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Long term sublethal stress in colonies of Saccharomyces cerevisiae deleted in WHI3
Krampotová, Ester ; Schierová, Michaela (advisor) ; Dušková, Michala (referee)
This diploma thesis is devoted to the topic of the development of natural non-pathogenic yeast colonies in non-laboratory diploid strain of Saccharomyces cerevisae with deletion WHI3, during long-term mild stress. The absence of Whi3 causes significantly higher senzitivity to medium composition relative to the parental strain, thus whi3Δ /whi3Δ colonies are a good model for study of the virulence induction due to environmental stress in pathogenic yeasts. Deletion of WHI3 in the BR-F strain results in a significant suppression of the ability to form structured colonies caused mainly by the reduced level of Flo11. The absence of Whi3 also has a negative effect on the expression of other genes involved in the stress response. The aim of the work is to determine whether changes in the expression of genes encoding stress proteins induced by sublethal doses of the inhibitor are dependent on Whi3, Yap6 and Mpt5 proteins. To induce stress in yeast, we used NaCl or CdSO4. The YAP6 and MPT5 genes encode regulatory proteins involved in the stress response, which expression is under control of Whi3. In whi3Δ /whi3Δ , the level of Hsp26, Tsa1, Pab1, and Gre2 was lower than in the parental strain. The Yap6 protein affects the response to the presence of Cd2+ in the medium, although according to SGD, only its...
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Molecular analysis of Cornelia de Lange syndrom
Vokáčová, Markéta ; Schierová, Michaela (advisor) ; Jedličková, Jana (referee)
Cornelia de Lange syndrome is a rare, very heterogeneous, genetic disorder classified as a cohesinopathy. It is caused by mutations in genes that encode proteins of the cohesin complex or its regulators. So far, 5 major genes, whose defects are responsible for the syndrome, have been discovered. The mutated gene determines the type of heredity and, above all, the extent of the pathology. Due to dysfunction of the cohesin complex, not only the cohesion of sister chromatids is impaired, but also the regulation of gene expression and the repair of DNA double-strand breaks. Knowledge of molecular aspects of the disease has been enhanced by ongoing experiments with animal models of Cornelia de Lange syndrome, cell cultures from patients, and the yeast Saccharomyces cerevisiae. The detailed analysis of the biological functions of the cohesin complex may help to develop therapeutic methods. Keywords: Cornelia de Lange syndrome, cohesin complex, cohesinopathy, transcriptome change, genome integrity, topologically associating domains, NIPBL
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Evolution of mixed cultures of the pathogenic yeasts Candida albicans and Candida guilliermondii
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Schierová, Michaela (referee)
The objective of this thesis was to investigate how cocultures of Candida albicans and Candida guilliermondii change overtime under control conditions and under the influence of fluconazole. These species are opportunistic fungal pathogens but widely differ in their susceptibility to antimycotic of interest - fluconazole. After a brief introduction to special commonalities, the mechanism of pathogenesis, and the treatment of infection, this work explores each organism's growth curves under selected conditions and the process of artificial evolution using the model of passaging of cocultures. Afterwards, these populations of C. albicans and C. guillieromondii were investigated using qPCR and chromogenic media. qPCR analysis revealed that under control conditions, C. albicans (CA) prevails; the possible reason behind this is a 20% shorter generation time, as revealed by the growth curve. In the presence of fluconazole, two trends occurred. One is related to the innate resistance of C. guilliermondii (CG), where CG was dominant by the end of passaging. The second trend led to CA being the dominant one, despite its susceptibility. This is a result of a heightened resistance, where minimal inhibitory concentration 50 (MIC50) increased almost 10-fold, possibly due to mutations. The change in populations...
