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Utilization of new generation sequencing methods to elucidate cystic fibrosis-like phenotype at patients with unclear illness of molecular type.
Matějčková, Iva ; Macek, Milan (advisor) ; Holá, Dana (referee)
Cystic fibrosis (CF) is genetically conditioned, autosomal recessive disease that occurs in the European population with a prevalence of about 1:2500 - 1:1800. In this disease we observe a mutation of the CTFR gene with subsequent fault in chloride channels. Such afflicted individuals usually suffer from chronic respiratory problems, pancreatic insufficiency, high concentration of chloride ions in sweat and obstructive azoospermia. Genetic testing of CFTR gene is indicated in individuals who meet the CF clinical picture and a positive sweat test (increased concentration of chlorides in the sweat). Genetic testing of the CFTR gene is usually done by using commercial kits detecting the most common mutations of the CFTR gene in the Czech Republic. If the testing results are negative, it is further performed an MLPA method that captures the larger deletions and duplications of gene, eventually a sequencing of all exons is. Despite the well-established algorithm of the testing, some patients suffering from symptoms of CF are left without genetic findings. Thanks to development of next generation sequencing, it is possible to make the diagnosis of CF more effective and uncover the variants that were not captured by previous methods.
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Molecular characterization of resistance to MLSb antibiotics in Staphylococcus aureus and SCV Staphylococcus aureus strains of cystic fibrosis patients
Vařeková, Eva ; Melter, Oto (advisor) ; Zikánová, Blanka (referee)
Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in Caucasians. Lower respiratory tract of CF patients is colonized by specific bacteria, often leading to chronic infection and lung tissue damage. In this thesis we characterized 338 isolates of S. aureus from 92 Czech CF patients isolated in 2011-2013. Using spa typing and PFGE we detected high clonal heterogenity of this collection with the exception of MRSA strains (resistant to oxacillin; 5% prevalence) which were clonally related. The prevalence of S. aureus MLSB resistance in our collection was high (69 %), which is a serious problem due to common usage of these antimicrobials in clinical practice. A half of the MLSB resistant strains lacked any known determinant of this resistance (ermA, ermC, ermT, msrA). Sequencing of the ribosomal genes revealed a high number of S. aureus strains carrying target site mutations resulting in MLSB resistance (37 %). This is new important information about the staphylococcal strains associated with chronic infections in Czech Republic. Focusing on mutability of analysed strains, we also detected several strains with point mutations or deletions in their mutator genes mutS a mutL. Hypermutability could be responsible for the high rate of ribosomal mutations and for the presence of...
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Possibel approaches to gene therapy of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Vaněk, Ondřej (referee)
1 Abstract Cystic fibrosis is an autosomal recesive disorder caused by a mutation in the CFTR gene, which leads to inefficiency or absence of CFTR chlorid channel. One way to induce production of CFTR protein in target cells, is to use gene therapy. The principle od gene therapy is to transfer DNA or mRNA molecules inside malfunctioning cells. The aim of this study was to optimise the detection of CFTR protein using the Western blot analysis. Then, using this method the effectiveness of CFTR-mRNA transfection was studied. To study the CFTR protein, a number of cell lines was used: a healthy human ephitelial cell line (NuLi-1), an ephitelial cell line with ΔF508 mutation (CuFi-1), and a human lung carcinoma cell line (A549). This study compared four different ways of cell lysis - lysis by sonication and lysis by three distinct lysis buffers. Lysis by RIPA buffer with protease inhibitors was determined for the detection of CFTR protein. Moreover, three different primary monoclonal antibodies were also tested. The CF3 antibody, which is specific to an extracellular epitope of CFTR protein, was found able to detect CFTR protein specifically. A couple of different glycosylated forms of CFTR protein was detected. The highest amount of CFTR protein was determined in the NuLi-1 cell line. CFTR protein was also...
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Prenatal diagnostics of cystic fibrosis and diseases associated with trinucleotide expansions - teaching at secondary schools
Nováková, Stanislava ; Ehler, Edvard (advisor) ; Pavlasová, Lenka (referee)
Cystic fibrosis and diseases associated with trinucleotide expansions are serious hereditary disease that serves in my Diploma thesis as role models suitable for teaching interesting topics of human genetics at secondary schools. By using appropriate methods of prenatal genetic treatment, it is possible to make a diagnosis of the developing fetus and to determine a corresponding prognosis in next prenatal and postnatal development in families at risk. The practical part of the thesis is devoted to the content analysis of biology schoolbooks for secondary schools and to the preparation of a prototype class of genetics at secondary schools. The aim of the content analysis of biology schoolbooks for secondary schools is the evaluation of various textbooks according to several preselected criteria. The aim of the presentation of the proposed prototype class was to determine, based on the responses obtained from the questionnaires, whether pupils of higher grades of secondary schools are interested in the subject matter of genetics, to find out what engaged their attention the most during the lessons or what they did not understand and what they considered as difficult. The lesson was conducted as a specialized seminar, the teaching method was a lecture. From the selected biology schoolbooks for...
