|
|
Individualization of the treatment of prostate cancer patients based on the immunocytochemical detection of circulating tumor cells
Škereňová, Markéta ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Svoboda, Marek (referee)
Introduction: Together with the introduction of new therapeutic options in castration- resistant prostate cancer (CRPC), an advance in individual disease characterization is required. Since common biopsy methods are not suitable for the majority of CRPC patients, one possible solution is the liquid biopsy that is, the analysis of circulating tumor cells (CTCs) isolated from the cancer patients' blood. Methods: A method based on the immunomagnetic enrichment of CTCs and subsequent PCR detection of tumor-associated genes (AdnaTest, Qiagen) was characterized and used for the detection of CTCs in 41 CRPC patients. Each patient was screened at the time of CRPC diagnosis and after the 3rd cycle of docetaxel therapy. A panel of genes associated with therapeutic decision-making was established and validated. Quantitative PCR (qPCR) method on a BioMark platform (Fluidigm, USA) was used to determine the expression of the gene panel in the CTC-enriched and primary tumor samples and the results were analyzed. Results: CTCs were found in 85% and 45% of CRPC patients before and during the therapy, respectively. The presence of CTCs, as well as EGFR and AR PCR fragments, was associated with a decreased sPSA response and lower survival. The gene expression of the CTC- enriched and primary tumor samples differed...
|
|
|
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...
|
|
|
Research of vitreous and vitreoretinal interface in microvascular retinal disorders focussed on eye complications of diabetes mellitus
Křížová, Libuše ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Sosna, Tomáš (referee)
In this work I present conclusions of clinical-laboratory research focused on the patients with diabetic macular edema (DME). We performed biochemical and immunochemical analyses of vitreous samples that were collected during the pars plana vitrectomy. Moreover, at patients with non-proliferative diabetic retinopathy (NPDR) we assessed morphological characteristics of DME using optical coherence tomography (OCT). According to our findings, the vitreous and serum concentrations of uric acid and glucose were significantly higher in patients with diabetic retinopathy and DME compared to controls. Also total ratio (serum/ vitreous concentration) of uric acid and glucose was in diabetics significantly higher than in controls. The most important determinant of increasing concentration of both uric acid and glucose in the vitreous was the grade of diabetic retinopathy. Moreover, we demonstrated significant correlation between vitreous concentration of uric acid and concentration of the vascular endothelial growth factor (VEGF) in patients with DME and NPDR. We found further, that the volume of the macula (cube volume - CV) computed with the software of Cirrus HD-OCT correlates in diabetics significantly with the vitreous VEGF concentration, but not with uric acid. This OCT parameter could be used to...
|
|
|
Novel biomarkers in patients with renal disease
Zakiyanov, Oskar ; Kalousová, Marta (advisor) ; Teplan, Vladimír (referee) ; Průša, Richard (referee)
Chronic kidney disease (CKD) and acute kidney injury (AKI) are major public health problems. It is important to be able to identify those at high risk of adverse outcome, CKD progression and associated cardiovascular disease. The aim of the thesis was to study novel promising biomarkers, their relationship to kidney function, chronic inflammation and/or cardiovascular risk - placental growth factor (PlGF), pregnancy associated plasma protein A (PAPP-A), matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), soluble receptor for advanced glycation end products (sRAGE), calcium binding protein S100A12 or extracellular newly identified RAGE binding protein (EN-RAGE), and high mobility group box protein-1 (HMGB-1) in patients with renal diseases including CKD, haemodialysis (HD), AKI patients, and healthy controls for comparison. First study revealed that PlGF is elevated in patients with decreased renal function. Second study demonstrated the association of MMP-2 and PAPP-A with proteinuria in patients with CKD. Moreover, serum MMP-2, MMP-9 and PAPP-A levels significantly differed in patients with various nephropathies. EN-RAGE levels are not elevated in patients with CKD, but are related to inflammatory status. PAPP-A, EN-RAGE and HMGB-1 levels are significantly elevated, but sRAGE and PlGF...
