Biologický význam metabolických produktů hemu a bilirubinu.

Předkládaná práce se zabývá studiem významu produktů katabolické dráhy hemu, zejména s ohledem na patogenezi, diagnostiku a léčbu nekonjugovaných hyperbilirubinémií (závažná novorozenecká žloutenka a Criglerův-Najjarův syndrom). Jedním z hlavních cílů bylo ozřejmění biologických účinků produktů bilirubinu, které vznikají při fototerapii těchto onemocnění a otestování nových léčebných přístupů a to jak na úrovni genové terapie, tak farmakoterapie. Novorozenecká žloutenka je jednou z nejběžnějších komplikací v neonatálním období. Zlatým standardem v její léčbě je fototerapie modrým světlem, jejíž použití však může být doprovázeno i závažnými nežádoucími efekty. Nutno podotknout, že fototerapie novorozenecké žloutenky je v některých zemích nadužívána, a že pacienti s Criglerovým- Najjarovým syndromem typu I jsou vystaveni celoživotní fototerapii (pokud nepodstoupí transplantaci jater). V rámci předkládané práce jsme na experimentálním in vitro modelu studovali biologické účinky fotoizomerů bilirubinu, které vznikají v průběhu terapie novorozenecké žloutenky. Dále jsme se za použití experimentálních modelů hyperbilirubinemických potkanů a myší zabývali možnostmi zavedení vhodné genové terapie, kterou by bylo možné bezpečně použít v léčbě Criglerova-Najjarova syndromu a omezit nebo zcela odstranit...Present work has been focused on the importance of the products of the heme catabolic pathway, in particular under conditions of unconjugated hyperbilirubinemias (neonatal jaundice and Crigler-Najjar syndrome (CNS)). The second part of the project was focused on the improvement of some pharmacological approaches used in the treatment of these diseases, as well as on studies of bilirubin products that are formed during the treatment by phototherapy (PT). Neonatal jaundice is one of the most common complications in neonates. Currently, there is no efficient pharmacotherapy and the treatment with blue light is used as a gold standard for severe neonatal jaundice. However, the absolute safety of PT has still not been confirmed. In this context, it is important to note that some neonatologists start the PT before serum bilirubin levels reach the recommended values and that patients with CNS type I (CNSI) are forced to be on life-long PT (unless undergoing liver transplantation). The focus of the present project was to study biological effects of bilirubin photoisomers (PI) in an in vitro model of the human neuroblastoma SH-SY5Y cells that are used for studies of the neuronal metabolism. In further studies performed on animal model of hyperbilirubinemic rats and mice, we investigated a suitable gene...Ústav lékařské biochemie a laboratorní diagnostiky 1. LF UK a VFNInstitute of Medical Biochemistry and Laboratory Medicine First Faculty of MedicineFirst Faculty of Medicine1. lékařská fakult

Pengembangan Perangkat Pembelajaran Flipped Classroom Pada Materi Getaran Harmonis

The aims of this research are to determine the validity of the product by experts and practitioners as well as to describe the attractiveness, the easiness and the expediency of the product. Development models used in this research is the ADDIE model which consists of five stages: analysis, design, development, implementation, and evaluation. The stage at this research explains only at the stage of development. The results obtained from expert validation test with an average score of 3.60 with the qualifying products is very valid, practitioners test results obtained an average score of 3.28 with very valid qualifying, and one by one test results of the product showed that the product was very attractive, very easy to use, and helpful. Penelitian ini bertujuan untuk mengetahui validitas produk oleh ahli dan praktisi serta untuk mendeskripsikan kemenarikan, kemudahan, dan kemanfaatan dari produk pengembangan. Model pengembangan yang digunakan dalam penelitian ini adalah model ADDIE yang terdiri dari lima tahapan, yaitu analisis, desain, pengembangan, implementasi, dan evaluasi. Pada penelitian ini, tahapan yang dilakukan hanya sampai pada tahap pengembangan. Hasil penelitian yang diperoleh dari uji validasi ahli yaitu rata-rata skor produk 3,60 dengan kualifikasi sangat valid, hasil uji praktisi diperoleh rata-rata skor 3,28 dengan kualifikasi sangat valid, dan hasil uji 1-1 menunjukkan bahwa produk memiliki kualitas sangat menarik, sangat mudah digunakan, dan bermanfaat

Heme oxygenase-1 may affect cell signalling via modulation of ganglioside composition