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The use of cell-free nucleic acids in maternal plasma for non-invasive prenatal diagnosis of monogenic diseases, placental insufficiency-related complications and Down syndrome
Veselovská, Lenka ; Hromadníková, Ilona (advisor) ; Schierová, Michaela (referee)
Since the discovery of cell-free fetal DNA in peripheral blood of pregnant women, cell-free nucleic acids in maternal plasma are explored in relation to non-invasive prenatal diagnosis of various fetal conditions and pregnancy complications. Non-invasive prenatal diagnosis of monogenic diseases represented by TSC1-linked tuberous sclerosis could be achieved by detection of paternally-inherited mutant allele in the pool of maternal alleles in plasma. Reliability of detection of mutant allele could be improved by simultaneous mutation haplotype analysis or detection of universal fetal marker. None of the 3 methods (allele- specific real-time PCR, SNaPshot minisequencing and quantitative fluorescent PCR) evaluated using artificial mixtures and maternal plasma samples reliably and accurately detected low-frequency allele distinguished by point mutation, SNP or microsatellite in TSC1 gene or in its close proximity. We developed a strategy for prediction of proportion of informative couples for panel of SNPs of interest that can be applied to any monogenic disease. Exploiting differential methylation of promoters of genes RASSF1A, HLCS and OLIG2 in maternal and fetal genome, we failed to establish functional fetal marker. MicroRNAs of placental origin released into plasma could serve as biomarkers of...
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Turner syndrome and its relation with X chromosome inactivation
Kašíková, Lenka ; Schierová, Michaela (advisor) ; Král, Jiří (referee)
Turner syndrome (TS) is a genetic anomaly occurring in women with worldwide frequency 1:2,000. Turner syndrome's possible causes include X chromosome monosomy, mosaic karyotype (45,X/46,XX; 45,X/46,XY; 45,X/47,XXX), X isochromosome (46,X,i(Xq)), ring X chromosome (45,X/46,X,r(X)), and other X chromosome aberrations (deletion and translocation). Patients with Turner syndrome (with the exception of ring chromosome abnormalities) aren't diagnosed with mental retardation. Turner syndrome is closely related to the regulation of gene expression level, particularly with X chromosome inactivation. Genes escaping the X chromosome inactivation process in 45,X women are expressed in half the dose of a healthy woman. An example of such a gene is SHOX, which I decided to focuse on in this thesis. SHOX gene haploinsufficiency causes major Turner syndrome phenotype manifesting of short body and bone abnormalities. The research of other genes with possible roles in Turner syndrome is complicated by the absence of adequate model organism, which could be used for TS study with possibility to extrapolate the results to humans. In mice, both the inactivation process itself is different and the phenotypic manifestation of X monosomy (39, X) is also much milder than in 45,X women. This difference could be explained by...
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The prevalence of chosen polymorphisms of luteinizing hormone receptor gene in Czech population and patients with ovarian hyperstimulation syndrome
Chrudimská, Jana ; Macek, Milan (advisor) ; Schierová, Michaela (referee)
Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic potentially life-threatening complication of assisted reproduction techniques (ART). It is caused by an increased sensitivity of ovaries to gonadotropins administered during the controlled ovarian hyperstimulation (COH). Thus, the degree of ovarian response can be gently tuned by genetic polymorphisms of gonadotropins and their receptors. The aim of this study is to ascertain the prevalence of polymorphisms Asn291Ser (rs1470652), Ser312Asn (rs2293275) and insLQ (insertion of leucine and glutamine, rs58356637) in the luteinizing hormone chorionic gonadotropin receptor (LHCGR) gene in 102 Czech fertile men, 149 fertile women and 58 patients with serious grade of OHSS. Detection of the Asn291Ser and Ser312Asn polymorphisms was performed using TaqMan SNP Genotyping Assays. The insLQ variation was detected by the capillary electrophoresis with fluorescence-labeled primers. This study ascertained the prevalence of studied variants in Czech fertile population. Obtained results are in concordance with the majority of data from other European populations. There is no difference in prevalence between control-men and control-women. No relation to the development of OHSS was disclosed. The number of analyzed samples is too small for haplotype analysis. These...
