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Identification and determination of metalloproteins and metalloproteinases in experimental models and in groups of patients
Kukačka, Jiří ; Průša, Richard (advisor) ; Zima, Tomáš (referee) ; Stoklasová, Alena (referee)
Introduction: Matrix metalloproteinases (MMPs) are zinc-containing metalloproteins which take part in many processes associated with extracellular matrix (ECM) remodeling. These enzymes participate in most processes degrading connective tissue during ontogenesis. Changes in MMP expression and activity can be observed in most inflammato ry, degenerative, and malign processes. Methalothionein also belongs to the group of zinc-dependent metalloproteins and it is involved in metal trafficing, detoxification or protection of cells against reactive oxygen species. Aim: The aim of th is study was to analyze MMP and metallothionein in various experimental models of caridac tissue remodeling (under hypoxia and methamphetamine administration) and in groups of patiens with dislipoproteinemia and traumatic brain injury. Methods: Laboratory rats were exposed to 1) hypoxia for 3 weeks and treated with MMP inhibitor 2) hypoxia and hypoxic hypercapnia for 4 days. 3) ln another experimental set methamphetamine was aplicated to rats for 9 weeks. 4) A group of patiens with dyslipoproteinemia was investigated before and after 1 month therapy by diet or hypolipidemics. 5) Blood samples were collected from patiens with traumatic brain injury during hospitalization. MMPs were analysed by zymography, immunochemical method, and...
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Significance of determination SAA of comparison with CRP and procalcitonin
Daňková, Michaela ; Zadina, Jiří (advisor) ; Průša, Richard (referee)
The purpose of the bachelor thesis is to evaluate serum amyloid (SAA) as a diagnostic marker in selected patient groups and to compare the results of SAA with those of C-reactive protein (CRP) and procalcitonin (PCT). SAA, CRP and PCT are acute-phase proteins, whose blood concentration significantly increases within a few hours after an inflammatory stimulus. The synthesis, caused by proinflammatory cytokines, takes place in the liver. CRP is the most and longest used acute-phase reactant in routine practice. As indicators of inflammation, SAA and especially procalcitonin have been used more lately. Although SAA is an acute-phase reactant as sensitive as CRP, it is not employed so often in the diagnosis since a method suitable for routine diagnosis was not developed until recently. Briefly describing the acute-phase proteins, the theoretical part elaborates on the characterization and comparison of SAA, CRP and PCT. It further outlines methods for the determination of individual analytes. The experimental part focuses on defining selected patient groups and the immunonephelometric method for measuring SAA by the Immage 800 biochemical analyzer. The data obtained in three selected patient groups have been statistically evaluated. The aim of the thesis has been to evaluate the correlation of inflammatory...
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Immunoflulorescent detection of endomysial antibodies (EmA) in the coeliac diagnosis
Martikán, Petr ; Kocna, Petr (advisor) ; Průša, Richard (referee)
The aim of this thesis was comparing of a several immunofluorescence methods and substrates for detection endomysial antibodies (EmA). The common method (Anti-Endomysium antibodies AEA) is used by UKLBD in VFN and by the some samples gives ambigious results owing to another autoantibodies (e.g. ANA, AMA, ASMA). The common method was compared with another sets (IFA-EmA-IgA - AEMAk 03a-10, ImmuGlo anti Endomysial Antipody EMA IFA Kit). Another aim of the work was how to work with this method and be able to evaluate samples by the fluorescence microscope. (...) This thesis brought me a lot of valuable experiences in laboratory and also teached me how to evaluate endomysium antibodies by indirect immunofluorescence method.