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Finalizing the full genome sequence of epidemic strain Burkholderia cenocepacia ST32 and identification of a prognostic marker for infections that are caused by the ST32 strain in patients with cystic fibrosis
Vavrová, Jolana ; Dřevínek, Pavel (advisor) ; Wiedermannová, Jana (referee)
Burkholderia cenocepcia is one of the serious infectious agents of respiratory tract among cystic fibrosis patients. There are problems mainly with strains which are capable of epidemic spread. The known epidemic in the Czech Republic was caused by ST32 strain in the past. In this work, there was completed whole genome sequence of referential isolate 1232 of B. cenocepacia ST32 in cooperation with bioinformatics by new generation sequencing techniques and by determining the problematic areas by a combination of Sanger sequencing bioinformatics approaches and manual assembling of sequence reads localized in these areas. The final version of the genome sequence was annotated by PGAAP and at the present time it is finalized. Second part of this work is dedicated to looking for a prognostic marker of infection caused by ST32 strain in patients with cystic fibrosis. We analysed the results of ST32 trancriptomic experiment and chose genes possibly connected with the cepacia syndrome - serious, mostly fatal state of infection. By quantitative PCR we compared their expression in isolates from 4 patients from time of cepacia syndrome and month before that. We checked the possibility of direct detection of the expression of these genes in clinical material. We identified genes for type III secretion system as...
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Chicken antibodies - tool for passive immunization of Cystic fibrosis patients
Hadrabová, Jana ; Hodek, Petr (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is a congenital disease which affects respiratory and gastrointestinal system. It is the autosomal recessive disorder and it arises from the CFTR (cystic fibrosis transmembrane conductance regulator) gene mutation. The CFTR is an ion channel for chloride ions transmission. If its function is limited, the pulmonary mucus becomes dense and microorganisms are able to infect the lungs and grow effectively. Most common and dangerous bacteria in CF patients lungs is Pseudomonas aeruginosa. Antibiotics are used as the first therapy, but the bacteria becomes resistant against them. The chronic inflammation arises and it proceeds to the pulmonary tissue destruction (fibrosis). New methods of CF patients therapy are searched especially because of antibiotics resistance. The promising approach could be the passive immunization with chicken antibodies specific against Pseudomonas aeruginosa virulence factors. The IgY solution is suitable to be converted to aerosol by nebuliser and the patient could inhale that. As the IgY immunoglobuline can be an inflammatory substance when inhaled, it should be tested what causes in healthy lungs. The Wistar rat was used as a suitable experimental model. At first the chicken antibodies were isolated, then the Fab fragment was prepared by digestion. The...
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Development of diagnosis and treatment of cystic fibrosis in context of physiotherapy
Jindrová, Barbora ; Smolíková, Libuše (advisor) ; Babková, Lenka (referee)
Bachelor thesis on development of diagnostics and treatment of cystic fibrosis in the context of physiotherapy is processed in the form of case study research. The work is divided into three parts. The first part contains basic information about cystic fibrosis, its etiology, pathology, associated diseases, clinical picture and treatment. The main part of the work is concentrated on the development of cystic fibrosis diagnostics and treatment of changes in focusing on the progress of physiotherapy techniques. Work also discuss how the result of these changes varied prognosis and health status of patients with cystic fibrosis. Then the work contains a description of the techniques used in physical therapy for cystic fibrosis. The last section contains a case study of two patients with cystic fibrosis.
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Molecular genetic diagnosis of cystic fibrosis
Matějčková, Iva ; Krkavcová, Miroslava (advisor) ; Kotlas, Jaroslav (referee)
Cystic fibrosis is a very common and serious disease. It is an autosomal recessive disease with an incidence of 1 : 2500 - 1 : 4500. With this disease, CTFR gene mutations occur which are responsible for the failure of chloride channels. It is a multi-organ handicap, which primarily infects the respiratory and gastrointestinal system, but also affects the hepatobiliary system, reproductive system, and sweat glands. Cystic fibrosis can be diagnosed by a sweat test, molecular genetic diagnosis, or more rarely, using transepitel potential differences, which has not been introduced in the Czech Republic yet. Since 2009, the Czech Republic applies neonatal screening CF, which helps make a faster diagnosis to then be able to proceed to the rapid introduction of appropriate treatment. The introduction of an early treatment has significantly improved the prognosis of the patient. Currently CF patients can live on average from thirty to forty years. For the CFTR gene genetic testing is used postnatal, prenatal or preimplantation material. From these samples, DNA gets isolated and is further investigated using genetic methods, which may be based on the principle of PCR, reverse hybridization, restriction analysis, DNA sequencing or MLPA. Powered by TCPDF (www.tcpdf.org)
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The detection of factors virulence and resistance genes bacterial pathogens in patients with cystic fibrosis
Jandová, Oldřiška ; Melter, Oto (advisor) ; Nyč, Otakar (referee)
Cystic fibrosis is an autosomal recessive genetic disorder, which is caused by mutation in CFTR gene (Cystic Fibrosis Transmembrane Conductance Regulator). This gene encodes protein with the same name, which is responsible for pathogenesis of CF. Cystic fibrosis is characteristic for frequent infection of respiratory system, which causes, destruction of lung tissue. These infections are characterized by occurrence of typical bacterial pathogenes, for example: S. aureus, P. aeruginosa etc. S. aureus is one of the most typical opportunistic pathogens, which causes serious difficulties in patients with the cystic fibrosis. Strains of S. aureus are characterized by production of multiple virulence factors and resistance to broad spectrum of antibiotics. Besides common mechanisms of resistance there is also possibility of emergence of so called Small Colony Variants in chronically infected patients. These resistant subpopulation is relatively common among S. aureus isolates of patients with CF. The aim of this work was to study isolates from three patients with cystic fibrosis, who are chronically infected by S. aureus. Our goal was to determine changes in the pattern of the antibiotic resistance and occurrence of virulence factors together with description of SCV strains. Powered by TCPDF (www.tcpdf.org)
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