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Biomarkers of neurodegeneration in serum and cerebrospinal fluid in patients with selected neurological diseases
Nosková, Libuše ; Fialová, Lenka (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Švarcová, Jana (referee)
Neurofilaments are the key structural component of the cytoskeleton of neurons, where they are essential for many functions. They consist of 3 subunits: light chain (NFL); medium chain (NfM) and heavy chain (NfH). Except neurofilament proteins there is also α-internexin in the central nervous system (CNS) or peripherin in the peripheral NS. Due to various pathophysiological processes, neurofilament proteins are released into the extracellular space, where they can interact with the components of the immune system. While the involvement of the immune system in the pathogenesis of neurodegenerative diseases is obvious, less knowledge about the antibody response to the neurofilament proteins is available. It is eligible to expand our knowledge in this area. Determination of free antibodies against neurofilaments together with their immune complexes with corresponding antigen provides us more detailed insight into the antibody immune response against neurofilaments. We have optimized the ELISA methods to determine free antibodies against light and heavy chain of neurofilaments together with their corresponding immunocomplexes in both serum and cerebrospinal fluid. Implementation of these methods is precondition for analysis of those parameters in serum and cerebrospinal fluid of patients with...
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CFTR-mRNA: the alternative for the gene therapy of cystic fibrosis
Pecková, Kateřina ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by mutation of the CFTR gene coding homonymous protein, whose main function is transport of chloride ions. In this thesis, gene therapy was used for correction of this defect. Two types of stable mRNA was synthetised - both contained at least 200 adenines on the 3' end, 25 % of pseudouridine, 25 % of 5-methylcytidine and classical cap (enzyme mRNA) or cap analogue 3'-O-Me-m7G(5')ppp(5')G (ARCA cap) on the 5' end. Cell lines isolated from healthy volunteer (NuLi-1) and those from patient suffering from cystic fibrosis with F508del mutation (CuFi-1) were used. The mRNA transfection efficiency was determined using different methods. Increased expression of the CFTR protein was confirmed by visualization of this protein by optimized immunofluorescence method in both cell lines while using both ARCA mRNA and enzyme mRNA. CFTR protein function was studied using fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinoline (MQAE), which is quenched by halogen ions. CFTR channel ion transport was verified using CFTR(inh)-172. This inhibitor specifically inhibits this channel through binding on the R domain of the CFTR protein. CFTR protein function was restored after 24h transfection of the CuFi-1 cell line by ARCA mRNA. The bacterial adhesion of Pseudomonas...
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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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Possible approaches to the treatment of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by a mutation in the CFTR gene. This leads to an absence or a malfunction of CFTR chloride channel, which is also a crutial regulator of other ion channels. This thesis was aimed at gene therapy of cystic fibrosis using CFTR- mRNA gene transfer. To determine the expression of CFTR protein, methods of indirect immunofluorescence and Western blot immunodetection were utilized. Also relative gene expression levels of CFTR gene were assessed by quantitative PCR. Experiments were carried out on a healthy lung epithelial cell line (NuLi-1), a lung epithelial cell line with F508del mutation (CuFi-1) and an embryonic kidney epithelial cell line (HEK293S). CFTR protein was visualized by previously mentioned methods using six primary antiobodies (432, 450, 570, 596, 769, CF3). Primary antibodies 570 a CF3 were found as optimal for the detection of CFTR protein by the method of indirect immunofluorescence, whereas for the detection of this protein by the method of Western blot only the CF3 antibody was suitable. It was also determined that CFTR gene is expressed in overall small levels. Its relative gene expression in the CuFi-1 cell line was approximately six times higher than in the NuLi-1 cell line. The efficiency of transfection of CuFi-1 cell line by CFTR-mRNA...
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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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Development of antibacterial antibodies for cystic fibrosis patients
Vašková, Michaela ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene (CF transmembrane conductance regulator). These mutations result in absent or defective CFTR chloride channel function. The susceptibility to bacterial respiratory infections due to the accumulation of thickened mucus and altered glycosylation in lungs is typical for this disease. Bacteria Pseudomonas aeruginosa (PA) is a major cause of these infections. Among other virulent factors, the pathogenicity of these bacteria is caused by fucose-specific PA-IIL lectin which plays a role as an adhesin. The effect of anti-PA-IIL egg yolk antibodies and multivalent fucose-based PA-IIL inhibitors on PA adherence to lung epithelial cells was studied in this work. Chicken antibodies were isolated from egg yolks before and after immunization with antigen PA-IIL. Specific anti-PA-IIL antibodies were obtained by affinity chromatography using a column with an immobilized PA-IIL. Reactivity of IgY was verified by ELISA. The presence of PA-IIL in the bacterial culture of Pseudomonas aeruginosa (PAK, ST 1763) and the ability of antibodies to recognize this bacterial lectin were verified by Western blotting followed by immunodetection. Appropriate culture conditions have also been found for the expression of this lectin. The...
