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Effects of heme arginate in HIV-1 acute infection and in latency reversal
Prakash, Shankaran ; Mělková, Zora (advisor) ; Hirsch, Ivan (referee) ; Hejnar, Jiří (referee)
The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...
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The biological role of the metabolic products of haem and bilirubin.
Jašprová, Jana ; Vítek, Libor (advisor) ; Farghali, Hassan (referee) ; Mičuda, Stanislav (referee)
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...
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Surface-enhanced Raman spectral detection of bilirubin and temporally synchronized monitoring of its photochemical transformations in selected solvents by SERS and electronic absorption spectroscopy
Hrnčířová, Jana ; Vlčková, Blanka (advisor) ; Procházka, Marek (referee)
Surface-enhanced Raman scattering (SERS)-active systems based on macroscopic Ag nanosponge aggregates as well as the conditions of SERS spectral measurements were optimized for selective and sensitive detection of a biomedically important, amphiphilic bile pigment bilirubin (BR) in alkaline aqueous solutions and in its solutions in a selected water miscible solvent, namely dimethylsulfoxide (DMSO) and/or water-immiscible solvent, namely CH2Cl2. Ag nanosponges assembled by using HCl as a pre-aggregation agent were found to be the optimal SERS-active systems for a reliable detection of BR in all the above-mentioned solvents. In all cases, the protonated form of adsorbed BR has been detected upon BR incorporation into Ag nanosponges, and its marker bands have been established by SERS spectral probing at excitation wavelengths in the 445-780 nm range. The sensitivity of SERS spectral detection was evaluated in terms of the concentration values of SERS spectral detection limits (SERS LODs) of BR incorporated into Ag nanosponges. In particular, the SERS LOD for BR incorporated from its alkaline aqueous solution is 1 x 10-8 M (at 532 nm excitation), for BR incorporated from its solution in DMSO, its value is also 1 x 10-8 M (at 532 and/or 633 nm excitations), and for incorporation from the solution of BR...
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Antioxidant and antiinflammatory effects of bilirubin.
Valášková, Petra ; Muchová, Lucie (advisor) ; Martínková, Markéta (referee) ; Dvořák, Karel (referee)
For a long time, bilirubin (BR) has been considered a waste molecule with potential toxic effects especially on the central nervous system. Later, it was found that BR exhibited cytoprotective effects and mildly elevated BR levels showed antioxidant, anti-inflammatory and immunomodulatory properties, however, exact mechanisms of the anti-inflammatory actions of BR have not been fully understood yet. The main aim of this study was to assess the protective effects of BR using experimental in vivo and in vitro models in relation to inflammation and oxidative stress. Partial goal was to establish validated analytical method for determination of BR and lumirubin. Gunn and heterozygous rats were treated with lipopolysaccharide (LPS, 6 mg/kg, IP) or vehicle (saline). After 12 hours, blood and organs were collected for analyses of inflammatory and hepatic injury markers. Primary rat hepatocytes were treated with BR and TNF-α, HepG2 and SH-SY5Y cell lines were treated with BR and chenodeoxycholic acid. LPS-treated Gunn rats had a significantly decreased inflammatory response and hepatic injury compared to LPS- treated normobilirubinemic controls. We found different profile of leukocytes subsets and decreased systemic mRNA expressions and concentrations of IL-6, TNF-α, IL-1β and IL-10 in Gunn rats. Hepatic mRNA...
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Induction of heme oxygenase and biological role of its metabolic products.
Šuk, Jakub ; Muchová, Lucie (advisor) ; Jirsa, Milan (referee) ; Neužil, Jiří (referee)
Heme oxygenase (HMOX) catalyzes first and rate-limiting step in heme degradation. By its action, carbon monoxide (CO), ferrous iron and biliverdin which is subsequently reduced to bilirubin are produced. Before discovery of HMOX reaction mechanism, CO was considered only a toxic waste product without any significant importance for human organism. Bilirubin, marker of liver dysfunction, has been also exposed to similar perception. But results from past decades show that HMOX and its metabolic products play an important role in number of physiological as well as defense against pathophysiological processes. The aim of this thesis was to clarify the role of HMOX and its metabolic products, presumably CO and bilirubin, in vivo and in vitro. We focused on the role of CO in a rat model of lipopolysaccharide-induced cholestasis. We were first to describe tissue distribution and pharmacokinetics of inhaled CO in this animal model and found out that CO inhalation is associated with anti-inflammatory and hepatoprotective effects. In a rat model of ethinylestradiol-induced cholestasis, we demonstrated the anticholestatic effect of HMOX. The induction of HMOX by its substrate heme increased the expression of liver transporters thereby increasing bile flow and simultaneously facilitated effective clearance of...
