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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...
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Effects of obesity on the course of Trypanosoma cruzi infection
Brima, Wunnie ; Zima, Tomáš (advisor) ; Šimánek, Vilím (referee) ; Škrha, Jan (referee)
Obesity is very widespread and detrimental to health. Obesity brings with it many changes including heightened immune function, and a higher prevalence of major cardiovascular disorders, cancer, diabetes, and Alzheimer disease. Obesity is also associated with shortened lifespan. The detrimental effects of obesity are linked to the "metabolic syndrome", a broad range of changes in metabolic processes and immune function. As a first approximation, we agree with this formulation but we will then proceed to document some of its weaknesses. (i) Crude mortality rates increase with increasing body mass index (BMI) but as the BMI approaches the normal range, mortality rates reverse (the now classic "J-shaped curve") so that individuals with reduced BMI have elevated mortality. (ii) A multiplicity of medical and surgical conditions have been reported where short term and medium term outcomes are better for overweight patients. These conditions are placed under the heading of "obesity paradox". (iii) The medical community has introduced a binary system for the metabolic syndrome ---- yes, patient has it or no, the patient does not have it, despite the fact that all of the changes that are considered components of the metabolic syndrome are continuous variables. Our work is focused on sharpening focus and improving...
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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...
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Changes in the iron metabolism and its regulation in patients with end-stage renal disease treated by hemodialysis and kidney transplantion
Turková Sedláčková, Terezie ; Racek, Jaroslav (advisor) ; Zima, Tomáš (referee) ; Teplan, Vladimír (referee)
Iron is a very important biogenous trace element, which is involved in many of cell processes in organism. For its character iron can be also involved in Fenton reaction, where a toxic hydroxyl radical is produced. The iron metabolism is very carefully regulated in order to avoid formation of hydroxyl radical. Iron Responsive Proteins-Iron Responsive Elements (IRPs-IREs) system is involved in the regulation of iron metabolism on cell level, small peptide hormon hepcidin is involved in the regulation on systemic level. Hepcidin was discovered in 2000 as a peptide with antimicrobial properties. It is a key regulator of iron metabolism, as was discovered later. The target of hepcidin is ferroportin-the only known iron cell exporter. The expression of hepcidin is downregulated by hypoxia and anemia and upregulated by iron overload and inflammation. Hemodialyzed patients suffer from anemia very often. This anemia is caused by many factors, e.g. inadequate production of erythropoietin, chronic inflammation, chronic oxidative stress, blood loss during hemodialysis process or lower lifetime of red blood cells. We realized three studies on the patients with end-stage renal disease in our laboratories. Our aim was to find a relationship of hepcidin and other parameters of iron metabolism, inflammation and...
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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...
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Distribution and number of wear debris particles around jouint replacements
Veselý, Filip ; Sosna, Antonín (advisor) ; Zima, Tomáš (referee) ; Koudela, Karel (referee)
Aseptic loosening due to polyethylene wear presents one of the most frequently occurring joint replacement complications requiring a revision surgery. Numerous scientific works and publications deal with the issue of morphology, determination and distribution of wear particles. Few studies, however, determine reliably amounts of particles in individual periprosthetic zones. The objective of this work was to evaluate the correlation between amounts of wear particles and periprosthetic osteolysis findings. Description of distribution of these particles in the surroundings of joint replacement presented further objectives of the work. Wear particles were isolated from granuloma obtained during revisions of total hip replacements. Amounts of wear particles were determined using IRc. These findings were correlated with X-ray examinations and intraoperative findings. Method of progressive enzymatic degradation of tissue was used in order to describe wear particles distribution in polyethylene granuloma and surrounding periprosthetic tissues. Results showed uneven incidence of wear particles in the surroundings of the total joint replacement. The incidence correlated with X-ray examinations and intraoperative findings. Distribution of particles was described as well as new determination methods and further...
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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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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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