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In vitro evaluation of novel Toll-like receptor ligands II.
Machalová, Vanda ; Trejtnar, František (advisor) ; Janočková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Vanda Machalová Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands II. The aim of this diploma thesis was to test the new substances at Toll-like receptors (TLR), namely TLR4 and TLR8 subtypes, as potential vaccine adjuvants. Adjuvants are required for new subunit vaccines to promote stimulation of a sufficient immune response. Stimulation of TLR receptors is safe and leads to the activation of both innate and adaptive immunity and to subsequent specific immune response. Testing was carried out on cell lines stable co-expressing the respective receptors, with colorimetric activity detection. In this work, both agonist and antagonist activity at TLR4 and TLR8 receptors was investigated and a half maximal effective concentration (EC50) of resiquimod was determined. Based on the results, the respective substances cannot be considered as potential adjuvants to the TLR8 receptor and DM014 exhibits the potential for agonist activity on the TLR4 receptor, whereas the substance DM015 can be used on further investigation of immunosuppression in autoimmune diseases and diseases associated with excessive activation of the...
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In vitro evaluation of novel Toll-like receptor ligands I.
Hudáková, Kristína ; Trejtnar, František (advisor) ; Soukup, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kristína Hudáková Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands I Vaccination against preventable infections prevents millions of deaths each year. Their immunity enhancing activity is strengthened by the presence of vaccine adjuvants. Development of vaccine adjuvants leads to improved safety profile and also can play a vital role in the research of new vaccines against pathogens against which the vaccines currently do not exist. The main aim of this diploma thesis was to verify the ability of rationally developed small molecule ligands to influence Toll-like receptors and thus their potential to be utilized as vaccine adjuvants. The assay was carried out using modified cell lines continually expressing the human TLR4 or TLR8 whose activation leads to production of secreted embryonic alkaline phosphatase. Ten analyzed substances labelled as DM 001 - DM 010 were examined for their agonistic and also antagonistic properties while interacting with the TLRs. Immunomodulatory activity of these tested samples was then determined by quantification of secreted alkaline phosphatase with the help of a colorimetric...
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Analysis of streptomycete isolates carrying a cyclizing aminolevulinate synthase gene
Rašmanová, Petra ; Petříčková, Kateřina (advisor) ; Felsberg, Jurgen (referee)
Streptomyces bacteria are well-known producents of many bioactive metabolites. Its secondary metabolism is a source of many important groups of active compounds that are recently investigated by means of many new methods based on bioinformatic analyses of genome data, modern LC-MS techniques, and metabolic modeling methods. This thesis originates from the genetic screening for a specific gene (als) for cyclizing 5-aminolevulinate synthase. Based on earlier studies, we consider this gene as a genetic tag of the producers of secondary metabolites containing the C5N unit (2-amino-3-hydroxycyklopent- 2-enon). Such metabolites include several groups with variable structures and biological activities, which include manumycins as well. Manumycins are small polyketides with a weak antibiotic aktivity, especially against gram-positive bacteria. However, its cancerostatic and anti-inflammatory effects are of greater importance. Streptomyces monomycini BCCO10 1552 and Streptomyces capoamus BCCO10 1636 strains were found positive for the presence of the als gene in the targeted genetic screening. By the als phylogeny, they cluster near the producents of manumycin compounds. This thesis aimed to determine whether these new natural isolates produce any compounds containing C5N unit, and to characterize them in...
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Colonization of human lung by non-pathogenic streptomycetes
Herbrík, Andrej ; Petříčková, Kateřina (advisor) ; Zikánová, Blanka (referee)
Streptomycetes, primary soil saprophytic microorganisms are at the center of interest in many research groups, mainly because of their ability to produce a wide range of biologically active substances useful in medicine, biotechnology and agriculture. The marginal, and little explored areas are the interactions that streptomycetes create with humans. Recent metagenomic studies have shown that streptomycetes colonize the skin, the respiratory and possibly the urogenital tract of humans. In addition to apparent pathogens such as S. somaliensis and S. sudanensis, the clinical impact of these streptomycetes on human health is unknown. For this reason, a unique collection of non-pathogenic streptomycetes isolated from human clinical specimens was developed. The isolates were collected by the National Reference Laboratory for Pathogenic Actinomycetes in Trutnov, Czech Republic. On the basis of pilot studies, an isolate labeled TR42 was selected from the Trutnov collection, showing a very broad spectrum of biological activities. This strain was isolated from sputum from a patient with unknown respiratory diagnosis. The TR42 strain exhibits considerable biotechnological potential and after following a thorough study, it could be a source of new biologically active substances with pharmaceutically...
