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Molecular causes and mechanisms of hereditary cholestasis and statin-induced myopathy
Neřoldová, Magdaléna ; Jirsa, Milan (advisor) ; Dvořáková, Lenka (referee) ; Bronský, Jiří (referee)
The discovery of the molecular basis of Rotor syndrome consisting in biallelic inactivating mutations in both SLCO1B1 and SLCO1B3 genes encoding hepatic transporters OATP1B1 and OATP1B3, together with the previously described association of the rs4149056 variant in OATP1B1 with statin-induced myopathy (SM), led us to hypothesis that private variants in OATP1B1 and OATP1B3 confer predisposition to SM. This hypothesis was not supported by our study of 88 patients in whom exome sequencing was performed. Interestingly, we detected candidate variants in several genes mutated in recessively inherited muscle disorders, namely CLCN1, whose carriage may predispose to SM. Pathogenic variants in several dozens of causative genes underlie hereditary cholestasis. We performed exome sequencing in 51 unexplained cases and revealed several unexpected diagnoses such as autosomal recessive polycystic polyposis, cutaneous porphyria or nephronophthisis. The most remarkable finding was that of yet unreported F11R deficiency due to a homozygous splice mutation found simultaneously in an index patient suffering from liver cirrhosis and her healthy sister. The F11R protein is involved in formation of tight intercellular junctions and mutations in F11r predispose to liver failure in mice. The finding of bialellic variants...
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Prevalence of neurodegenerative disorders in patients with esophageal achalasia
Jerie, Martin ; Vojtěch, Zdeněk (advisor) ; Šonka, Karel (referee) ; Bronský, Jiří (referee)
Introduction: Achalasia is a primary motility disorder of the esophagus due to degeneration neurons in myenteric plexus. Although the exact pathogenesis is unknown, autoimmune and neurodegenerative processes seem to be involved. We thus hypothesized that the prevalence of neurodegenerative and/or neuroinflammatory disorders (NDD) with autoimmune component could be higher among patients with achalasia and vice versa as the background pathogenetic mechanisms might be similar. Methods: This was a prospective, observational, comparative questionnaire-based study. Patients with achalasia from a gastroenterology center and patients with NDD from neurology centers in the Czech Republic were enrolled. Patients from achalasia group were then examined by neurologist and neurological patients by gastroenterologist, including further testing to confirm or rule out either NDD and achalasia, respectively. We assessed the prevalence of both achalasia and NDD and compared them with prevalences in general population. Results: A total of 150 patients with achalasia and 112 patients with NDD were enrolled. We observed an increased prevalence of NDD among patients with achalasia (6.0 % (9/150) as compared to the estimated 2.0 % prevalence of NDD in general population, p=0.003). In the NDD group, 32 out of 112 patients...
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Biochemical predictors of the treatment failure in pediatric IBD patients
Zárubová, Kristýna ; Bronský, Jiří (advisor) ; Hrdý, Jiří (referee) ; Petrášová, Miroslava (referee)
Up to 20 % of patients with IBD (inflammatory bowel disease) is diagnosed in childhood, the course of the disease tends to often be more severe and the treatment more difficult than in case of adults. Therefore, it is necessary to commence or eventually change the treatment in order to prevent development of complications. Early detection of relapse plays significant role for further course of the disease and general prognosis of the patient. Changes to laboratory parameters (e.g. to the level of fecal calprotectin) can significantly prevent the development of clinical symptoms and may indicate the necessity for control endoscopy, further escalation or change in therapy. The primary focus of this thesis are patients who underwent surgical bowel resection, who constitute one of the risk groups. Studies show that as many as 70 % of patients suffering from Crohn's disease reach the point of bowel surgery within the first 10 years of the diagnosis. According to the results, a promising predictor of relapse of the disease seems to be serum albumin, which may relate to the overall nutritional status of the organism. We have also examined the effectivity of using fecal calprotectin and CRP as relapse predictors. Testing directly in the resected bowel did not prove usability of calprotectin as a predictor,...
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Metabolism of immunosuppressants in children with inflammatory bowel disease
Pospíšilová, Kristýna ; Bronský, Jiří (advisor) ; Leníček, Martin (referee) ; Dědek, Petr (referee)
Metabolism of immunosuppressants in children with inflammatory bowel disease Abstract Thiopurines are still used as the main immunosuppressants in the maintenance therapy of paediatric Crohn's disease. According to official guidelines, in case of failure, biological therapy (mainly infliximab or adalimumab) is generally commenced. The present thesis focuses on the possibilities of therapy optimization in children in whom the conventional Crohn's disease therapy has failed based on the metabolism of administered immunosuppressive medications. We confirmed that the knowledge of thiopurine metabolites concentration in red blood cells may help to monitor patients' adherence to therapy. However, according to our observations in children on combination therapy of infliximab and thiopurine lower concentrations of 6- thioguanine (the active metabolite of thiopurines) seems to be effective compared to what has been expected. We presented a web application designed to partially substitute for the thiopurine metabolite measurements based on easily available laboratory data. Last, but not least, we confirmed that the clinical effect and safety profile of both adalimumab and infliximab are similar in the treatment of paediatric Crohn's disease. Only in case of family history of atopic dermatitis adalimumab may be preferred.
