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The role of mitochondrial SCoAS substrate-level phosphorylation in the bloodstream form \kur{T. brucei}
HUSOVÁ, Michaela
Mitochondrial metabolism of Trypanosoma brucei is considered highly reduced because of its dysfunctional electron transport chain and tricarboxylic acid cycle. But previously published paper suggests significant mitochondrial ATP pool created by substrate phosphorylation via succinyl coenzyme A synthetase, which plays crucial role in T. brucei survival. This thesis is therefore focused on substrate phosphorylation and on influence of succinyl coenzyme A synthetase heterodimer RNAi on T. brucei cells.
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Study of adipokinetic hormone role in insects stressed by entomopathogenic nematodes
IBRAHIM, Emad Ahmed Sayed
In this thesis, the effect of infection elicited by entomopathogenic nematodes (EPNs) Steinernema carpocapsae on Pyrrhocoris apterus and Drosophila melanogaster models were evaluated, and a role of adipokinetic hormones (AKHs) during the infection was characterized. These were monitored by determination of mortality, and various biochemical and physiological characteristics such as AKH levels both in the central nervous system (CNS) and in hemolymph, AKH gene expression in CNS, level of anti-oxidative stress markers, general metabolism and level of nutrients in normal and genetically modified insects. At P. apterus the mortality tests revealed that application of AKH increases the efficacy of EPN treatment. This result was confirmed using the firebugs with AKH receptor deficiency. Further, the increase of AKH expression and AKH levels in CNS and hemolymph seemed to be coordinated after the nematode treatment. At the D. melanogaster model also, the effect of adenosine into the above-mentioned characteristics was included. For this, mutants in AKH (AHK1), adenosine receptor (AdoR1) genes, and in both these genes together (AHK1 AdoR1 double mutant) were employed. Altogether, the results confirmed the involvement of AKH, and partially also adenosine into the antistress defense reactions elicited by the nematobacterial infection. Finally, the last part of the study was focused on examination of the vitellogenin (Vg) role in the defense reaction in firebug body P. apterus affected by two entomopathogenic organisms, the nematode S. carpocapsae and the fungus Isaria fumosorosea. The results revealed that Vg proteins play an important role in the defense against both types of the infections and are also able to kill entomopathogenic bacteria Xenorhabdus nematophila, that are symbionts of S. carpocapsae and that increase toxicity of this nematode.
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Circadian clock genes in the circadian clock and photoperiodic timer in Pyrrhocoris apterus
CHODÁKOVÁ, Lenka
This thesis focuses on the circadian clock genes and their involvement in the photoperiodic time measurement in the linden bug, Pyrrhocoris apterus. Application of the molecular biology methods enabled us to propose the architecture of circadian clockwork. We also investigated the role of several previously undescribed players in the circadian clock. Furthermore, by using molecular biology methods we focused on the involvement of core circadian clock genes in the photoperiodism.
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Adipokinetic hormone activities in insect body infected by entomopathogenic nematode
IBRAHIM, Emad Ahmed Sayed
The role of adipokinetic hormone (AKH) in the firebug Pyrrhocoris apterus adults infected by the entomopathogenic nematode (EPN) Steinernema carpocapsae was examined in this study. It was found that co-application of EPN and AKH enhanced firebug mortality about 2.5 times within 24 h (from 20 to 51% in EPN vs. EPN + AKH treatments), and resulted in metabolism intensification, as carbon dioxide production in firebugs increased about 2.1 and 1.6 times compared to control- and EPN-treated insects, respectively. Accordingly, firebugs with reduced expression of AKH receptors showed a significantly lower mortality (by 1.6 to 2.9-folds), and lower general metabolism after EPN + AKH treatments. In addition, EPN application increased Akh gene expression in the corpora cardiaca (1.6 times), AKH level in the corpora cardiaca (1.3 times) and haemolymph (1.7 times), and lipid and carbohydrate amounts in the haemolymph. Thus, the outcomes of the present study demonstrate involvement of AKH into the antistress reaction elicited by the nematobacterial infection. The exact mechanism by which AKH acts is unknown, but results suggested that the increase of metabolism and nutrient amounts in haemolymph might play a role.
