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Efekt ADGF-A RNAi na imunitní odpověď \kur{Drosophily melanogaster} po infekci parazitickou vosou
ŠNEBERGEROVÁ, Pavla
In previous studies the extracellular adenosine has been proved as an important signaling molecule controlling dynamics of the immune response in Drosophila melanogaster. In mammals the adenosine signaling and control is more complex process. Since there is a certain genetic and evolutionary conservation between mammals and invertebrates, Drosophila represents simple system how to study such a complex regulation in vivo. Mutations of adenosine receptor in all cells as well as downregulation of ENT2 in immune cells caused decrease of resistance of the immune system against Drosophila parazitoid. The aim of the thesis is to determine whether RNAi targeted against mRNA of adenosine deaminase ADGF-A, which degrades e-Ado under natural conditions, would have any effect on immune response.
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Functional analysis of novel F\dindex{1}-ATPase subunit in \kur{Trypanosoma brucei}
VÁCHOVÁ, Hana
Although F1-ATPase is extremely conserved among organisms, a putative subunit p18 was identified in Trypanosoma brucei F1-ATPase complex. To explore its function in the procylic, bloodstream and dyskinetoplastic trypanosomes, three different RNAi cell lines were created. Upon p18 silencing the F1-moiety structural integrity was impaired suggesting that p18 is indeed a bona fide subunit of this complex. Since F1-ATPase is crucial for the bloodstream form survival, its potential inhibitor from the 4-oxopiperidine-3,5-dicarboxylates class (JK-11) was examined. JK-11 inhibited growth of the bloodstream trypanosomes, decreased mitochondrial membrane potential and reduced ATPase and ATP synthase activity in mitochondrial lysates. Our results suggest that JK-11 may act on FoF1-ATP synthase/ATPase and its inhibition may contribute to the cytotoxicity of this drug.
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Iron-Sulfur Cluster Assembly in Trypanosoma brucei
HAINDRICH, Alexander Christoph
In this thesis we investigated genes of the Cytosolic Iron sulfur cluster assembly (CIA) pathway in T. brucei procyclic and blood-stream form for their possible functional redundancy. For this, RNAi double knockdown plasmids were generated containing knockdown partners which were selected based on the proposed model of the CIA pathway in S. cerevisiae. The generated plasmids were transfected into T. brucei cells, and growth effects on the transfectants upon tetracycline induced RNAi was measured.
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Experimental examination of EFL and MATX eukaryotic horizontal gene transfers: co-existence of mutually exclusive transcripts predates functional rescue
RŮŽIČKA, Petr
Many eukaryotic genes do not follow simple vertical inheritance. Elongation factor 1? (EF-1?) and methionine adenosyl transferase (MAT) are enzymes with complicated evolutionary histories and, interestingly, the two cases have several features in common. These essential enzymes occur as two relatively divergent paralogs (EF-1?/EFL, MAT/MATX) that have patchy distributions in eukaryotic lineages that are nearly mutually exclusive. To explain such distributions, we must invoke either multiple eukaryote-to-eukaryote horizontal gene transfers (HGTs) followed by functional replacement, or presence of both paralogs in the common ancestor followed by longterm co-existence and differential losses in various eukaryotic lineages. To understand the evolution of these paralogs, we have performed in vivo experiments in Trypanosoma brucei addressing the consequences of long-term co-expression and functional replacement. In the first experiment of its kind, we have demonstrated that EF-1? and MAT can be simultaneously expressed with EFL and MATX, respectively, without affecting the growth of the flagellates. After the endogenous MAT or EF-1? was down-regulated by RNA interference, MATX immediately substituted for its paralog, while EFL was not able to substitute for EF-1?, leading to mortality. We conclude that MATX is naturally capable of evolving patchy paralog distribution via HGTs and/or long term co-expression and differential losses. The capability of EFL to spread by HGT is lower and so the patchy distribution of EF-1?/EFL paralogs was probably shaped mainly by deep paralogy followed by long term co-existence and differential losses.
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Functional analysis of prohibitin in \kur{Trypanosoma brucei}
TÝČ, Jiří
In this study the importance of prohibitin1 and prohibitin2 genes for Trypanosoma brucei was examined. RNA interference showed both of them essential for parasites to survive. Knocking down of these genes resulted in altered morphology of the mitochondrion, changes in membrane potential and shut down of mitochondrial translation. No changes were observed in levels of Reactive Oxygen Species and respiration. Both prohibitines are part of big complex present in the mitochondrion.
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Potentials and limits of RNA interference in the tick \kur{Ixodes ricinus}
MUSIL, František
The function of chitin binding protein (CBP) and two isoforms of cathepsin B (cathB1, cathB2) were tested by using RNA interference in the tick I. ricinus. Two different methods have been used to deliver dsRNA for RNAi in ticks {--} injection and capillary feeding. The synthesized dsRNA was used to find out the impact of RNAi in the tick tissues, which were tested by RT-PCR and Western blot. The expression of CBP was successfully silenced by RNAi in the salivary glands. The silencing of cathB1 and cathB2 in the gut was less effective, but still limited tick`s ability to feed.
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The transcriptional regulation by the nuclear receptor NHR-25 in \kur{Caenorhabditis elegans}
MERGLOVÁ, Linda
NHR-25 is a one of few conserved nuclear receptors in C. elegans and its family is involved in many developmental processes not only in the worm but also in flies, fish, mice and humans. Yet, the cellular mechanism of the action of this gene family is poorly understood. In C. elegans, it is likely to function as a transcription factor but its direct target gene has not been identified to date. In this study, I utilized the defined NHR-25 binding (target) sequence and GFP as a marker to visualize a possibility that NHR-25 regulates transcription of other gene(s) in vivo. I have also tried the "candidate approach" of two genes utilizing existing transgenic worm strains carrying promoter::GFP fusion transgenes expressed in epithelial cells to see if those genes can be regulated by NHR-25. According to the results it seems, that there could be an interaction between one of these genes and nhr-25 and that NHR-25 can play some role in endocytosis.
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