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The use of RNA interference for the modification of DNA topoisomerase II levels in cancer cells and its influence on the antineoplastic effect of anthracyclines.
Klieber, Robin ; Jirkovská, Anna (advisor) ; Lenčová, Olga (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Bc. Robin Klieber Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: The use of RNA interference for the modification of DNA topoisomerase II levels in cancer cells and its influence on the antineoplastic effect of anthracyclines. Topoisomerase II (TOP II) is an enzyme that alters the topological state of the DNA double helix during physiological processes through the formation of transient DNA double strand breaks. Two TOP II isoforms are known: TOP IIα is essential for proper separation of chromosomes in mitotic cells, whereas TOP IIβ is primarily associated with gene transcription. Anthracycline antibiotics (ANT) belong to the group of topoisomerase poisons that stabilize the covalent complex of TOP II and DNA. This prevents the religation of the DNA double strand breaks and thus causes irreversible DNA damage leading to programmed cell death. Although ANTs are frequently administered in various antineoplastic protocols (hematooncological malignancies, hormone-dependent tumors and others), the therapy still possess a high risk of irreversible cardiotoxicity. The mechanism of cardiotoxicity remains unraveled. However, it has been previously discussed that TOP IIβ inhibition could play a...
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Studium dráhy pro syntézu mastných kyselin u klíštěte \kur{Ixodes ricinus}
ZEMANOVÁ, Aneta
Aim of this work was to study fatty acid synthesis pathway in the tick Ixodes ricinus. I have characterized tissues and stage expression profiles of three genes; acc (acetyl coenzyme A carboxylase), fas (fatty acid synthetase) and fabp (fatty acid binding protein) by qRT-PCR. Then expression of these genes was silenced by RNA interference and phenotypes observed during and after feeding in nymphs and adults. The pathway seems to be crutial for the ticks.
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Analýza exprese inhibitorů serinových proteáz v klíštěti \kur{Ixodes ricinus} pomocí kvantitativní real-time PCR
HAUSEROVÁ, Simona
Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.Tick saliva contains a lot of biological active substances helping them to succesfully complete their feeding which is neccesary for their next development. Both proteinaceous and non-proteinaceous molecules including protease inhibitors are present in tick saliva. The biggest family of these proteases are serpins. Serpins are involved in many biological processes as blood coagulation, fibrinolysis, apoptosis or inflammation. The aim of this diploma work was to determine expression profiles of 10 serpins from nymphs of Ixodes ricinus fed for different times using quantitative real time PCR. For chosen genes (IRS 10, IRS 20) dsRNA for silencing of the gene was prepared and using RNA interference the role of these genes during tick (I. ricinus nymphs) feeding and transmission of Borrelia afzelii spirochetes, a vector of Lyme borreliosis, was evaluated.
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Nanofiber dressing consisting of antisense rna-functionalized nanodiamonds for therapy of non-healing wounds in diabetic individuals
Neuhoferová, Eva ; Petráková, V. ; Vocetková, K. ; Kindermann, Marek ; Křivohlavá, Romana ; Benson, Veronika
Non-healing wounds are serious complication in diabetic patients and represent an attractive challenge for development of suitable carrier system possessing constant and localized release of therapeutic biomolecule into the wound without any undesired side effects. Given the fact that these non-healing wounds are result of impaired balance in metalloproteinases synthesized by immune cells residing the wounds, gene therapy offering knock down of such enzymes is of great interest. \nHere we challenged a development of functional and biocompatible wound dressing enabling controlled release of trackable carrier loaded with therapeutic siRNA. Our dressing consists of scaffold from degradable polymer nanofibers enriched with fluorescent nanodiamond particles (FND). We have previously shown the nanodiamond particles are great carriers for antisense RNAs. Their advantages represent high biocompatibility, stable luminescence giving us the possibility to track the carrier system in the wound, and effective release of antisense RNA in the wound. Embedding of nanodiamond-siRNA systems into nanofiber scaffold enables continuous release of siRNA and maintaining the stable siRNA concentration in the wound site resulting in a promotion of wound healing. \nWe developed FND-siRNA complexes specific to MMP-9 that efficiently inhibit the expression of target MMP-9 mRNA. The complexes were embedded into core/shell nanofibers from PVA and PCL, visualized by confocal microscopy, and characterized by electron microscopy. Real-time PCR was used to assess the silencing effect of siRNA that has been delivered to target murine fibroblasts by FND released from nanofiber dressing. Nanofiber system with embedded FNDs was applied on wounds in diabetic animal models to evaluate its suitability regarding short and long term toxicity, efficacy, and handling in vivo. \n
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Induction of endogenous RNAi in mammalian cells
Demeter, Tomáš ; Svoboda, Petr (advisor) ; Vopálenský, Václav (referee)
Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...
