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Biocompatible protein cages for encapsulation and internatization of small interfering RNA
Mokrý, Michal ; Balvan, Jan (referee) ; Heger, Zbyněk (advisor)
This thesis is focused on creation of apoferritin nanocarrier with encapsulated small interfering RNA marked with fluorescent dye. Main objectives are optimization of pH and amount of siRNA encapsulated into apoferritin cavity and physicochemical characteristics of created nanocarrier. First part deals with theoretical knowledge necessary for understanding concept of this thesis. Second part describes used methods and evaluated results. Created apoferritin nanocarriers were optimal in size with great hemocompatibility, but long-term stability didn’t meet our expectations.
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Vliv povrchových modifikací lipidových nanočástic na jejich vlastnosti
Mašková, Vendula
The aim of the thesis was to study the effect of surface modifications of liposomal nanoparticles on their physicochemical properties and to evaluate their uptake by cells in vitro. For this purpose, two types of liposomal nanoparticles (LNPs) that differed in their sensitivity to pH changes, i.e. pH-sensitive LNPs containing the lipid CPA (cholesteryl-PEG350-aminoxylipid) in their composition, were prepared for comparison, to which PEG (polyethylene glycol propionaldehyde) was attached by an oxime bond, and pH-insensitive LNPs containing DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol). The siRNAs were encapsulated into these nanoparticles and the storage stability, pH sensitivity, cleavage of pH-sensitive linkage, protein corona formation and their uptake by HepG2 cells were studied. The pH sensitivity of the linkages between PEG and CPA, or between PEG and DSPE, was verified using MALDI-TOF mass spectrometry at decreasing pH. Nanoparticle size, PDI and zeta potential were also monitored during the pH sensitivity study. According to the results, pH insensitive LNPs indeed appeared to be inert to pH change and pH sensitive LNPs showed changes in parameters at pH 6.0. Furthermore, the stability of both types of LNPs during long-term storage under different temperature conditions was assessed based on the change in size and PDI. The prepared LNPs appeared to be size uniform and stable throughout the experiment even at different temperatures. The exceptions were the fluorescently labelled LNPs, which exhibited poorer stability within the parameters studied. Finally, the effect of surface modifications on the reduction of protein corona formation was confirmed. The prepared LNPs also efficiently entered the cells, and within 24 hours there was a release of siRNA into the cytoplasm from pH-sensitive LNPs and a probable accumulation of siRNA in endosomes in the case of pH-insensitive LNPs. Based on the results of this work, it can be concluded that the prepared pH-sensitive LNPs exhibit desirable behavior in biological systems, and thus have potential as a suitable carrier for siRNA transport in biological applications.
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AGO-hook domains in RNA-directed DNA methylation in Arabidopsis thaliana
Teznerová, Kateřina ; Čermák, Vojtěch (advisor) ; Moravec, Tomáš (referee)
RNA-directed DNA methylation (RdDM) is an important pathway that regulates gene expression by inducing DNA methylation and is involved in regulation of gene expression and defence against invading DNA elements (especially transposons). Argonaut (AGO) proteins with small RNAs (sRNAs) that have sequence complementarity to the target DNA play a key role in the RdDM pathway. Domains called AGO-hooks are able to interact with Argonaut proteins. In plants, two proteins with AGO-hook domains are involved in the RdDM pathway: NRPE1 and SPT5L. Recently, a third protein, SPT6L, has been discovered at the investigator's site to be part of the Pol V complex (as well as the two proteins mentioned above). The role of SPT6L has not yet been described, but we hypothesize that it also plays a role in the RdDM pathway. This work focuses on the study of all three AGO-hook domains in Pol V complex and their involve in the RdDM pathway in Arabidopsis thaliana, from the preparation of mutants lacking different combinations of these AGO-hook domains to the study of their role and substitution in DNA methylation at different loci. Key words AGO-hook, Arabidopsis thaliana, NRPE1, SPT5L, SPT6L, siRNA, epigenetic chromatin labelling, Argonaut protein
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The role of small RNAs in transgenerational plant stress memory
Jaklová, Veronika ; Marková, Hana (advisor) ; Lipavská, Helena (referee)
Plants are constantly affected by various abiotic and biotic stresses, which cause a whole range of reactions.The resultcanbe increasedplantresistance tovariousstressfactorssuchasherbivoryattack or lack of water. Additionally, this resistance can also be passed on to subsequent generations through epigeneticmechanisms.Small RNAsservingas signaling moleculesof the plant's rapid response tostress can play a large part in the formationof intergenerationalandmultigenerationalstressmemory.MiRNAs are mainly regulators of gene expression,throughtheirinhibitory and degradative activitiesthey control the transcription of genes and the translation of a large number of proteins. SiRNAs could participate in the transfer of transcriptional memory through the mechanism of RNA-directed DNA methylation (RdDM). DNA methylation and histone modifications together act as chromatin marks that can be epigeneticallytransferredtosubsequentgenerations.Basedonthis,plants derivedfromstressedparent plants show large changes in gene expression compared to plants with non-stressed parents. These changesthenpersistfor varyinglengthsof time,dependingon whethergene expressionisagain induced bythe stressfactoror not.Interestinunderstandingthe mechanismsof transgenerationalstressmemory has recently grown considerably, and this...
