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The relation between microbial diversity and biodegradation of organic pollutants in soils
Adámek, Michael ; Svobodová, Kateřina (advisor) ; Matyska Lišková, Petra (referee)
This work sums up the knowledge of the currently studied microorganisms capable of degradation of organic compounds contaminating soil and of the impact of biodiversity on biodegradation efficiency. As documented in many studies effective soil remediation can be achieved by mixed-species consortia isolated from polluted soils. However, use of these cultures for bioaugumentation requires further research on their influence on the biodiversity of autochthonic soil microflora. Though bioaugumentation provably affects bioremediation effectivity no ideal carrier for microbes has been found yet that would provide survival of the introduced organisms in the competitive soil environment. Next, selection of suitable bioaugumentation agents should be based on previous analyses of autochthonic microbes in the targeted contaminated soils. Further, this work shows that the presence of key species might be more important for the biodegradation efficiency than biodiversity of soil microflora. Biodiversity is more related to the functionality of soil ecosystems which can be affected by the presence of contaminants leading to positive selection of taxa capable of pollutant degradation. However, there are just a few studies on the relationship between biodiversity and degradation of pollutants. Its further research...
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Structure and characteristics of mutant hPrp31 in Retinitis pigmentosa
Těšina, Petr ; Staněk, David (advisor) ; Lišková, Petra (referee)
Retinitis pigmentosa is a hereditary eye disease causing progressive loss of photoreceptor cells, which leads to an irreversible sight handicap and eventually complete blindness. It is a major cause of visual handicap or blindness with very heterogenous genetic background. There are four genes accounting for retinitis pigmentosa that encode splicing factors necessary for spliceosomal assembly and function. Unlike any of the other known genes associated with this disease, these are all expressed ubiquitously throughout the human body. Intriguingly, the mutant forms of these vital splicing factors cause cell-type specific disease affecting only photoreceptor cones and rods. Molecular mechanisms underlying this cell-type specific effect remain elusive. One of these splicing factors is the hPrp31 protein. Its mutant form known as AD29 is the focal point of this thesis. Some of the effects of this mutation on the cellular level have been discovered recently. A creation of the expression vector followed by expression and purification of the truncated hPrp31 protein carrying the AD29 mutation is presented in this thesis. The purified product has been used for production of a αhPrp31 polyclonal rabbit antibody, whose applicability to western blot and immunofluorescence staining has been verified. Moreover...
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Mechanism of inducible gene expression of resistance protein Vga(A)LC from Staphylococcus haemolyticus.
Novotná, Michaela ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
The staphylococcal protein VgaA belongs to ARE ABCF family, which confers resistance to ribosome binding antibiotics by the target protection mechanism. VgaA confers resistance to lincosamides, streptogramins A and pleuromutilins and thus provides the so-called LSAP resistance phenotype. The expression of resistance genes often reduces fitness in the absence of an antibiotic, therefore the expression of resistance genes is often tightly controlled and triggered only in response to the presence of an antibiotic to which the protein confers resistance. The inducible expression has also been observed for the vgaA gene, nevertheless, its mechanism has not been elucidated. In the diploma thesis, it was shown that the vgaALC gene from Staphylococcus haemolyticus is regulated by ribosome-mediated attenuation. The mechanism is based on the detection of translation inhibitors via a ribosome translating a special regulatory open reading frame (uORF), which is part of an attenuator located in the 5' untranslated region of the mRNA. The vgaALC gene is regulated at the transcriptional level in response to LSAP antibiotics. Antibiotic specificity of induction is affected not only by the nature of the peptide encoded by uORF but also by the antibiotic specificity of the resistance protein. Fluorescence microscopy...
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Anterior segment dysgenesis disorders and their molecular genetic cause
Moravíková, Jana ; Lišková, Petra (advisor) ; Krulová, Magdaléna (referee)
Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...
