, it is important to analyze truncating mutations in the PALB2 gene in BRCA1/2-negative patients from families with a strong history of hereditary breast cancer. The frequency of PALB2 mutations may be comparable to the frequency of mutations in the BRCA2 gene in Czech hereditary...
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Targetting prostate tumor cells by polyomavirus virus-like particles
Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee)
The aim of this thesis is to investigate the targeting potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as vectors for directed cell delivery of therapeutic or diagnostic compounds. Major capsid protein VP1 of MPyV is able to selfassemble into the noninfectious VLPs. Our main goal is to retarget these VLPs from its native receptor to the prostatic cancer cells by changing the receptor binding site in the surface-exposed loop of VP1. We introduced a peptide ligand CTITSKRTC, which binds prostate-specific membrane antigen (PSMA), by insertion or substitution into BC loop of VP1. These modifications did not change the stability of the particles and genetic substitution prevented the native receptor binding. PSMA-specific binding of modified VLPs was tested by pull-down assay and surface plasmon resonance. In order to further utilize these VLPs, we tested several approaches for preparation of VLPs as vehicles for compounds delivery into eukaryotic cells. Although the method for encapsidation of the DNA into the VLPs in cellular nuclear extracts, which mimic the in vivo conditions, did not enabled us to produce pseudocapsids, we successfully optimized procedure for dissassembly and reassembly of purified particles. This method will be use for encapsidation of molecules into the...
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Action of polyomavirus protein in host cell cycle
Suchanová, Jiřina ; Král, Jiří (referee) ; Drda Morávková, Alena (advisor)
The polyomaviruses belong to a class of small DNA viruses that are capable to induce tumor growth within the cells infected by the virus. The course of their life cycle is entirely dependent on utilization of the infected cell mechanisms that have to be very precisely regulated. Above all, this thesis concentrates on investigating the factors that influence the course of the infected cell cycle, and also the ways in which the virus is able to interact with its control mechanisms. The polyomavirus early transcripts (T-antigens) have a main impact on the progression of the individual phases of the cell cycle. The biggest T-antigen (so called "Large T-antigen") is responsible for both the interaction with the main regulators of the cell cycle (proteins p53 and pRB) and a viral replication, immortalization of the cell and blocking of the mitotic phase of the cell cycle. The mitosis is very unfavorable to the virus because it utilizes a great amount of limited material of the cell organism that the virus needs for its own replication. According to the latest research the virus induces several changes within the cell cycle. For example, it creates a second S phase instead of the mitotic phase in the cell. It causes the accumulation of the infected cells in the late G2 phase as opposed the accumulation of...
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