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Optimalizace přípravy lipozomálních nosičů pro cílený transport terapeutických nukleových kyselin
Maráková, Ester
This bachelor thesis titled 'Optimization of the preparation of liposomal carriers for targeted transport of therapeutic nucleic acids' deals with the use of liposomal nanocarriers and the mechanism of RNA interference by means of siRNA in targeted therapy. The aim of this work was to optimize the preparation of complex of liposomes, polyethylene glycol and siRNA for further research, and to write an expert treatise of the issue. In the experimental part of this work, cationic liposomes were prepared from a mixture of lipids in a molar ratio of 20:50:10:20 (DODAG, DOPE, cholesterol, CPA·HCl), into which a control siRNA was encapsulated after their extrusion. Further, two types of polyethylene glycol, PEG2000 and PEG2120, were attached to the liposomal surface. The experimental part of this work consisted of three main experiments: monitoring the effect of encapsulated siRNA amount, monitoring the effect of PEGylation rate and a stability study of liposomes stored for 3 months. At the end of this work, selected samples of liposomes were imaged by scanning electron microscope. In each experiment, size, PDI and ζ potential were measured using dynamic light scattering. In the experiment, where the effect of volume of encapsulated siRNA was studied, we observed a slight increase in size with increasing amount of encapsulated siRNA. Based on results in the second-mentioned experiment, liposomes with the PEGylation rate of 5% containing PEG2000 and the PEGylation rate of 2% containing PEG2120 were selected for the stability study as well as phosphate-buffered saline (PBS) (the effect of PEGylation rate was also compared with samples prepared in 20 mM HEPES + 135 mM NaCl buffer). In a long-term storage study, we were able to demonstrate the potential of liposomes to be used as stable carriers for therapeutics. However, given that the structures of these complexes appeared to be disintegrated after 28 days, further optimization steps, like use of fast protein liquid chromatography (FPLC) or use of lipids with PEG already incorporated in their structure are needed in order to reduce the error rate.
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Improvement of quality, function and transplantation outcomes of pancreatic islets using RNA interference
Kosinová, Lucie ; Kříž, Jan (advisor) ; Štechová, Kateřina (referee) ; Petrák, Ondřej (referee)
Transplantation of insulin producing tissue is the only therapeutic method so far allowing type 1 diabetic patients to achieve long-term independence of insulin administration. Transplantation of isolated pancreatic islets (PI) into liver represents a safer but less effective alternative to whole-organ transplantation. This is caused by a significant loss of transplanted islets within a short time after transplantation due to the instant blood-mediated inflammatory reaction (IBMIR). This reaction is triggered by molecules of tissue factor, abundantly expressed on the surface of PI cells. Tissue factor activates directly the coagulation pathway leading to platelet aggregation, complement activation, and infiltration of islet graft by leukocytes which results in a destruction of up to 60 % of transplanted tissue, and a formation of ischemic areas of downstream lying liver tissue. Tissue factor also stimulates angiogenesis which makes it necessary for revascularization of PI after transplantation. Inhibition of tissue factor gene in isolated PI using RNA interference provides an opportunity for short-term suppression of tissue factor expression, leading to protection of PI in the early post-transplantation period without hindering the connection of capillaries to the recipient vascular system later...
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RNA directed DNA methylation in Arabidopsis thaliana
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Moravec, Tomáš (referee)
The differential transcriptional activity of the genome is provided by epigenetic modifications, which include DNA methylation, alteration of histone N-terminal amino acids and changes in histone variants. RNA interference is a regulatory process, in which transcriptional or post-transcriptional silencing of exogenous or endogenous sequences is mediated by the action of small RNAs derived from these sequences. The 24-nucleotide siRNAs, forming a fraction of small RNAs, direct de novo DNA methylation and participate in the maintenance of DNA methylation (RNA-directed DNA methylation; RdDM), which facilitates transcriptional silencing of heterochromatin and transposable elements representing a large part of plant genomes. The presence of two RNA polymerases involved in this pathway is characteristic for flowering plants, which were discovered for the first time in the genome of Arabidopsis thaliana, which has also become the main plant model for the study of RdDM. Polymerase IV transcribes siRNA precursors; siRNAs are subsequently associated with AGO4 proteins and guide methylation enzymes to the target sequences via complementarity with polymerase V transcripts.
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RNA interference in plants
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Kulich, Ivan (referee)
The process of RNA interference allows cells to regulate functions of their genes. This process is usually initiated by the presence of double-stranded RNA within a cell. Such double-stranded RNA is diced by a specific protein called Dicer into duplexes of small RNAs, usually 20-25 nucleotides long. Single-stranded small RNAs, released from the duplexes, are the heart of RNA interference and they can be categorize into several groups according to their biogenesis. There are two groups of small RNAs in plants: miRNA and siRNA. Small RNAs can associate with a protein called Argonaut and guide it to the target molecule on the bases of sequence complementarity. The Argonaut-small RNA complex can act on itself or it can interact with other proteins in a wide spectrum of processes. The complex can slice the target mRNA (which can be handled by the sole Argonaut and small RNA), it can suppress translation or it can direct chromatin modifications. The phenomena of RNA interference can be found in almost all Eukaryotes where it can serve many functions, for example it can control cell differentiation, participate in stress responses, direct changes in chromatin and defend the organism against viruses. A diverse set of operating modes of RNA interference can be found in plants, which we are only at the...
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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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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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