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Genetic diseases of the liver
Šepsová, Marika ; Doleželová, Eva (advisor) ; Tripská, Katarína (referee)
Author: Marika Šepsová Title: Genetic diseases of the liver Form: Bachelor Thesis University: Charles University, Faculty of Pharmacy in Hradec Králové Degree: Medical Laboratory Technician Liver function may be affected by various factors including genetic diseases. The aim of this bachelor thesis is to collect and summarize current information about genetic diseases of the liver. Diseases with the highest incidence in the population are Dubin-Johnson syndrome, Rotor syndrome, Crigler-Najjar syndrome and Gilbert's syndrome. They are known as inherited hyperbilirubinemia characterized by an impairment in bilirubin metabolism. These genetic diseases are very rare with exception of Gilbert's syndrome. However, despite low prevalence and incidence it is necessary not to prolong their diagnosis. Most of them do not have any complications and do not require any treatment. The exception is Crigler-Najjar's syndrome, as untreated can have fatal consequences. Wilson's disease and hereditary hemochromatosis are inherited disorders of metal metabolism. Wilson's disease is a rare disease caused by an impairment in copper metabolism. Inherited hemochromatosis causes excessive iron deposition in the liver and other organs. Alpha-1 antitrypsin deficiency is characterized by impaired alpha-1 antitrypsin protein...
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Effects of heme arginate in HIV-1 acute infection and in latency reversal
Prakash, Shankaran ; Mělková, Zora (advisor) ; Hirsch, Ivan (referee) ; Hejnar, Jiří (referee)
The available antiretroviral compounds can effectively suppress the replication of HIV-1 and block the disease progression. However it is impossible to eradicate the virus from the organism as the HIV-1 integrated in the genome is not affected by the existing anti-HIV-1 drugs. Therefore, new latency reversing agents are being actively developed as part of "shock and kill" therapy to reactivate the provirus and clear the reservoir. Normosang (heme arginate; HA) is a human hemin- containing compound used to treat acute porphyria. Heme is physiologically catabolised by heme oxygenases to form iron (Fe2+ ), carbon monoxide (CO) and biliverdin that is further converted to bilirubin by biliverdin reductase. In this study, we have demonstrated that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA synergised with phorbol myristyl acetate (PMA) and reactivated the HIV-1 provirus in ACH-2 cells and the HIV-1 "mini-virus" in Jurkat cell clones A2 and H12. HIV-1 ''mini-virus'' was reactivated also by HA-alone. Further, we have studied the effects of heme degradation products on latent HIV-1 reactivation when added individually. We employed addition of ascorbate to generate Fe2+ , resulting in an increased...
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Application of Mass Spectrometry for Analysis of Biologically Active and Clinically Significant Compounds.
Štícha, Martin ; Jelínek, Ivan (advisor) ; Smrček, Stanislav (referee) ; Tůma, Petr (referee)
- 8 - ABSTRACT (EN) The thesis is submitted as a commented set of reviewed publications documenting and depicting the possibilities of mass spectrometry in the field of chemical, biological and pharmaceutical research; namely for the purposes of structure elucidation of selected organometallic complexes, analyses of drugs and their metabolites, monitoring of important biological markers. In course of experimental work, the following objectives were studied and solved: Proposal and realization of micro-scale preparation of selected rhenium complexes with aromatic ligands, utilizing tetrabutyammonium tetrachlorooxorhenate as a starting material; preparation and structure characterization of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3- dihydroxynaphtalene as ligands by means of ESI/MS, APPI/MS and LDI-MS; ESI/MS and UV/Vis study of kinetic behavior of complexes arising from the reaction of tetrabutylamonnium tetrachlooxorhenate with pyrogallol and catechol as ligands. Special aim was devoted to the study of subsequent chemical transformation of primarily formed Re(V) complexes; structure characterization of selected ferrocene complexes with copper, gold and silver by means of ESI/MS. Proposal of methodology of structure characterization and quantification of the...
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The biological role of the metabolic products of haem and bilirubin.
Jašprová, Jana ; Vítek, Libor (advisor) ; Farghali, Hassan (referee) ; Mičuda, Stanislav (referee)
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...
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Antiproliferative effects of heme catabolic pathway's products
Koníčková, Renata ; Vítek, Libor (advisor) ; Haluzík, Martin (referee) ; Farghali, Hassan (referee)
Presented work is focused on heme metabolism with the main interest in bile pigments. Recent data indicate that bilirubin is not only a waste product of the heme catabolic pathway, but also emphasize its important biological impacts, including possible antiproliferative effects. Until today metabolism of bilirubin has not been completely elucidated, which has prevented detailed evaluation of its potential anticancer action. The aim of this study was to clarify some aspects of heme catabolism with respect for antiproliferative properties of its products. Based on the fact that bilirubin potently affects carcinogenesis of the intestine, we initially investigated not properly known bilirubin metabolism by intestinal bacteria. We studied bilirubin neurotoxic effects in hyperbilirubinemic Gunn rats - its distribution in the brain tissue and its degradation during pathological conditions, such as severe newborn jaundice or Crigler-Najjar syndrome. Possible approaches to improve the treatment of severe unconjugated hyperbilirubinemias, combination of the phototherapy and human albumin administration were also investigated. The main reason of these studies was the fact that mechanisms of neurotoxic effects of bilirubin are predominantly identical with those, by which bilirubin inhibits cancer cells growth....
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