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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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The Design of siRNAs and the Influence of Modifications on Their Stability and Efficiency
Kuldanová, Kateřina ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
Design of small interfering RNA (siRNA) is a basic step to successful RNA interference (RNAi). However, it is not trivial to design a suitable siRNA duplex at all, because there are some problematic areas like stability of siRNA duplexes in the presence of nucleases, efficiency of target mRNA elimination, sequence-specific elimination of non-target mRNAs, cytotoxicity and immunogenicity for example, which can influence the aliveness of siRNA duplexes and consequently also their utility in medicine and other fields. The understanding of the principles of siRNA duplexes function during RNAi is a necessary step to solution of the obstacles on the way to efficient drugs. The mean of optimisation of described troublesome properties is an alteration of siRNA duplexes design by use of different modifications. Phosphodiester backbone, bases and also ribose can be modified. Some modifications beneficial for siRNA duplexes and consequently RNA interference are known in every one of these groups. This work presents existing pieces of knowledge about some of the most known modifications, just like phosphorothioate substitution, 2′-deoxy-2′-fluoronucleotides, 2′-O-methyl ribonucleotides and LNA nucleotides are, as well as about a higher number of less known modifications like boranophosphate substitution,...
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Variability of the IRES elements of the hepatatis C virus
Zeman, Jakub ; Vopálenský, Václav (advisor) ; Španielová, Hana (referee)
Hepatitis C virus (HCV) has an internal ribosomal binding site (IRES) located near the 5ʹ end of its genome. The HCV IRES is capable of direct binding to the 40S small ribosomal unit and eukaryotic initiation factor eIF3, and can initiate translation after the assembly of the whole 80S ribosome. Various molecular types can act as IRES inhibitors. Small molecule compounds seem to be the most promising agent for use in the clinic. The main objective of the thesis was to develop a system for searching for small molecule compound inhibitors of HCV IRES in a library of chemical compounds. Several variants of vector carrying bicistronic cassettes were prepared. After validating their functionality by transient transfection of mammalian cell cultures, mammalian stable cell lines were established. These stable cell lines will allow for automatization of the search for small molecule compound inhibitors of HCV IRES. Our second objective was to study the variability of HCV IRES sequences in patient samples. The samples were analysed by temperature gradient gel electrophoresis (TGGE). Select specimen were sequenced, cloned into a vector with bicistronic cassette and analysed by flow cytometry. In this was we evaluated the effect of specific mutations in the HCV IRES sequence on the level of IRES dependent...
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Size matters - siRNAs biogenesis and function in Arabidopsis
Přibylová, Adéla ; Fischer, Lukáš (advisor) ; Honys, David (referee)
RNA interference (RNAi) play a key role in various biological processes including regulation of gens and transposons, phylogenetic of part plant body, stress response, chromatin remodeling and antiviral mechanism. The ground of RNAi is short RNA molecules (small RNA, sRNA). In plants they are produced in range from 21 to 24 nucleotides (nt) and on the basis of being complementary they recognize target molecule of RNAi. It is possible to divide small RNA in two basic classes: microRNAs (miRNA) and small interfering RNAs (siRNA). To product and put small RNA into activate needs proteins from several gene family. DICER-LIKE (DCL) proteins create small RNAs from double-strand RNA precursors, which are often created by RNA dependent RNA polymerase (RDR) activity. With these small RNAs interact ARGONAUTE (AGO) proteins and together create RNA-Induced Silencing Complex (RISC). Those complexes play a key role in recognizing target molecule in active phase of RNAi. Structure and biogenesis of sRNAs has decisive influence on RISC complex and its next way in biogenesis. RNAi cause effect on post-transcriptional level (PTGS), as degradation of target molecule or repression of translation. And on transcriptional level (TGS) as sRNA intermediate histone and DNA methylation.
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RNA interference in plants
Čermák, Vojtěch ; Kulich, Ivan (referee) ; Fischer, Lukáš (advisor)
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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