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Inducible RNAi against essential genes of nitrogen metabolism as a tool for control of GM plants
Kobercová, Eliška ; Fischer, Lukáš (advisor) ; Tylová, Edita (referee)
Uncontrolled spreading of genetically modified (GM) plants is one of the main concerns about their cultivation. Inducible RNA interference against an essential gene could be a tool for control of GM plants. After spraying with a chemical inducer, the essential gene will be silenced so the treated GM plant will die. For testing this strategy we chose two key enzymes of nitrogen metabolism, glutamate synthase (GOGAT) and glutamine synthetase (GS). GS processes ammonium ions into glutamine, then GOGAT transfers the amide group from glutamine to 2-oxoglutarate to form two glutamates. GS/GOGAT cycle is the main pathway for assimilation of ammonium ions, which could be toxic to plants in a higher concentration. Disruption of ammonium assimilation during photorespiration causes a strong inhibition of photosynthesis. The aim of this work was to describe the effects of silencing GOGAT and GS genes in Arabidopsis thaliana. To induce silencing, RNAi hairpin constructs under a control of constitutive or estradiol-inducible promoter were prepared. In selected independent transformants with the inducible hairpin against GOGAT, chlorosis and reduced growth were observed after the estradiol treatment in in vitro conditions. However, the spraying with estradiol was tricky, at the whole plant level, the induction of...
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Use of RNAi and CRISPR systems in genetic modifications of parasitic protists
Kaiserová, Veronika ; Votýpka, Jan (advisor) ; Stojanovová, Darja (referee)
In organisms, RNA interference serves as a defence mechanism against foreign nucleic acids. RNAi has a negative effect on translation, via the binding of small non-coding molecules to the complementary region of mRNA, resulting in its degradation. CRISPR, a new method of genetic engineering, is based upon modulating genetic expression via creating double-stranded breaks in target DNA, aided by a ribonucleoprotein complex, consisting of the prokaryotic endonuclease Cas9 and sgRNA. Both of the aforementioned methods can be utilised in functional analysis of proteins and the characterisation of metabolic pathways in organisms of interest. This work summarises the current state of knowledge regarding RNAi and CRISPR and their use in genome editing of parasitic protists.
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Employing an RNA interference method (RNAi) to sudy oncogenic properties of Kaposi's sarcoma-associated herpesvirus (KSHV)
Riegerová, Petra ; Lubyová, Barbora (advisor) ; Hirsch, Ivan (referee) ; Pávová, Marcela (referee)
Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus that has been associated with all epidemiological forms of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV undergoes two phases of life cycle (latent and lytic replication). During latency, the viral genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed, namely LANA (latency- associated nuclear antigen), Kaposin, vFLIP (viral FLICE inhibitory protein), vCyclin, and vIRF3/LANA2 (viral interferon regulatory factor 3). These viral genes are responsible for regulation of host cell proliferation, prevention of apoptosis, facilitation of immune evasion, and maintenance of the extrachromosomal viral genome during cell divisions. vIRF3 is a multifunctional nuclear protein that is constitutively expressed in KSHV positive PEL cells and Castleman's disease tumors, which expression causes dramatic changes of critical host pathways that are involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. In our study, we have demonstrated and elucidated predicted mechanism, by which vIRF3 enhances transcription activity of c-Myc. Moreover, we have clarified the previously unappreciated...
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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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