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Study of lectin-antibody interaction by using surface plasmon resonance
Zákopčaník, Marek ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is an autosomal recessive disease affecting mostly Caucasian race. The disease is caused by mutations in CFTR gene. The most affected organ system is respiratory tract, because the changes lead to insufficient natural defence function of lungs, causing the lungs being susceptible to bacterial infections, which are the most common cause of death of CF patients. Among these bacteria is also Burkholderia cepacia, which causes rapid degradation of pulmonary tissue. Eradication of this bacterial infection is complicated by a number of antibiotic resistances and biofilm formation. Chicken antibodies against bacterial lectins are potential means of preventing bacterial lung infection of patients with CF. Using the affinity chromatography with BC2L-A immobilized column a specific antibody fraction was obtained. The immunoreactivity of the mixed antibodies, affinity purified antibodies and unbounded fraction was compared by ELISA. The comparison of immunoreactivity has emerged that the concentration of the specific antibodies has grown more than 10 times. Interaction of lectin-antibody was studied with SPR method. In the first arrangement, the binding strength of the active lectin to glycosylation of the unbounded fraction antibodies was measured. The dissociation constant of this...
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Possible approaches to the treatment of cystic fibrosis
Král, Jan ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is a genetic disease caused by a mutation in the CFTR gene. This leads to an absence or a malfunction of CFTR chloride channel, which is also a crutial regulator of other ion channels. This thesis was aimed at gene therapy of cystic fibrosis using CFTR- mRNA gene transfer. To determine the expression of CFTR protein, methods of indirect immunofluorescence and Western blot immunodetection were utilized. Also relative gene expression levels of CFTR gene were assessed by quantitative PCR. Experiments were carried out on a healthy lung epithelial cell line (NuLi-1), a lung epithelial cell line with F508del mutation (CuFi-1) and an embryonic kidney epithelial cell line (HEK293S). CFTR protein was visualized by previously mentioned methods using six primary antiobodies (432, 450, 570, 596, 769, CF3). Primary antibodies 570 a CF3 were found as optimal for the detection of CFTR protein by the method of indirect immunofluorescence, whereas for the detection of this protein by the method of Western blot only the CF3 antibody was suitable. It was also determined that CFTR gene is expressed in overall small levels. Its relative gene expression in the CuFi-1 cell line was approximately six times higher than in the NuLi-1 cell line. The efficiency of transfection of CuFi-1 cell line by CFTR-mRNA...
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Gene therapy of cystic fibrosis
Baráčková, Petra ; Bořek Dohalská, Lucie (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the membrane protein called cystic fibrosis transmembrane conductance regulator (CFTR). It is the most frequent genetic disease in Caucasian populations. It currently affects approximately 75,000 individuals worldwide. CF as a monogenic disease represents an appropriate target for the gene therapy. Since the sequencing of the CFTR gene in 1989, a large number of preclinical and clinical studies have been performed examining the various principles of this therapy. The aim of this review study is to summarize the findings and introduce the latest strategies of the gene therapy of cystic fibrosis, which represent a promising basis for definitive cure of this disease. The evolving technologies of genomic engineering, deepening knowledge in pathogenesis of this disease and the results of preclinical and clinical trials motivate researches to further testing of the gene therapy strategies, which have the potential to eliminate the cause of the disease and cure it by intervening in human genome. Keywords: cystic fibrosis, CFTR gene, CFTR protein, gene therapy
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Antibody against lectin PA-IIL as a tool preventing bacterial infections of cystic fibrosis patients
Vašková, Michaela ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Cystic fibrosis is an autosomal recessive disease caused by mutation of the CFTR gene. This mutation results in the damage of protein with ion channel function. As a consequence of this damage, electrolyte equilibrium is impaired. The respiratory tract is affected the most because of the mucus thickening and changes in the glycosylation of lung epithelium cell surface structures. These changes lead to an insufficient defense function of lungs and greater susceptibility to bacterial infections. The most common pathogen of respiratory tract of CF patients is Pseudomonas aeruginosa. This bacterium contains numerous virulence factors and has the ability to form a biofilm that protects it against the host defense mechanisms and antibiotic effect. Antibiotic treatment is also complicated by the development of bacterium resistance. Chicken antibodies have a considerable potential as a tool for preventing bacterial lung infections of CF patients. The influence of specific and nonspecific antibodies on the adherence of Pseudomonas aeruginosa (PAK, ST 1763) ex vivo was monitored on a model system of lung epithelia cells from a CF patient (CuFi-1) and a healthy individual (NuLi-1). Bacterial and pulmonary cells were labeled with PKH fluorescent dyes to allow their spectrofluorimetric determination. It has...
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