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Monitoring of biochemici markers of septic states
Brodská, Helena ; Kazda, Antonín (advisor) ; Zazula, Roman (referee) ; Průša, Richard (referee)
The topic of systemic inflammatory response is lately often discussed across the whole field of medicine. Early diagnosis and the following adequate therapy minimize the risks of the development of systemic inflammatory response syndrome (SIRS). Therefore there is a general urge to find an appropriate marker for diagnosing and monitoring of the development of SIRS. Each year new markers appear, markers that fill up the mosaic of a pathophysiological picture, but we still do not have a marker, that would fulfill the demands of specificity, sensitivity and availability. In the recent ten years, one of the most promising markers seems to be the PCT. (...) In two studies with patients in end states renal diseases (ESRD) the non infectious elevations appear as marker of severity of clinical state. In the last study we confirm the relationship between PCT and severity of clinical state and the antioxidative defence, respectively the plasmatic selenium level. In the above mentioned studies I would like to present the wide range of this marker, which in the meantime is one of the most used markers of the SIRS. I would like to stress the importance of a correct indication and of an accurate interpretation in accordance with all the other parameters, but most of all with the clinical state. PCT, being almost ten...
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Investigation of thyroid disorder in pregnancy and reference intervals in evaluation of maternal thyroid function
Springer, Drahomíra ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Zamrazil, Václav (referee)
The importance of maternal thyroxine for the development of the fetus brain early in pregnancy has received increasing acceptance. It has more recently become evident that maternal hypothyroxinemia results in the birth of children with decreased mental and psychomotor development. In our group of 7,530 women in 9-11 week of pregnancy were determined TSH, anti TPOAb and FT4. For evaluation of results was necessary to set reference intervals for pregnant women. The TSH reference interval was determined to be 0.06 - 3.67 mU/l and for FT4 9.8 - 23.43 pmol/l was used. The limit for anti-TPO positivity was determined to be 143 kU/l. A raised concentration of TSH was found in 5.14% of women; and a suppression of TSH was found in 2.90% of women and 11.5% of pregnant women were found positive. Serum concentrations of FT4 were lower in TPOAb positive as compared to TPOAb negative women and differences of FT4 in euthyroid women with suppressed, normal and elevated TSH were found. (...) In Czech Republic, case finding screening is able to disclose only about 20% of asymptomatic mild or deep hypothyroidism or women with positive anti TPO in pregnancy. Foreign study report 70% of these high-risk pregnant women, consequently is evident need of general screening of pregnancy thyroid failure in Czech Republic. Moreover the...
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Oxidative and carbonyl stress in kidney diseases
Kratochvílová, Markéta ; Tesař, Vladimír (advisor) ; Průša, Richard (referee) ; Zadražil, Josef (referee)
Aims: 1. Determination of AGEs (Advanced Glycation End products) in patients with various types of nephropathy. 2. Association AGEs with nutritional parameters and anemia. 3. Influence of renal parameters on sRAGE (soluble form of Receptor for Advanced Glycation End products) levels. 4. Technics and proceeding methods of the podocytes cultivation. 5. Determination of urine podocytes. Methods: We determined fluorescent AGEs by spectrofluorometry, sRAGE by Enzyme-Linked ImmunoSorbent Assay (ELISA). Podocytes were passaged and identified immunocytochemically. Podocytes in urine were specified by flow cytometry method. Results: 1. We did not find significant differences in AGEs serum levels among various types of nephropathy, even though the pathogenesis differs. 2. The albumin and prealbumin levels positively and haemoglobin levels negatively correlate with AGEs in patients with CKD grade 1-5, without necessity of dialysis. 3. Serum sRAGE levels are increased in patients with decreased renal function independently on the course of renal disease. 4. We implemented the methods and technics of podocyte cultivation. 5. Urine podocytes observation and confirmation that podocyturia relates to disease activity. Conclusion: We confirmed that AGEs serum levels depend more on renal function than the type of...