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Significance of protein phosphorylation for bacterial cell
Gregorová, Michaela ; Branny, Pavel (advisor) ; Lišková, Petra (referee)
Phosphorylation - most common post-translational modification has an important role in many cellular processes of bacteria. Bacteria contain enzymes that are in charge of adding phosphoryl group (kinases) or enzymes with reciprocal activity (phosphatases). Reversible phosphorylation and dephosphorylation of proteins are fundamental for signal transduction from the environment to the cell. These modifications can affect enzymatic activity, protein stability, localization as well as interaction with another protein. Due to the complexity of these phosphorylation networks, bacterial cells are capable to adapt very effectively to changing environmental conditions.
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Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
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Epidemiology and preventive measures in inherited retinal dystrophies in the Czech Republic.
Kousal, Bohdan ; Lišková, Petra (advisor) ; Vlková, Eva (referee) ; Mahelková, Gabriela (referee)
Introduction: Inherited retinal diseases (IRDs) are one of the most common causes of incurable blindness in children and young adults. In the Czech Republic, prior to the start of our work, these disorders had not been the subject of a systematic research. The aim of the study was to identify, clinically characterize and molecular genetically analyse Czech patients with monogenic IRDs and based on the knowledge gained subsequently implement preventive and therapeutic measures to clinical practice. Material and methods: We have performed a comprehensive clinical examination, genealogical analysis and molecular genetic investigation in patients with IRDs and their family members. Detailed ocular examination included spectral domain optical coherence tomography, high-resolution fundus photography and autofluorescence imaging. DNA was isolated from venous blood samples or buccal cells. Causal variants were searched for using Sanger and massively parallel sequencing, and their pathogenicity was evaluated in the context of previously published data, bioinformatical analysis and segregation in available family members. Results: In total, 103 individuals from 76 Czech families diagnosed with IRDs were characterized and their data published. Specifically, we have described clinical and molecular genetic...
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Inherited corneal disorders: options for early detection, influencing the onset and progression.
Skalická, Pavlína ; Lišková, Petra (advisor) ; Netuková, Magdaléna (referee) ; Vlková, Eva (referee)
Introduction: The development of molecular genetic methods has in many fields necessitated their inclusion in routine clinical practice, including ophthalmology. The main aim of this thesis was detailed clinical characterization of Czech patients with suspected inherited corneal disorders, followed by genetic testing to determine or specify their clinical diagnosis and subsequently to use the knowledge gained in clinical and genetic counselling and to apply preventive measures in order to avoid loss of vision. Material and Methods: Individuals included in this research were either followed up or newly referred to the Cornea clinic of the Department of Ophthalmology, First Faculty of Medicine, Charles University and General University Hospital in Prague. Detailed clinical examination included corneal tomography, specular microscopy, spectral domain optical coherence tomography, biometry and genealogical analysis. DNA was extracted from peripheral blood leucocytes or buccal cells. Disease-causing variants were searched for using Sanger or massively parallel sequencing, variant pathogenicity was assessed in silico using various algorithms and by segregation analyses within the families. In some cases assessment of the functional impact on the pre-mRNA splicing process was performed. In patients with...
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The function of ABCF proteins in bacteria
Mičke, Bianka ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Translation belongs to the most basic processes which happens in the living cells. It is the last step of proteosynthesis when genetic information encoded by the mRNA is transformed into the protein on a ribosome. Organisms have developed a wide range of mechanisms that can regulate it's needs. I focused on one of them - ABCF proteins. This protein group is a member of the ABC transporters superfamily but they haven't a transmembrane domain and their purpose is protect the ribosomes from antibiotics that bind 50S ribosomal subunit or interact with the ribosomes and influence ribosomal functions. Today, we can divide ABCF proteins into the two functional groups: antibiotic resistence proteins (ARE) and proteins with the regulatory functions. The translational regulatory function has been confirmed There is 45 ABCF protein subfamilies spread through the bacteries and eukaryotes but many essential informations like the structure and exact function of them are still missing. My bachelor thesis is analysis and summary of facts that are known about the bacterial ABCF proteins. Key words: ABCF proteins, antibiotic resistence, ARE, translational regulation, ribosome, translation, translational factors
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