|
|
|
Laboratory diagnostics of kidney function
Šálek, Tomáš ; Palička, Vladimír (advisor) ; Jabor, Antonín (referee) ; Průša, Richard (referee)
Laboratory diagnostics of kidney function The work deal with issue of glomerular filtration rate (GFR). GFR is the most important indicator of kidney function. Its decline is associated with increased morbidity and mortality. Chronic kidney disease (CKD) is defined as decrease of GFR bellow 1.0 ml/s/1,73m2 for more than three months. Patients with CKD receive nephroprotective drugs and have reduced drugs excreted by kidneys if needed. Early detection of CKD is of clinical importance. We compare GFR estimated from serum creatinine (eGFRcreatinine) and from serum cystatin C (eGFRcystatinC) in four cohorts of patients: 101 MGUS patients, 13 methanol intoxicated patients, 1515 diabetics and 352 patients with CKD. Creatinine was measured by enzymatic method traceable to international reference material NST SRM 967 in all patients. Cystatin C was determined by standardized immunoturbidimetric method traceable to DA ERM 471. This standardized method was not available only for MGUS patients. eGFR was always calculated according to the best available validated equation. MGUS patients were calculated according to MDRD equation for eGFRcreatinine and according to Grubb equation for eGFRcystatinC. Methanol intoxicated patients were estimated according to Lund-MalmÖ equation for eGFRcreatinine and according to...
|
|
|
New markers of liver fibrosis
Skřebská, Lenka ; Čepová, Jana (advisor) ; Průša, Richard (referee)
This material is talking about problems of liver fibrosis. It Is a very fundamental liver's disease which is bringing about a chronic harm of the liver's tissue. An effect of it is a process defect of fibrogenesis and fibrolysis which has to be balanced. A diagnosis of liver fibrosis on time is important for start an efficient therapy. At this time we are getting diagnosis of the liver fibrosis by liver's biopsy which is bringing many risks for patients and it is disadvantageus for them. This situation is running us to search for a new non-invasive methods. It is a lot of calculations they are using a specific and unspecific markers to determine fundamental measures of specific fibrotisation and nonspecific too. A goal of this work it is to get a contrast between a new automatic imunochemicle ELF test which is using specific markers for the liver fibrosis and using calculations APRI and the Hepascore until now. Powered by TCPDF (www.tcpdf.org)
|
|
|
The Heme Catabolic Pathway in Chronic Hepatitis C
Subhanová, Iva ; Zima, Tomáš (advisor) ; Průša, Richard (referee) ; Kráslová, Ivana (referee)
This thesis focuses on the importance of the heme catabolic pathway in chronic hepatitis C (HCV). The aim is mainly to investigate, whether expresion/activity of key enzymes of the heme catabolic pathway, heme oxygenase (HMOX) and biliverdin reductase (BLVRA) in the liver and blood (study A) or promoter variations of HMOX1 and UDP- glucuronosyltransferase (UGT1A1) (study B) may be associated with the progression of fibrosis and may also predict antiviral treatment outcome in patients chronically infected with HCV. We set up a new sensitive method to quantify HMOX activity by reduction gas chromatography. We developed and extensively validated RealTime PCR assay for HMOX and BLVRA expression in the liver and peripheral blood leucocytes (PBL). The (GT)n and (TA)n dinucleotide variations in HMOX1 and UGT1A1 gene promoters, respectively, were determined by fragment analysis. No association was detected between either expression of HMOX/BLVRA or the HMOX1/ UGT1A1 promoter variants and the individual histological stages of liver disease in the HCV positive patients. A marked difference in BLVRA expression in PBL between the sustained responders (SVR) and patients with treatment failure (NVR) was detected before antiviral treatment and during the follow-up. Our data suggests, that BLVRA basal expression...
|
|
|
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...
|
|
|
Determination of the concentration of alpha-1-antitrypsin in the stool by immunoassay method
Plačková, Marie ; Kocna, Petr (advisor) ; Průša, Richard (referee)
Determination of the concentration of α1 - antitrypsin in the stool is a diagnostic indicator of inflammatory diseases of the small and the large intestine, especially malabsorption syndrome. α1 - antitrypsin belongs to the family of plasma proteins with antiproteinase effect. α1 - antitrypsin is synthesized in liver, in small amount in macrophage and is a protease inhibitor of serine proteases sercected from neutrophils. α1 - antitrypsin is acute phase protein. Higher α1 - antitrypsin values are in early phase of inflammation associated with raised CRP and other pozitive acute phase proteins. Fecal α1 - antitrypsin clearance is a sensitive and specific marker of protein loss. For α1 - antitrypsin determination in stool samples ELISA method can be used. ELISA is noncompetetive immunoassay used to detect presence of antibody or an antigen in a sample. The aim of this work was to compare two ELISA sets (Immundiagnostik and Ridascreen) used for determination α1 - antitrypsin in the stool. Then examine stability of α1 - antitrypsin in the stool and in extract prepared from stool in various storing conditions temperature and time. After this establish this method as routine in laboratory. 20 patient stool samples were examined to compare ELISA sets. Samples were suggested to be α1 - antitrypsin...
|
|
|
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...
|
guest ::