Heme oxygenase 1 (Hmox1), a ubiquitous enzyme degrading heme to carbon monoxide, iron, and biliverdin, is one of the cytoprotective enzymes induced in response to a variety of stimuli, including cellular oxidative stress. Gangliosides, sialic acid-containing glycosphingolipids expressed in all cells, are involved in cell recognition, signalling, and membrane stabilization. Their expression is often altered under many pathological and physiological conditions including cell death, proliferation, and differentiation. The aim of this study was to assess the possible role of Hmox1 in ganglioside metabolism in relation to oxidative stress. The content of liver and brain gangliosides, their cellular distribution, and mRNA as well as protein expression of key glycosyltransferases were determined in Hmox1 knockout mice as well as their wild-type littermates. To elucidate the possible underlying mechanisms between Hmox1 and ganglioside metabolism, hepatoblastoma HepG2 and neuroblastoma SH-SY5Y cell lines were used for in vitro experiments. Mice lacking Hmox1 exhibited a significant increase in concentrations of liver and brain gangliosides and in mRNA expression of the key enzymes of ganglioside metabolism. A marked shift of GM1 ganglioside from the subsinusoidal part of the intracellular compartment into sinusoidal membranes of hepatocytes was shown in Hmox1 knockout mice. Induction of oxidative stress by chenodeoxycholic acid in vitro resulted in a significant increase in GM3, GM2, and GD1a gangliosides in SH-SY5Y cells and GM3 and GM2 in the HepG2 cell line. These changes were abolished with administration of bilirubin, a potent antioxidant agent. These observations were closely related to oxidative stress-mediated changes in sialyltransferase expression regulated at least partially through the protein kinase C pathway. We conclude that oxidative stress is an important factor modulating synthesis and distribution of gangliosides in vivo and in vitro which might affect ganglioside signalling in higher organisms

The biological role of the metabolic products of haem and bilirubin.

Present work has been focused on the importance of the products of the heme catabolic pathway, in particular under conditions of unconjugated hyperbilirubinemias (neonatal jaundice and Crigler-Najjar syndrome (CNS)). The second part of the project was focused on the improvement of some pharmacological approaches used in the treatment of these diseases, as well as on studies of bilirubin products that are formed during the treatment by phototherapy (PT). Neonatal jaundice is one of the most common complications in neonates. Currently, there is no efficient pharmacotherapy and the treatment with blue light is used as a gold standard for severe neonatal jaundice. However, the absolute safety of PT has still not been confirmed. In this context, it is important to note that some neonatologists start the PT before serum bilirubin levels reach the recommended values and that patients with CNS type I (CNSI) are forced to be on life-long PT (unless undergoing liver transplantation). The focus of the present project was to study biological effects of bilirubin photoisomers (PI) in an in vitro model of the human neuroblastoma SH-SY5Y cells that are used for studies of the neuronal metabolism. In further studies performed on animal model of hyperbilirubinemic rats and mice, we investigated a suitable gene..

The biological role of the metabolic products of haem and bilirubin.

Present work has been focused on the importance of the products of the heme catabolic pathway, in particular under conditions of unconjugated hyperbilirubinemias (neonatal jaundice and Crigler-Najjar syndrome (CNS)). The second part of the project was focused on the improvement of some pharmacological approaches used in the treatment of these diseases, as well as on studies of bilirubin products that are formed during the treatment by phototherapy (PT). Neonatal jaundice is one of the most common complications in neonates. Currently, there is no efficient pharmacotherapy and the treatment with blue light is used as a gold standard for severe neonatal jaundice. However, the absolute safety of PT has still not been confirmed. In this context, it is important to note that some neonatologists start the PT before serum bilirubin levels reach the recommended values and that patients with CNS type I (CNSI) are forced to be on life-long PT (unless undergoing liver transplantation). The focus of the present project was to study biological effects of bilirubin photoisomers (PI) in an in vitro model of the human neuroblastoma SH-SY5Y cells that are used for studies of the neuronal metabolism. In further studies performed on animal model of hyperbilirubinemic rats and mice, we investigated a suitable gene..

Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice

Bilirubin is considered to be one of the most potent endogenous antioxidants in humans. Its serum concentrations are predominantly affected by the activity of hepatic bilirubin UDP-glucuronosyl transferase (UGT1A1). Our objective was to analyze the potential bilirubin-modulating effects of natural polyphenols from milk thistle (Silybum marianum), a hepatoprotective herb. Human hepatoblastoma HepG2 cells were exposed to major polyphenolic compounds isolated from milk thistle. Based on in vitro studies, 2,3-dehydrosilybins A and B were selected as the most efficient compounds and applied either intraperitoneally or orally for seven days to C57BL/6 mice. After, UGT1A1 mRNA expression, serum, intrahepatic bilirubin concentrations, and lipoperoxidation in the liver tissue were analyzed. All natural polyphenols used increased intracellular concentration of bilirubin in HepG2 cells to a similar extent as atazanavir, a known bilirubinemia-enhancing agent. Intraperitoneal application of 2,3-dehydrosilybins A and B (the most efficient flavonoids from in vitro studies) to mice (50 mg/kg) led to a significant downregulation of UGT1A1 mRNA expression (46±3% of controls, p<0.005) in the liver and also to a significant increase of the intracellular bilirubin concentration (0.98±0.03vs.1.21±0.02 nmol/mg, p<0.05). Simultaneously, a significant decrease of lipoperoxidation (61±2% of controls, p<0.005) was detected in the liver tissue of treated animals, and similar results were also observed after oral treatment. Importantly, both application routes also led to a significant elevation of serum bilirubin concentrations (125±3% and 160±22% of the controls after intraperitoneal and oral administration, respectively, p<0.005 in both cases). In conclusion, polyphenolic compounds contained in silymarin, in particular 2,3-dehydrosilybins A and B, affect hepatic and serum bilirubin concentrations, as well as lipoperoxidation in the liver. This phenomenon might contribute to the hepatoprotective effects of silymarin

