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Development of model system for study of bacterial adhesion on lung epithelium of CF patients
Nosková, Libuše ; Hodek, Petr (advisor) ; Švédová, Martina (referee)
Cystic fibrosis is an inherited disease bearing a number of health difficulties. The main complication is a chronic colonization and infection of respiratory tract with specific microorganisms - especially Pseudomonas aeruginosa. The lung infection with this microorganism is the most common cause of all of death in these patients. The colonization of respiratory tract is mediated by the series of adhesive structures such as lectin PA-IIL. Currently, the most widely used therapy is an antibiotic treatment. Due to the increasing resistance to antibiotics another methods for treatment are being searched. One possibility is a passive immunization of patients with chicken antibodies. For this purpose, we prepared antibodies against one of the adhesive structures of P. aeruginosa - recombinant lectin PA-IIL. These antibodies be able to recognize a native lectin PA-IIL, expressed by P. aeruginosa. To test the ability of antibodies to prevent adhesion of bacteria on the lung epithelial cells a suitable model system was necessary to develop. The basal components of this system include epithelial cells and P. aeruginosa. Epithelial cells from airways of cystic fibrosis patient were isolated by two methods. One method is based on the direct isolation from the dissected tissue and the second one is a brushing...
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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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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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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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The introduction of diagnosis of new bacterial pathogens Ralstonia a Achromobacter isolated from patients with cystic fibrosis and determining their sensitivity to antibiotics
Michálková, Alice ; Melter, Oto (advisor) ; Bébrová, Eliška (referee)
Cystic fibrosis (mucoviscidosis) is an incurable genetic disease caused by mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator Gene). The most common causes of increased mortality and morbidity of patients include bacterial respiratory infections which may occur even due to less frequent pathogens. Bacterial species of the Ralstonia and Achromobacter genera are not considered pathogenic for healthy people, but they have been established as pathogens in the sputum of patients with CF. However, due to their phenotypic similarity to other bacterial pathogens encountered in patients with CF, microbiologists often do not pay attention to them. The aim of this thesis was to propose some methods of identification of the genus Ralstonia, to determine both quantitative and qualitative susceptibility towards antibiotics, and to make a bibliographical search focused on the issue of the Achromobacter genus. Strains of Ralstonia spp. were identified using phenotypic and genotypic methods and were tested for the susceptibility towards antibiotics. The thesis also proposes a new method of genotypic indentification of R. respiraculi.
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New approaches to determination pathophysiological changes in patients with cystic fibrosis
Doušová, Tereza ; Dřevínek, Pavel (advisor) ; Kreslová, Marcela (referee) ; Gayillyová, Renata (referee)
New approaches to determination pathophysiological changes in patients with cystic fibrosis Cystic fibrosis (CF) is a life-limiting disease caused by mutation in the cystic fibrosis transmembrane regulator (CFTR) gene. To date, more than 2,000 mutations in the CFTR gene have been described, of which only 360 are directly related to CF. In a group of patients carrying mutations of unknown or variable clinical significance, it may be difficult not only to diagnose CF but also to facilitate clinical studies to determine the efficacy of new low - molecular weight compounds targeting disrupted CFTR protein. These so-called CFTR modulators have opened a new era in causal treatment of CF. To maximize the effect of these new therapies, not only the patient's genotype, but also the individual rate of response is crucial. In recent years, intestinal organoids have been used as an ex vivo model to determine the degree of CFTR function and at the same time to predict the therapeutic response to available therapeutic molecules. In our project, using the patient's native tissue and cultures of intestinal organoids derived from this tissue, we demonstrated varying degrees of CFTR residual function in a total of 14 patients with CF (0-39.7% of healthy control function). We characterized de novo mutation of the CFTR gene in...
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Cardiovascular Risks in Chronic Airway Disease in Childhood
Kreslová, Marcela ; Sýkora, Josef (advisor) ; Fila, Libor (referee) ; Kopřiva, František (referee)
1 Cardiovascular risks in chronic airway disease in childhood The aim of this thesis was to evaluate cardiovascular risk by using a combined diagnostic approach by measuring RHI and specific biochemical markers in patients with chronic respiratory disease, where we could assume a possible risk of CVD. A total of 119 probands were examined, including 22 patients with cystic fibrosis (CF) and 52 asthma patients. We evaluated RHI using a new plethysmographic method that has a number of advantages over the ultrasonographic methods used in other studies, including non-invasiveness, high sensitivity, low biological variability and objectivity due to automatic processing. Of the biochemical parameters, we measured 4 biomarkers in relation to endothelial dysfunction (ED): hsCRP, ADMA, E-selectin, and VCAM-1. We compared RHI and biomarkers in CF and asthma patients with healthy controls and sought mutual correlations. We did not prove a statistically significant difference in RHI between the test groups with CF children but we confirmed the decreasing trend of RHI since adolescence and significantly lower RHI values in CF adults, confirming the progressive development of atherogenesis and worsening of ED with age. Biochemical parameters showed significantly higher levels of hsCRP, sVCAM-1 and E-selectin in CF...
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Interaction of bacterial lectins with human lung epithelium
Vyhnalová, Kateřina ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Recessive autosomal disease cystic fibrosis (CF) is caused by a mutation in the CFTR gene ("regulator of cystic fibrosis transmembrane conductance"), which encodes the same named chloride channel. This mutation leads to incorrect ion transport, which causes the formation of an excessively viscous mucus on the surface of the airways and subsequently to the susceptibility to bacterial diseases. This disease mainly affects the respiratory system, where infections are associated with various causes of death in patients with CF. The most common pathogen causing infections is Pseudomonas aeruginosa (PA), which uses many virulence factors, such as pili or adhesins. Lectin PA-IIL, from the group of PA adhesins, is characterized by a high affinity for L-fucose, so it contributes to the adhesion of PA to the low sialylated epithelium of CF patients. In this work the interactions between PA-IIL and lung epithelium were investigated. The cell lines CuFi-1 (CF patient) and NuLi-1 (healthy individual), which were examined ex vivo, were used. A part of these cell lines were exposed to neuraminidase. The PA-IIL lectin was isolated from the E. coli cell line pET25_PAIIL and subsequently fluorescently labeled with DyLight 488. The activity of mentioned lectin was verified by red blood cell agglutination. The...
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