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New regulatory hormones of breast milk
Mitrová, Katarína ; Bronský, Jiří (advisor) ; Frühauf, Pavel (referee) ; Kohout, Pavel (referee)
Background: Breastfeeding is supposed to exert long-term protection of the infant from the development of civilisation diseases later in life. Adiponectin, leptin and AFABP are candidate proteins in nutritional programming of the infant. Aim: To determine intraindividual changes of breast milk levels of regulatory hormones (adiponectin, leptin, AFABP) and adipophilin in breast milk during 12 months of lactation. Methods: Proteins were measured using high sensitive ELISA method in 72 healthy mothers within 2 days of beginning of lactation (day 0) and 1, 3, 6 and 12 months following delivery. Results: Adiponectin levels in breast milk on day 0 (D0) were 22.8 ± 0.8 (mean ± S.E.M.), in 1 month (M1) 22.0 ± 0.6, in 3 months (M3) 20.5 ± 0.6, in 6 months (M6) 21.4 ± 0.8, and in 12 months (M12) 25.7±1.4 ng/ml. AFABP levels were 12.3 ± 2.0, 6.2 ± 1.3, 1.3 ± 0.2, 2.5 ± 1.0 and 4.6 ± 1.9 ng/ml, respectively. Leptin levels were 0.3 ± 0.04, 0.2 ± 0.03, 0.1 ± 0.01, 0.1 ± 0.02 and 0.2 ± 0.04 ng/ml, respectively. Adipophilin levels in D0 were 1.98 ± 0.12, in M1 2.83 ± 0.21, in M3 2.39 ± 0.17, in M6 2.57 ± 0.16, and in M12 3.25 ± 0.21 μg/ml. We found significantly higher levels of adiponectin in M12 in comparison to M3 and M6 (p = 0.0026), higher levels of AFABP in D0 and M1 when compared to M3, M6 and M12 (p < 0.0001) and...
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Examining the significance of peptides regulating the intake of food and the nutritional state of children and adolescents
Bronský, Jiří ; Průša, Richard (advisor) ; Pelouch, Václav (referee) ; Hyánek, Josef (referee)
Introduction: Regulation of food intake and nutritional status is mediated by complex interactions of regulatory peptides of the central nervous system, gastrointestinal tract and adipose tissue. These systems are connected by feedback loops which inform the centre about amount of ingested food and energy reserves in the organism. Dysfunction of any of these regulatory areas may lead to changes in nutritional status of the organism. Methods: We used radioimmunoassay to measure plasma levels of orexin A, total ghrelin and serum levels of leptin and enzyme immunoassay to measure serum levels of adiponectin in healthy subjects and in children with obesity, anorexia nervosa, Crohn's disease and celiac disease and we evaluated the influence of nutritional therapy on these levels. Moreover, we evaluated relationship of these regulatory peptides to other biochemical and anthropometrical factors of nutritional status. We also measured plasma levels of total and unreduced amylin by enzyme immunoassay with immunofluorescence detection in adult patients with osteoporosis, type II diabetes mellitus and in the control group. Results: During reduction of body weight in obese children and adolescents, there were statistically significant changes of plasma orexin A levels and total ghrelin levels, but we haven't seen any...
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Quantification of macronutrients in human milk after premature birth depending on the gestational and postnatal age of infant
Burianová, Iva ; Bronský, Jiří (advisor) ; Kokštein, Zdeněk (referee) ; Dvořáková, Hana Marie (referee)
(jednostránkový souhrn) v angličtině Growth restriction of premature newborns is a serious complication for the further development of the infant. It is not yet definitely clear to which extent it is associated with the low content of certain macronutrients of human milk. Maternal characteristics may be connected with human milk macronutrients, but no definite conclusions have been made to date. This study aimed to evaluate the content of macronutrients in human milk samples after preterm delivery during the first 9 weeks of lactation and determine the relationship of maternal-associated factors on the content of macronutrients in human milk after preterm delivery. Milk samples were collected from mothers after premature birth between 24+0-35+6 weeks. Macronutrients and energy content were analyzed by mid-infrared transmission spectroscopy. A total 1917 human milk samples from were analyzed. Median of protein content decreased from 1.6 g/dL in group A and 1.5 g/dL in group B in the first week of life, to 1.1 g/dL in both groups at the end of week 3, and then remained stable up to week 9. Content of carbohydrates and fat was stable during the whole observation, with interindividual differences. Similarly to colostrum, higher protein content and lower carbohydrates content were observed in...