Chlorophyll-Mediated Changes in the Redox Status of Pancreatic Cancer Cells Are Associated with Its Anticancer Effects

Nutritional factors which exhibit antioxidant properties, such as those contained in green plants, may be protective against cancer. Chlorophyll and other tetrapyrrolic compounds which are structurally related to heme and bilirubin (a bile pigment with antioxidant activity) are among those molecules which are purportedly responsible for these effects. Therefore, the aim of our study was to assess both the antiproliferative and antioxidative effects of chlorophylls (chlorophyll a/b, chlorophyllin, and pheophytin a) in experimental pancreatic cancer. Chlorophylls have been shown to produce antiproliferative effects in pancreatic cancer cell lines (PaTu-8902, MiaPaCa-2, and BxPC-3) in a dose-dependent manner (10–125 μmol/L). Chlorophylls also have been observed to inhibit heme oxygenase (HMOX) mRNA expression and HMOX enzymatic activity, substantially affecting the redox environment of pancreatic cancer cells, including the production of mitochondrial/whole-cell reactive oxygen species, and alter the ratio of reduced-to-oxidized glutathione. Importantly, chlorophyll-mediated suppression of pancreatic cancer cell viability has been replicated in in vivo experiments, where the administration of chlorophyll a resulted in the significant reduction of pancreatic tumor size in xenotransplanted nude mice. In conclusion, this data suggests that chlorophyll-mediated changes on the redox status of pancreatic cancer cells might be responsible for their antiproliferative and anticancer effects and thus contribute to the decreased incidence of cancer among individuals who consume green vegetables

Physico-chemical characterization of bilirubin-10-sulfonate and comparison of its acid–base behavior with unconjugated bilirubin

Abstract Unconjugated bilirubin (UCB) is the end-product of heme catabolism in the intravascular compartment. Although beneficial for human health when mildly elevated in the body, when present at greater than a critical threshold concentration, UCB exerts toxic effects that are related to its physico-chemical properties, particularly affecting the central nervous system. The aim of the present study was to characterize bilirubin-10-sulfonate (ranarubin), a naturally occurring bile pigment, including determination of its mixed acidity constants (pK a *). Thanks to the presence of the sulfonic acid moiety, this compound is more polar compared to UCB, which might theoretically solve the problem with an accurate determination of the UCB pK a * values of its propionic acid carboxylic groups. Bilirubin-10-sulfonate was synthesized by modification of a previously described procedure; and its properties were studied by mass spectrometry (MS), nuclear magnetic resonance (NMR), infrared (IR), and circular dichroism (CD) spectroscopy. Determination of pK a * values of bilirubin-10-sulfonate and UCB was performed by capillary electrophoresis with low pigment concentrations in polar buffers. The identity of the synthesized bilirubin-10-sulfonate was confirmed by MS, and the pigment was further characterized by NMR, IR, and CD spectroscopy. The pK a values of carboxylic acid moieties of bilirubin-10-sulfonate were determined to be 5.02, whereas those of UCB were determined to be 9.01. The physico-chemical properties of bilirubin-10-sulfonate were partially characterized with low pK a * values compared to those of UCB, indicating that bilirubin-10-sulfonate cannot be used as a surrogate pigment for UCB chemical studies. In addition, using a different methodological approach, the pK a * values of UCB were found to be in a mildly alkaline region, confirming the conclusions of a recent critical re-evaluation of this specific issue

Potential of therapeutic bile acids in the treatment of neonatal Hyperbilirubinemia.

Neonatal hyperbilirubinemia or jaundice is associated with kernicterus, resulting in permanent neurological damage or even death. Conventional phototherapy does not prevent hyperbilirubinemia or eliminate the need for exchange transfusion. Here we investigated the potential of therapeutic bile acids ursodeoxycholic acid (UDCA) and obeticholic acid (OCA, 6-α-ethyl-CDCA), a farnesoid-X-receptor (FXR) agonist, as preventive treatment options for neonatal hyperbilirubinemia using the hUGT1*1 humanized mice and Ugt1a-deficient Gunn rats. Treatment of hUGT1*1 mice with UDCA or OCA at postnatal days 10-14 effectively decreased bilirubin in plasma (by 82% and 62%) and brain (by 72% and 69%), respectively. Mechanistically, our findings indicate that these effects are mediated through induction of protein levels of hUGT1A1 in the intestine, but not in liver. We further demonstrate that in Ugt1a-deficient Gunn rats, UDCA but not OCA significantly decreases plasma bilirubin, indicating that at least some of the hypobilirubinemic effects of UDCA are independent of UGT1A1. Finally, using the synthetic, non-bile acid, FXR-agonist GW4064, we show that some of these effects are mediated through direct or indirect activation of FXR. Together, our study shows that therapeutic bile acids UDCA and OCA effectively reduce both plasma and brain bilirubin, highlighting their potential in the treatment of neonatal hyperbilirubinemia