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Microtubules in the fungal pathogen Ustilago maydis have important roles, which include polar budding, morphogenesis and nuclear migration. They also serve as tracks for molecular motors, responsible for intracellular transport of organelles and membrane trafficking. Moreover, microtubules are indispensable during both interphase and cell division, and they play a crucial role in long-distance microtubule-based transport, which occurs in neurons or fungal hypha. Therefore, in order to carry out their functions correctly they need to be well organised and stabilised, which is achieved mainly by various microtubule-associated proteins. In this thesis, different aspects of microtubule (MT) cytoskeleton organisation in U. maydis were investigated, using bioinformatics and experimental approaches. In the first part of the thesis I studied the microtubule-associated protein (MAP) repertoire in U. maydis, which has never been done before in a comprehensive way. For this purpose, searches across five eukaryotic model organisms were conducted to identify all of their known MAPs, to query the U. maydis database. In addition, all of the proteins were checked for their domain architecture, to help decide if an orthologue had been found. As a result, 66 potential MAP orthologues were identified. The second part of this thesis focused on identifying novel factors involved in the organisation of the microtubule cytoskeleton using a specially designed genetic screen. This work involved five microtubule-organisation defect (MOD) mutants, generated by UV-mutagenesis, which were characterised by inability to produce long hyphae as well as by short, fragmented microtubules. To find which genes were responsible for this phenotype, the genomes of all mutants were sequenced and compared with a wild-type genome, and mutations in many genes were found. The analysis revealed potential candidate genes responsible for the specific phenotype of the mutants. However, most probably, UV-generated point mutations in more than one gene played a part in the defective microtubule array. In the final part of this thesis, the function of two beta-tubulin isotypes in U. maydis was analysed. Using conditional mutants, I demonstrated that there are subtle functional differences between the two beta tubulins.

The genus Geosmithia contains 11 described and several tens of undescribed species of fungi living nearly exclusively in galleries of subcorticolous insects, especially bark beetles. In this work, a phylogenetic analysis of the genus was made using DNA sequences of four protein-coding genes, namely TEF-1, RPB2, Mcm7 and Tsr1. The analysis has confirmed that ecological strategies of these fungi (such as association with conifers or broad leaved trees or symbiosis with ambrosia beetles) have evolved several times in this genus. 51 species are recognized based on the obtained phylogenetic tree according to Genealogical Concordance Phylogenetic Species Recognition. I have also tested utility of the above mentioned genes to serve as "barcode" for identification of closely related Geosmithia species.

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-cancer activities. In this study, the beta-subunit of C-PC (ref to as C-PC/beta) was expressed and purified from bacteria E. coli BL-21. The recombinant C-PC/beta has been demonstrated to have anticancer properties. Under the treatment of 5 microM of the recombinant C-PC/beta, four different cancer cell lines accrued a high proliferation inhibition and apoptotic induction. The C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found. Under the treatment of the C-PC/beta, depolymerization of microtubulin and actin-filament was observed. The cells underwent apoptosis with increase of Caspase-3 and Caspase-8 activities. Cell cycle was arrested at G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Inhibition of cancer cell proliferation and induction of apoptosis may potentate C-PC/beta as a promising cancer prevention or therapy agent.

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including anti-inflammatory and anti-cancer activities. In this study, the beta-subunit of C-PC (ref to as C-PC/beta) was expressed and purified from bacteria E. coli BL-21. The recombinant C-PC/beta has been demonstrated to have anticancer properties. Under the treatment of 5 microM of the recombinant C-PC/beta, four different cancer cell lines accrued a high proliferation inhibition and apoptotic induction. The C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found. Under the treatment of the C-PC/beta, depolymerization of microtubulin and actin-filament was observed. The cells underwent apoptosis with increase of Caspase-3 and Caspase-8 activities. Cell cycle was arrested at G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Inhibition of cancer cell proliferation and induction of apoptosis may potentate C-PC/beta as a promising cancer prevention or therapy agent.

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Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating xylogenesis, a number of physical and biochemical limitations to microscopic and molecular experimentation. Efforts have been made to understand molecular aspects of xylogenesis and have involved functional gene testing using transgenic approaches. These methods involve the production of plantlets from a variety of plant tissues using in vitro full plant regeneration techniques. Although these systems are effective, the time taken from transformation event, to plant establishment and growth, then finally to secondary wood production can take up to several years and requires high labor and technical inputs. (For complete abstract open document)

A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...