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Study of protein changes in patients with nephrotic syndrome and Anderson-Fabry disease
Vojtová, Lucie ; Zima, Tomáš (advisor) ; Teplan, Vladimír (referee) ; Průša, Richard (referee)
Background: Heavy proteinuria may be caused by either increased glomerulal basement membrane permeability or membrane or podocyte structural damage, and also by impairment of secretion-reabsorption tubular processes. In this study, 60 patients with nephrotic proteinuria and other diagnoses (lupus nephritis, membranous nephropathy, IgA nephropathy, Wegener's granulomatosis) and 20 patients with Anderson-Fabry disease (AFD),which is an X-linked genetic disorder with deficient a-galactosidase A activity, were analysed by the 2D electrophoresis method. The main aim of this work was to investigate possible differences in urine proteins in nephropaties, between healthy controls and AFD patients and to identify abnormal proteins as potential biomarkers of disease. Methods: The urine proteins were devided by isoelectric focusing method using polyacrylamide strips (pH 3-10 linear). The second dimensional SDS electrophoresis was performed in 12 % polyacrylamide gel. The proteins were visualized by silver method and selected proteins were identified by MALDI-TOF MS. The gels were evaluated by Phoretix 2D expression software 2005. Results: We found out that without adding protease inhibitors we can detect proteolysis, with increased quantity of proteins manifested in the area about 10 kDa and decreased quantity...
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Optimizing of the regime of marker's examination of clinically important infections in blood donors
Dušková, Daniela ; Tesař, Vladimír (advisor) ; Borovanský, Jan (referee) ; Průša, Richard (referee)
Project title: Optimalization of the regime of marker's examination of clinically important infections in blood donors Project author: Daniela Dušková, M.D. Project supervisor: prof. Vladimír Tesař, M.D., Dr.Sc., MBA, FASN The aim of this project is to contribute to the discussion about introducing the methods of molecular biology into the routine blood donor testing in the transfusions departments in the Czech Republic. The theoretical part includes a brief history and some turning points in transfusion medicine. The next part within the theoretical section is dedicated to the problems of infectious diseases concerning transfusion and the general examination processes used during the selection of blood donors. The end of the theoretical part concentrates on existing possibilities of markers' examination of clinically important infections in blood donors, including the list of processes performed in the Czech Republic, the European Union and other countries. The practical part describes this study, ie. the routine screening test of blood donors using the CMIA method (a routine method) and using RT-Real Time PCR method (a molecular biology method) for detecting infectious markers (HCV, HBV, HIV). Within this part, the principle of both methods and the process of actual examinations are described in...
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Pregnancy proteins - molecular biological and biochemical analysis
Muravská, Alexandra ; Kalousová, Marta (advisor) ; Černá, Marie (referee) ; Průša, Richard (referee)
The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...
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Anti-cytoskeletal antibodies in patients with multiple sclerosis and other neurological diseases
Švarcová, Jana ; Matouš-Malbohan, Ivan (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Průša, Richard (referee)
This thesis focuses on the role of anti-cytoskeletal antibodies in various neurodegenerative diseases. Autoantibodies against different axonal cytoskeletal proteins, such as the light (NF-L) and medium (NF-M) subunits of neurofilament and tubulin (TU), in serum and cerebrospinal fluid may be generated in response to the release of cytoskeleton from damaged neurons. The aim of this study was to assess autoimmune involvement in amyotrophic lateral sclerosis (ALS) as well as to evaluate antibody light and medium neurofilament subunit in serum and cerebrospinal fluid (CSF) of patients with ALS. Furthermore, we were interested in the relationships among these antibodies in the serum and in the CSF as well as between the two anti-NF antibody subtypes. Secondly, the aim was to compare the levels of anti-tubulin antibodies (anti-TU) in cerebrospinal fluid and serum in multiple sclerosis (MS) disease, using bovine tubulin as the antigen in one enzyme-linked immunosorbent assay (ELISA) method (anti-TUb antibodies) and a synthetic neuron-specific octapeptide of tubulin in a second ELISA method (anti-TUs antibodies). In the observed groups of ALS patients, serum levels of anti-NF-L antibodies were higher in ALS patients than in controls and serum anti-NF-L antibodies and intrathecal anti- NF-M antibodies were...
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