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Viannia development in the vector
Hlaváčová, Jana ; Volf, Petr (advisor) ; Dostálová, Anna (referee)
Leishmania of the subgenus Viannia are protozoan parasites transmitted by phlebotomine sandflies (Diptera: Phlebotominae). They occur in tropical and subtropical areas in South America, where they cause cutaneous and mucocutaneous leishmaniasis. In this thesis, we studied developmental pattern of Viannia group and factors affecting its development within the sand fly gut. First, we investigated Leishmania braziliensis development within the Lutzomyia longipalpis digestive tract. Using GFP-labeled strain we demonstrated peripylar development: promastigotes escaped from the endoperitrophic space, colonized the hindgut and then migrated anteriorly. Four morphological forms were found within the Lu. longipalpis digestive tract: elongated nectomonads, short nectomonads, metacyclic promastigotes and paramastigotes. Furthermore, using the histological methods we demonstrated parasite attachment in pylorus region, while there were only free promastigotes in the midgut; neither form was found attached to the midgut epithelium. The next part was devoted to the effect of temperature on Viannia in Lu. longipalpis. We compared development of two closely related species L. peruviana and L. braziliensis at 20 řC and 26 řC. Leishmania braziliensis developed well in both temperatures tested, L. peruviana developed...
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The influence of RDR6 activity and mode of RNAi induction on dynamics and mechanism of silencing of the reporter GFP gene in tobacco cell line BY-2
Motylová, Šárka ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee)
RNA interference (RNAi) is a process mediated by small RNAs (sRNA), which is significantly involved in the regulation of gene expression in plants. Diverse RNAi pathways can be divided into two basic mechanisms, which are post-transcriptional and transcriptional gene silencing (PTGS and TGS). Production of sRNAs is dependent on the presence of a double-stranded RNA molecule (dsRNA), which is cleaved by one of DCL proteins to produce sRNAs usually of 21-24 nt in length. One strand of the sRNA is subsequently loaded onto AGO protein. During PTGS, the AGO-sRNA complex interacts with the target RNA based on its sequence complementarity to the sRNA and cleaves it or blocks its translation. In the case of TGS, AGO interacts with plant-specific RNA Pol V and its transcripts, which are again complementary to the sRNA. This interaction allows assembling of a protein complex facilitating DNA and histone methylation inhibiting RNA Pol II transcription. There are numerous ways the dsRNA can arise. A significant part of dsRNA cell production is dependent on synthesising the complementary strand of the dsRNA by RDR6 (RNA-dependent RNA polymerase 6). RDR6 is also involved in the process of the secondary sRNA formation. The significance of RDR6 during PTGS was examined using a GFP reporter gene either during...
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Dynamics and variability of induced transgene silencing in tobacco cell line BY-2
Čermák, Vojtěch ; Fischer, Lukáš (advisor) ; Pečinka, Aleš (referee) ; Lafon Placette, Clément (referee)
RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...
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MicroRNAs encoded by polyomaviruses.
Zachovalová, Veronika ; Bruštíková, Kateřina (advisor) ; Malík, Radek (referee)
MicroRNAs are small regulating molecules of RNA that are encoded by orgamism's genome. Biogenesis of microRNA takes place partly in the nucleus and partly in the cytoplasm. Result of this biogenesis is a 22 nt long microRNA molecule. They are able to silence the genes thanks to sequence- specific degradation of a target mRNA or thanks to the repression of translation of target, complementary mRNA. In mammalian cells the mechanism of translational repression is more common. During this mechanism the microRNA molecule is not entirely complementary to 3'UTR of its target mRNA. Polyomaviruses are small, non-enveloped dsDNA viruses with a circular genome and icosahedral capsid composed of VP1 protein pentamers. These viruses belong in a group called onkoviruses, which can transform infected cells and contribute to development of serious illnesses such as Merkell cell carcinoma. Their genome encodes regulating proteins called T antigens, structural capsid proteins and also microRNAs. My main focus in this thesis will be SV40, MPyV, MCPyV, BKPyV and JCPyV encoded microRNA molecules. Key words: polyomaviruses, small interfering RNA, microRNA, siRNA, RNA interference, mouse polyomavirus, BK virus, JC virus, SV40
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Study of RNAi mechanisms in tobacco BY-2 cell line and potato plants
Tyč, Dimitrij ; Fischer, Lukáš (advisor) ; Kovařík, Aleš (referee) ; Moravec, Tomáš (referee)
Knowledge of the processes of RNA interference, the regulation of gene expression by small RNAs (sRNAs), has grown at an unprecedented rate over the last 30 years. Some of the findings were literally revolutionary, as they revealed events that overturned many long-held notions. Many phenomena have been shown to be highly conserved and common to organisms of different species, but others are specific to certain lineages or have not yet been fully explored. There is also a lack of knowledge about the interconnection of numerous pathways - for example between silencing at the transcriptional (TGS, leading to the promoter methylation) and post-transcriptional levels (PTGS, affecting mRNA stability or translation). The present work summarizes the findings of two published and two unpublished works and attempts to describe some of the less known sites of RNA interference using various plant model organisms. Research on Solanum tuberosum transgenic lines has revealed the ability of 5-azacytidine to restore the expression of transcriptionally silenced transgenes at the whole plant level. De novo regeneration from leaves of such plants can lead to re-silencing of reactivated transgenes and thus serves as a selection method to exclude lines prone to spontaneous silencing. The nature of changes in the...
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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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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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