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In vitro evaluation of novel Toll-like receptor ligands I.
Hudáková, Kristína ; Trejtnar, František (advisor) ; Soukup, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kristína Hudáková Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands I Vaccination against preventable infections prevents millions of deaths each year. Their immunity enhancing activity is strengthened by the presence of vaccine adjuvants. Development of vaccine adjuvants leads to improved safety profile and also can play a vital role in the research of new vaccines against pathogens against which the vaccines currently do not exist. The main aim of this diploma thesis was to verify the ability of rationally developed small molecule ligands to influence Toll-like receptors and thus their potential to be utilized as vaccine adjuvants. The assay was carried out using modified cell lines continually expressing the human TLR4 or TLR8 whose activation leads to production of secreted embryonic alkaline phosphatase. Ten analyzed substances labelled as DM 001 - DM 010 were examined for their agonistic and also antagonistic properties while interacting with the TLRs. Immunomodulatory activity of these tested samples was then determined by quantification of secreted alkaline phosphatase with the help of a colorimetric...
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In vitro evaluation of novel Toll-like receptor ligands III
Tamášiová, Linda ; Trejtnar, František (advisor) ; Pejchal, Jaroslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Linda Tamášiová Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands III Research on new ligands with potential immunomodulatory activity is a tool for the development of new immunological adjuvants for use in vaccines, or as separate immunomodulators in the treatment of various diseases. The aim of this thesis was to analyze the ability of new ligands to stimulate toll-like receptors 4 (TLR4) and to assess the potential for further use of these substances based on established immunomodulatory activity. The analysis was performed on modified TLR4-expressing cell lines whose activation was subsequently detected using a colorimetric-enzymatic reaction. Sixteen new substances were tested for TLR4 receptor activation in comparison with a standard agonist. The results showed a significant TLR4 agonistic activity in several of the test substances, suggesting that they are activating ligands of the receptor tested. However, due to their low solubility, some of these substances are not suitable candidates for further use and testing. Taking all parameters into consideration, among of all of the evaluated substances that...
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In vitro evaluation of novel Toll-like receptor ligands II.
Machalová, Vanda ; Trejtnar, František (advisor) ; Janočková, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Vanda Machalová Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands II. The aim of this diploma thesis was to test the new substances at Toll-like receptors (TLR), namely TLR4 and TLR8 subtypes, as potential vaccine adjuvants. Adjuvants are required for new subunit vaccines to promote stimulation of a sufficient immune response. Stimulation of TLR receptors is safe and leads to the activation of both innate and adaptive immunity and to subsequent specific immune response. Testing was carried out on cell lines stable co-expressing the respective receptors, with colorimetric activity detection. In this work, both agonist and antagonist activity at TLR4 and TLR8 receptors was investigated and a half maximal effective concentration (EC50) of resiquimod was determined. Based on the results, the respective substances cannot be considered as potential adjuvants to the TLR8 receptor and DM014 exhibits the potential for agonist activity on the TLR4 receptor, whereas the substance DM015 can be used on further investigation of immunosuppression in autoimmune diseases and diseases associated with excessive activation of the...
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In vitro evaluation of novel Toll-like receptor ligands I.
Hudáková, Kristína ; Trejtnar, František (advisor) ; Soukup, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kristína Hudáková Supervisor: doc. PharmDr. František Trejtnar, CSc. Title of diploma thesis: In vitro evaluation of novel Toll-like receptor ligands I Vaccination against preventable infections prevents millions of deaths each year. Their immunity enhancing activity is strengthened by the presence of vaccine adjuvants. Development of vaccine adjuvants leads to improved safety profile and also can play a vital role in the research of new vaccines against pathogens against which the vaccines currently do not exist. The main aim of this diploma thesis was to verify the ability of rationally developed small molecule ligands to influence Toll-like receptors and thus their potential to be utilized as vaccine adjuvants. The assay was carried out using modified cell lines continually expressing the human TLR4 or TLR8 whose activation leads to production of secreted embryonic alkaline phosphatase. Ten analyzed substances labelled as DM 001 - DM 010 were examined for their agonistic and also antagonistic properties while interacting with the TLRs. Immunomodulatory activity of these tested samples was then determined by quantification of secreted alkaline phosphatase with the help of a colorimetric...
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