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Inaktivace ICAM-1 genu metodou oligonukleotidů modifikovaných lysinem
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Gene silencing of ICAM-1 by lysine modified oligonucleotides
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Adverse reactions after childhood vaccination in the Czech Republic
Kocourková, Aneta ; Dáňová, Jana (advisor) ; Marešová, Vilma (referee) ; Částková, Jitka (referee)
1 ABSTRACT In the Czech Republic a complex study of adverse reactions after vaccination in children has not been presented yet. Even though reporting of serious adverse reactions to the State Institute for Drug Control (SUKL) is mandatory by law, it is seldom performed. Moreover, there is no analysis correlating adverse reactions that occurred at doctors` surgeries with actual reported adverse reactions to SUKL. The main focus of presented work was to analyze and map adverse reactions appearing after vaccinations from the children's vaccination schedule in the Czech Republic. The study was conducted in the form of retrospective-prospective questionnaires concentrating on adverse reactions, their occurrence, character, seriousness, duration and frequency. Furthermore, the questionnaires also focused on socio-demographic data such as gender, age, residency, parents' education, birth weight. Also, the refusal of the compulsory vaccination was assessed. Occurrence of adverse reactions after vaccination in the Czech Republic was analyzed by questionnaires in pediatricians' surgeries (active surveillance). Obtained data were correlated with data available at SUKL (passive surveillance). The study data showed higher number of collected adverse reactions that number of adverse reactions reported to national program...
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Adverse reactions after childhood vaccination in the Czech Republic
Kocourková, Aneta ; Dáňová, Jana (advisor) ; Marešová, Vilma (referee) ; Částková, Jitka (referee)
1 ABSTRACT In the Czech Republic a complex study of adverse reactions after vaccination in children has not been presented yet. Even though reporting of serious adverse reactions to the State Institute for Drug Control (SUKL) is mandatory by law, it is seldom performed. Moreover, there is no analysis correlating adverse reactions that occurred at doctors` surgeries with actual reported adverse reactions to SUKL. The main focus of presented work was to analyze and map adverse reactions appearing after vaccinations from the children's vaccination schedule in the Czech Republic. The study was conducted in the form of retrospective-prospective questionnaires concentrating on adverse reactions, their occurrence, character, seriousness, duration and frequency. Furthermore, the questionnaires also focused on socio-demographic data such as gender, age, residency, parents' education, birth weight. Also, the refusal of the compulsory vaccination was assessed. Occurrence of adverse reactions after vaccination in the Czech Republic was analyzed by questionnaires in pediatricians' surgeries (active surveillance). Obtained data were correlated with data available at SUKL (passive surveillance). The study data showed higher number of collected adverse reactions that number of adverse reactions reported to national program...
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Inaktivace ICAM-1 genu metodou oligonukleotidů modifikovaných lysinem
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Gene silencing of ICAM-1 by lysine modified oligonucleotides
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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