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Prediction of small RNAs in selected bacterial producers of bioplastics
Heřmánková, Kristýna ; Vítková, Helena (referee) ; Sedlář, Karel (advisor)
Využití bakterií v biotechnologii vyžaduje důkladné pochopení schopností použitých bakterií pro maximalizaci výtěžku. To platí například pro výrobu bioplastů, kde konkurenceschopnost s levnějšími plasty na bázi fosilních paliv závisí také na ceně procesu pro získání požadovaného výtěžku. Nahlédnutí do regulačních mechanismů bakterií, jako je regulace pomocí malých RNA, která zajišťuje rychlou reakci především na stresové podmínky, může pomoct odhalit jejich vlastnosti a posoudit tak jejich využitelnost v biotechnologiích. Tato diplomová práce se zabývá zpracováním bakteriálních transkriptomických dat, které byly sekvenovány pomocí RNA-Seq. Práce je zaměřena zejména na predikci malých RNA u poskytnuté bakterie, slibného kandidáta pro využití v biotechnologii. Cílem je získat přehled o malých RNA v této bakterii, zejména analyzovat potenciální regulaci syntézy PHA malými RNA, které by mohly rozšířit přehled o využitelnosti bakterie pro produkci PHA.
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Biofyzikální studium malých RNA
Šmerková, Kristýna
Thanks to the prove of connection between the aberrant occurrence of small RNA and various diseases and their potential in diagnostics and treatment led to discovery of new methods and materials facilitating their detection and targeted transport during gene therapy. This work summarizes present knowledge about chosen groups of small RNA, their significance in medical science and the possibilities of their detection. This work primarily concentrates on combination of magnetic separation with electrochemical detection. Magnetic particles (MPs) with different surface modifications were used for isolation. Non-specific isolation was carried out using silanol-coated MPs; streptavidin-coated MPs modified with specific biotinylated probe were used for specific separation. Square wave voltammetry (SWV) was used as a very sensitive electrochemical detection method. Optimized method based on specific magnetic separation with SWV was able to reach nanomolar detection limit (4 nM) with microRNA. The method was applied on human embryonic cells for specific isolation and detection of miR-124. The CdTe quantum dots (QDs) were studied as a nanomaterial tool for nucleic acid detection. The QDs were modified with streptavidin for their bioconjugation with biotinylated molecules were used. Interaction of QDs with nucleic acids was studied using capillary electrophoresis.
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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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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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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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Biofyzikální studium malých RNA
Šmerková, Kristýna
Thanks to the prove of connection between the aberrant occurrence of small RNA and various diseases and their potential in diagnostics and treatment led to discovery of new methods and materials facilitating their detection and targeted transport during gene therapy. This work summarizes present knowledge about chosen groups of small RNA, their significance in medical science and the possibilities of their detection. This work primarily concentrates on combination of magnetic separation with electrochemical detection. Magnetic particles (MPs) with different surface modifications were used for isolation. Non-specific isolation was carried out using silanol-coated MPs; streptavidin-coated MPs modified with specific biotinylated probe were used for specific separation. Square wave voltammetry (SWV) was used as a very sensitive electrochemical detection method. Optimized method based on specific magnetic separation with SWV was able to reach nanomolar detection limit (4 nM) with microRNA. The method was applied on human embryonic cells for specific isolation and detection of miR-124. The CdTe quantum dots (QDs) were studied as a nanomaterial tool for nucleic acid detection. The QDs were modified with streptavidin for their bioconjugation with biotinylated molecules were used. Interaction of QDs with nucleic acids was studied using capillary electrophoresis.
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