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Function and localization of the SUN family of proteins in yeast populations
Kuznetsov, Evgeny ; Palková, Zdena (advisor) ; Janderová, Blanka (referee) ; Malcová, Ivana (referee)
The SUN family of proteins (Uth1p, Sun4p, Sim1p, Nca3p) is a group of fungal proteins similar to cell wall glucanases and highly homologous in their 256 long C- terminal amino acid domain. Our previous studies on yeast colony development revealed that members of the SUN family of proteins may be involved in the aging process and may play important role for survival during the development and grow of multicellular yeast populations. Our lab implemented a microarray analysis of expression changes in Saccharomyces cerevisiae colonies, which showed significant changes in the expression level of the SUN family member - UTH1. In addition, a strain with a disrupted UTH1 gene displayed a poorer grow and rate of survival in yeast colonies in comparison to the wild type. However, in this work, we focused on identifying and better understanding the functions of particular SUN proteins and determitation of their exact localization. Interestingly, some SUN family proteins have dual localization (Uth1p, Sun4p) to the mitochondria and cell wall and may thus be involved in mitochondrial and cell wall function. In this thesis, the "Results and discussion" section is divided into two parts as follows: the first part addresses questions concerning localization, oxygen-depended regulation and the possible involvement...
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Analysis of Rpg1/eIF3a mutation in Saccharomyces cerevisiae.
Luxová, Pavla ; Malcová, Ivana (advisor) ; Binarová, Pavla (referee)
Rpg1/Tif32/eIF3a is an essential and the largest subunit of translation initiation factor eIF3 in yeast Saccharomyces cerevisiae. Besides interactions within the eIF3 complex it has been shown to interact with microtubules. Preliminary data of the laboratory obtained using strains of the W303 genetic background indicated that there is a synthetic phenotype between rpg1-2 mutant and microtubule inhibitor nocodazole. Aim of this work to elucidate this "microtubule phenotype" of the rpg1-2 mutant and its dependency on used genetic background. I confirmed that independently on genetic background (W303, BY, SEY) all mutants rpg1-1, rpg1-2 and rpg1-3 were temperature- sensitive. I found that in contrast to published data on rpg1 mutants of the W303 background these mutants of the BY and the SEY backgrounds do not arrest the cell cycle in G1 phase during cultivation at the restrictive temperature (37řC, 4 hours). In addition, all three mutants did not show an increased sensitivity to benomyl and none of them affects microtubule rearrangement after a release of cells from the nocodazole treatment. I constructed new strains with a combination of the BUB1 gene deletion with the particular rpg1 mutation. Phenotypic analyses of new double mutants revealed that simultaneous dis-function of Bub1 and Rpg1 results...
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The influence of intron sequences on splicing effectivity in Saccharomyces cerevisiae
Oplová, Michaela ; Půta, František (advisor) ; Malcová, Ivana (referee)
Pre-mRNA splicing is a highly regulated cellular process. The tight cooperation of spliceosome and other splicing factors that enable pre-mRNA cis-elements interpretation results in precise pre-mRNA splicing regulation. Short conserved splicing sequences within introns represent an elementary and indispensable element for intron removal from primary transcript, yet they are not sufficient signals for efficient splicing events. Additional pre-mRNA features affect complex splicing regulation. We took advantage of strains with slightly disrupted spliceosome (prp45(1-169)) to study the effect of ACT1 and MAF1 intronic sequences on splicing efficiency. Here we show, that ACT1 intron region between branch point (BP) and 3' splice site (3'ss) maintains splicing efficiency in mutant cells. However, the specific element within this region was not determined. In addition, results implicate an alternative BP in splicing efficiency modulation in yeast Saccharomyces cerevisiae. Interestingly, this alternative BP is localized in ACT1 intron outside of the BP-3'ss region. Furthermore, splicing factors with potential influence on 3'ss selection were studied. Heterodimer composed of Slu7p and Prp18p participates in 3'ss positioning to the active site of the spliceosome. Splicing analysis of substrates with two...
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Role of formins in the organization and dynamics of intracellular structures in Arabidopsis thaliana
Rosero Alpala, Elvia Amparo ; Cvrčková, Fatima (advisor) ; Baluška, František (referee) ; Malcová, Ivana (referee)
On the basis of detailed phenotypic examination of fh1 and fh2 mutants we observed that the main housekeeping Arabidopsis thaliana formin AtFH1 (At3g25500) and its closest relative, AtFH2 (At2g43800) are involved in both actin filaments and microtubule dynamics. fh1 mutants showed increased sensitivity to the actin polymerization inhibitor Latrunculin B (LatB). Formin mutants had cotyledon pavement cells which exhibited more pronounced lobes compared to the wild type, and alterations in vascular tissue patterning were found. The double fh1 fh2 homozygote was not obtained, suggesting that at least one functional formin gene is required for proper gametophyte development. Methods used to observe and quantify both architecture and dynamics of the cortical cytoskeleton from confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM) were standarized and allowed to find that mutants exhibited more abundant but less dynamic F- actin bundles and more dynamic microtubules than wild type seedlings, fh1 mutant phenotype observed in roots was further aggravated by a (heterozygous) fh2 mutation. The formin inhibitor SMIFH2 mimicked the alterations observed in fh1 mutants in plants, it has been the first report of this inhibitor in plants. Defects in membrane trafficking were...