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The effect of Vps34p in yeast colony
Červenka, Jakub ; Schierová, Michaela (advisor) ; Převorovský, Martin (referee)
The phosphatidylinositol-3-kinase (PI3K) signalling pathway is evolutionarily conserved in all eukaryotes and its main function is the regulation of autophagy and protein sorting to the vacuole/lysosome. In the pathogenic yeast species Candida albicans and Cryptococcus neoformans the PI3K signalling pathway is required for virulence. In the yeast Saccharomyces cerevisiae the PI3K signalling pathway consists of two proteins - phosphatidylinositol-3-kinase, Vps34p and its regulator Vps15p. In this diploma thesis I analyse the role of the PI3K signalling pathway in the growth and development of colonies of natural and laboratory strains. I proved that VPS34 or VPS15 deletion in haploid laboratory strains has a significant influence on colony size and invasive growth (in strain ΣSh vps15Δ). Deletion of VPS34 or VPS15 also increases sensitivity of cells to oxidative stress and detergents. Attempts to delete VPS34 in natural strains were unsuccesful, probably because VPS34 is essential in these strains. Constitutive expression of VPS34 does not affect cell resistance in inhibitory tests, the size and differentiation of colonies or ammonia signalling but differences are notable in giant colony morphology and in patterns of invasiveness of the medium. Tagging of the C-terminal of Vps34p with GFP affects...
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Mutational analysis of the TRPC6 gene in patients with nephrotic syndrome
Obeidová, Lena ; Reiterová, Jana (advisor) ; Schierová, Michaela (referee)
Focal segmental glomerulosclerosis is one of the commonest cause of the nephrotic syndrome in adults patients. It is a damage of glomerulus characterized by leakage of proteins to urine and oedemas which usually develops into the end-stage renal disease within 10 years. Recently have been described familial forms of this disease which arise from injury to proteins making up filtration barrier of kidney. In 2005 non-selective ion channel TRPC6 was assigned among these proteins. In this thesis I focused on summarizing existing knowledge of the nephrotic syndrome, focal segmental glomerulosclerosis and involvement of TRPC6 in their origin. Second part of this work is devoted to the screening analysis of TRPC6 gene to discover possible mutations and polymorfisms in 47 patients with histologically proven focal segmental glomerulosclerosis or minimal change disease. The used methods were high resolution melting and direct sequencing. In the group of patients was detected no pathogenic mutation, only 2 known polymorfisms P15S and A404V and few changes which do not result in alteration of amino acid. So it seems TRPC6 gene mutations are a rare cause of the focal segmental glomerulosclerosis in adult patients in the Czech Republic.
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Quality control of molecular biology methods in transplantation medicine
Kotrbatý, Jiří ; Kolesár, Libor (advisor) ; Schierová, Michaela (referee)
This work is focused on the description of quality control of molecular-biological methods used in transplant medicine, ie methods of HLA typing. Quality control is part of the quality management system. It consists of several components: validation, verification, internal quality control and external quality assessment. Each medical laboratory, including laboratories engaged in HLA typing, these elements must be applied to its routine operation. Validation is done before the introduction of methods and verifies whether the method is correct for the intended use. Verification is to verify that the validated method is used correctly in laboratory conditions. Internal quality control is based on an analysis of known positive and negative samples and evaluate the accuracy and precision of the whole analytical process. The external evaluation of the quality of the different laboratories analyze the same sample and the results are compared to each other. Key words: HLA typing, validation, verification, internal quality control, external quality assessment
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The role of JAK-STAT signalling pathway in cellular senescence
Černohorská, Markéta ; Hodný, Zdeněk (advisor) ; Schierová, Michaela (referee)
Proliferating human cells cultivated in vitro after certain number of population doublings withdraw from the cell cycle and enter a specific state termed replicative cellular senescence. Lately, several other forms of senescence independent of the proliferative history and telomere shortening were described. This is called premature senescence, and can be elicited by exposure of cells to aberrant mitogenic or oncogenic signals, to oxidative stress or to variety of chemically and functionally unrelated DNA damaging agents. Senescent cells alter their morphology and expression pattern. This complex phenotype is characterized by enlarged cytoplasm, activation of cell cycle inhibitors, expression of tumor supressors and profound changes in cell secretory phenotype. These cytokines/chemokines induce many different cascades, for example Jak/STAT signaling pathway, that are activated in response to viral infection or inflammation. Senescent cells were found also in vivo in the tumor tissue that produces altered cytokines itself. This diploma thesis inquires into the role of interferon-Jak/STAT signaling pathway in premature cellular senescence induced by genotoxic agents that are often used in chemotherapy. Obtained results might help to understand the complexity of tumorogenesis and senescence. Powered by TCPDF...
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