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The use of immunochemical methods in the diagnostic of cancer and degenerative disease
Hanousková, Lenka ; Kotaška, Karel (advisor) ; Bronský, Jiří (referee) ; Štern, Petr (referee)
Introduction: Immunochemical methods have significant importance in biomedical research. The benefit of immunochemical methods is their high sensitivity and accuracy. The aim of the work was to use immunochemical methods in investigation of cancer biomarkers and degenerative diseases. A total of four studies were performed. Two studies were focused on the search for new biomarkers of prostate cancer, the third study was focused on evaluation of FGF23 in patients with hyperparathyroidism, and the fourth study looked at the assessment of hyaluronic acid in synovial fluid of patients with knee gonarthrosis. Methods and patients: Study 1: Concentrations of Chromogranin A, Endoglin, TIMP-1 and thymidine kinase 1 (TK1) in serum and Endoglin, SPINK-1, Annexin, TIMP-1 in urine were measured in 58 patients with the (mean age 68 years, range 45 - 82 years) with clinically diagnosed prostate cancer and in 30 healthy individuals with the (mean age 64 years, range: 55 - 78 years) without clinically relevant urological history and PSA values in the reference range. Subsequently, serum TK1 levels were measured in 169 patients (mean age 62 years, range 45- 82 years) with prostate cancer and in a control group of 39 healthy men (mean age 61 years, range 54-78 years). The TK1 assay was also used to determine the...
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Bilirubin secretory pathway and its disorders.
Sticová, Eva ; Jirsa, Milan (advisor) ; Bronský, Jiří (referee) ; Jirásek, Tomáš (referee)
Identification and functional characterization of numerous transport systems at the sinusoidal and canalicular membrane of hepatocytes have significantly expanded our understanding of bilirubin metabolism and contributed to elucidation of molecular basis of hereditary jaundice. Moreover, dysregulation of hepatobiliary transport systems could explain jaundice in many acquired liver disorders. This thesis is focused on the new aspects of bilirubin handling in hepatocytes based on elucidation of the molecular basis of Rotor syndrome. The first study is focused on the antioxidative properties of bilirubin in liver tissue in a model of obstructive cholestasis. In the second part of the thesis we present several novel mutations in ABCC2, the gene associated with Dubin-Johnson syndrome, identified in patients selected for the Rotor locus mapping study. In the key third study concerned with Rotor syndrome we demonstrated that biallelic inactivating mutations causing complete absence of transport proteins OATP1B1 and OATP1B3 result in disruption of hepatic reuptake of bilirubin, which is the molecular basis of Rotor-type jaundice. These results indicate that apart from secretion of conjugated bilirubin into bile, a significant fraction of bilirubin glucuronide is secreted via MRP3 into sinusoidal blood and...
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Optimization of biologic therapy in children with inflammatory bowel disease (IBD) using modern biomarkers
Ohem, Jan ; Bronský, Jiří (advisor) ; Kotaška, Karel (referee) ; Bajerová, Kateřina (referee)
Optimization of biologic therapy in children with inflammatory bowel disease (IBD) using modern biomarkers Abstract to thesis Study programme: Biochemistry and Pathobiochemistry Introduction: In adults, infliximab (IFX) levels correlate with disease activity and antibodies to IFX (ATIs) predict treatment failure. We aimed to determine the association of IFX levels and ATIs with disease activity in paediatric population. Methods: This study was performed as a prospective observational study. We prospectively collected blood, stool, and clinical data from 65 patients (age 10.5-15.1 years) with Crohn's disease (CD) before IFX administration, and measured IFX trough levels, ATIs, and faecal calprotectin levels (CPT). We used multivariate analysis to identify the predictors of IFX levels. IFX and ATIs levels were meassured using ELISA. Results: Lower levels of IFX were associated with ATIs positivity (OR [odds ratio] 0.027, CI [confidence interval] 0.009-0.077). Higher C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR) and CPT levels were found in patients with lower IFX levels. The optimal combination of specificity (50%) and sensitivity (74%) for disease activity was calculated for IFX levels ≥ 1.1 µg/ml using CRP level < 5 mg/l as a marker of laboratory remission. In a model that used CPT ≤...
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