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Molecular base of plant HSP90-MT interaction
Benáková, Martina ; Krtková, Jana (advisor) ; Malcová, Ivana (referee)
Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...
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Cell polarity establishment and changes during Saccharomyces cerevisiae cell cycle
Luxová, Pavla ; Malcová, Ivana (advisor) ; Šťovíček, Vratislav (referee)
Cell polarity can be defined as an asymmetric organization and distribution of biomolecules, cellular organelles and structures which are important for many cellular processes. Cell polarity establishment is essential for the proper development of all organisms. This work focuses on main mechanisms of cell polarity establishment, its maintenance and changes during Saccharomyces cerevisiae cell cycle. Budding yeast is one of the preferred model organism. Bud site selection is determined by the spatial landmarks which are accumulated at the previous division site. The spatial landmarks are recognized by Rho GTPases which act on their effectors and thus affect the actin cytoskeleton and septins. These structures are essential for polarized bud growth that is coordinated with the cell cycle. Newborn cells arising after the bud separation from the mother cell at the end of each cycle are able to undergo many more division cycles than their mothers what is a new challenge to study cell polarity in terms of cell aging.
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Postranslational modifications in DNA damage response
Moudrý, Pavel ; Hodný, Zdeněk (advisor) ; Malcová, Ivana (referee) ; Mistrík, Martin (referee)
Our genetic material is continually challenged by varieties of factors and processes that represent enormous threat to our ability to transmit genetic information to our offspring and to our survival as well. Cells possess many mechanisms capable of detecting and repairing damaged DNA, collectively known as DNA damage response (DDR). DDR activities must be tightly regulated in a spatiotemporal and DNA lesion-specific manner to optimize repair and prevent alterations in DNA during normal cellular processes. Defects in DNA repair drive genomic instability and may ultimately lead to cancer and neurodegenerative diseases. This work is a compilation of 4 projects focused on molecular mechanisms of DNA damage response and assembly of multiprotein signalling and repair complexes at sites of DNA damage. We identified nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import and DDR. We showed that NUP153 knockdown prevents 53BP1 from entering the nuclei in newly forming daughter cells, which translates to decreased IR-induced 53BP1 foci formation, delayed DNA repair, impaired survival after IR and exacerbates replication stress-induced DNA damage. Furthermore, we identified ubiquitin-activating enzyme UBA1 as the apical E1 enzyme required for response to IR and replication...
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The interaction of Prp22 and Prp45 proteins in budding yeast spliceosome
Senohrábková, Lenka ; Půta, František (advisor) ; Malcová, Ivana (referee)
Protein Prp22 is a DEAH box RNA helicase, which plays two distinct roles in pre-mRNA splicing: it participates in second transesterification step (ATP independent function) and it releases mature mRNA from the spliceosome (ATP dependent function). Prp45p, yeast ortholog of the human transcription co-regulator SNW/SKIP, is an essential splicing factor, it is included in spliceosome throughout the splicing reaction. Mutant prp45(1-169) genetically interacts with some alleles of NTC complex and second step splicing factors, one of them is also gene PRP22. Here we present, that mutants prp22(-158T) and prp22(-327A), which are synthetically lethal with prp45(1-169), express lower amount of Prp22p due to the mutation in upstream regulation region. Mutants prp22(-158T), prp22(300PPI) and prp22(-327A) affect splicing of pre-mRNA with mutation in 5'ss with respect to sequence of the second exon. N-terminal mutants prp22(∆301) and prp22(∆350) are synthetically lethal with prp45(1-169). Synthetic lethality is possibly caused by lower efficiency of Prp22 recruitment to the spliceosomes, which is no more viable for cells.
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Plasma-membrane alkali-metal-cation transporters involved in salt tolerance of pathogenic Candida species
Krauke, Yannick ; Sychrová, Hana (advisor) ; Malcová, Ivana (referee) ; Heidingsfeld, Olga (referee)
of Ph.D.Thesis Conclusions All the aims of the thesis were achieved.The toleranceto alkali metalcationsof four pathogenic Candida species was studied in detail and revealed differencesamong the yeasts. These differences in sall tolerance remained the same under various growth conditions.For the first time, the internalsodium and potassiumconcentrationsof several Candidaspecieswere estimatedunder highsalt-stressgrolvth.The internalK./Na* ratiowas not in relationwith the salt tolerancerevealingdifferentadaptationmechanismsto salt stress in Candida species. A first study on combinatoryuse of fluconazoleand NaCl revealed severe synergisticeffects of both compounds, leading to grovvthinhibitionand increased internalNa* concentrationsin C. albicans.The molecularbasis of this synergismremainsto be established. Ihe C. dubliniensis, C. glabrata and C. parapsilosis Cnhl NalH- antiporters were cloned and functionallycharacterizedupon heterologousexpression in S. cerevlslae to understandthe mechanismsinvolvedin the differentsalttolerancesof Candidaspecies.The three antiportersdifferedin theiractivityfor alkalimetalcations,which roughlycorrelatedwith the observed differences in salt tolerance among the species. Additionally,during the characterizationof heteroiogouslyexpressed antiporters,two antiporter chimeras...
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