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Reporter gene studies for nanoparticle mediated DNA and siRNA delivery.
Kovářová, Barbora ; Jirkovská, Anna (advisor) ; Hofman, Jakub (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...
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IN VITRO assays for investigating nucleic acid delivery.
Mihaličoková, Dajana ; Jirkovská, Anna (advisor) ; Matoušková, Petra (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Dajana Mihaličoková Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ.Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: In vitro assays for investigating nucleic acid assay Keywords: transfection, splice correction, BCA assay, polyplexes One of the most important tasks of biochemical research is to find out the right way how to cure cancer, genetic disorders and other illnesses which are still not curable. Towards this, gene therapy is emerging as a potential treatment owing to its ability to deliver genetic material inside the cell. Reporer gene based transfection process can be used to study gene expression. Transfection is mediated by vectors, either of viral or non-viral origin. Non-viral vectors offer several advantages over the viral counterparts like easier to synthesize, relatively cheap and the most important is their non-immunogenicity. Cationic polymers based on polyethylenimine form complexes with plasmid DNA reffered to as...
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Electrophysiological characterization of Kir2.1 membrane channel
Měsíčková, Klára ; Chmelíková, Larisa (referee) ; Svoboda, Ondřej (advisor)
The topic of this thesis is electrophysiological characterization of Kir2.1 membrane channel. Inward rectifier potassium channel Kir2.1 is located in muscular, heart and nerve cells and its dysfunction causes various diseases. Practical part of this stage is focused on cultivation of the HEK293T cell line that is used to transfection of the plasmid Kir2.1 and subsequent measurement of the ionic current through the electrophysiological method patch-clamp in whole-cell mode.
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Dipeptidyl peptidase-IV and Fibroblast activation protein in gliomagenesis.
Trylčová, Jana ; Šedo, Aleksi (advisor) ; Mandys, Václav (referee) ; Mareš, Vladislav (referee)
"Dipeptidyl peptidase-IV Activity and/or Structure Homologues"(DASH) represent a newly defined group of multifunctional molecules, typically bearing dipeptidyl peptidase-IV- like hydrolytic activity. Dipeptidyl peptidase-IV (DPP-IV) cleaves out X-Pro dipeptides from the N-terminus of peptides. Other molecules carrying similar enzyme activity, such as Fibroblast activation protein (FAP), DPP-II, DPP8 and DPP9 or even DPP-IV structure-like but hydrolytically inactive molecules (DPP6 and DPP10) also belong to this group. Recent knowledge suggest a substantial role of DASH in cancer pathogenesis. The aim of this study is a preparation of a biological model and its use for understanding the mechanisms of interaction(s) between transformed glial cells and stroma in the processes of origin and development of tumors derived from neuroectoderm. Stable transfected human glioblastoma cell lines with inducible gene expression of DPP-IV, Fibroblast activation protein and their enzymatically inactive mutated forms, were prepared within the project. Prepared cell lines are used as a tool for studying not only the "autocrine" importance of DPP-IV and FAP for the expressing cells in in-vitro, but also for their potential "paracrine" effect(s) within the tumor microenvironment after homotopic implantation into the...
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Reporter gene studies for nanoparticle mediated DNA and siRNA delivery.
Kovářová, Barbora ; Jirkovská, Anna (advisor) ; Hofman, Jakub (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biochemical Sciences University of Vienna, Faculty center for Pharmacy, Department of Pharmaceutical Chemistry, Laboratory of MacroMolecular Cancer Therapeutics Candidate: Barbora Kovářová Supervisor (Charles University): PharmDr. Anna Jirkovská, Ph.D. Supervisor (University of Vienna): Univ. Prof. Dipl. Ing. Dr. Manfred Ogris Co-supervisor (University of Vienna): Dr. Haider Sami, Ph.D. Title of diploma thesis: Reporter gene studies for nanoparticle mediated DNA and siRNA delivery Keywords: transfection, plasmid DNA, siRNA, nanoparticles Gene therapy is a promising field offering potential in several currently incurable diseases. Gene therapy is mediated by modulation of gene expression in specific cells by delivering exogenous nucleic acids. One of current tasks of nucleic acid delivery is exploring several synthetic vectors which would have a potential to overcome the disadvantages of commonly used viral vectors. The present study focused on different types of polyethyleneimine-based nanoparticles for plasmid DNA (pDNA) and small interfering RNA (siRNA) delivery. Integration of imaging contrast agents with gene delivery vehicles is advantageous for tracking the gene delivery process both in vivo and in vitro. Gadolinium...
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Preparation and characterization of diamond-based nanocarriers for transfection of siRNA
Majer, Jan ; Cígler, Petr (advisor) ; Fišer, Radovan (referee)
Although nanodiamonds were discovered and produced tens of years ago, they have been utilized in medical and biological fields just recently, particularly in drug and gene delivery into a cell and in bioimaging methods. Nanodiamonds can be modified with specific positively charged moieties for complexation with negatively charged nucleic acids. These complexes afterwards overcome extracellular and intracellular barriers and transport the nucleic acid either into cytosol or into the nucleus. Owing to fluorescence centres nitrogen- vacancy, which can be formed in the nanodiamonds, nanodiamonds exhibit excelling optical properties, as they emit stable fluorescence without "photoblinking" or "photobleaching". This thesis reviews properties, synthesis and modifications of nanodiamonds and other selected nanoparticles and their in vitro applications. This thesis also compares their cytotoxicity and gene knockdown efficiency.
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Preparation and characterization of synthetic mRNA coding for pancreatic transcription factors
Loukotová, Šárka ; Hodek, Petr (advisor) ; Jirák, Daniel (referee)
Diabetes mellitus type I is severe autoimmune disease which is caused by destruction of insulin-producing β-cells in pancreas. Diabetic patients are dependent on external usage of insulin during their whole life. Nowadays the only treatment of diabetes type I is transplantation of entire pancreas or isolated Langerhans islets. Due to the fact that this kind of treatment is very demanding and limited availability of suitable donors, the researchers are intensively working on development of new alternative ways how to produce the insulin-producing cells. One of the possible approaches on producing insulin-positive cells is transdifferentiation of pancreatic exocrine cells via transcription factors. In this diploma thesis, the transdifferentiation of exocrine cells AR42J was carried out with in vitro synthesized mRNA encoding transcription factors Pdx1, Ngn3 and MafA. The primary mRNA structure was optimized in order to prepare highly stable mRNA which is correctly translated into the protein. The main stabilizing elements in mRNA structure include 3' and 5' untranslated region derived from highly stable β-globin mRNA. In order to verify the function of synthetic mRNA the immunofluorescence staining of transcription factors has been investigated. Synthetic mRNAs encoding transcription factors Pdx1,...
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Preparation of Xenopus tropicalis transgenic testicular stem cell culture.
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Drobná Krejčí, Eliška (referee)
Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...
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