|
|
Micronuclei and their connection with intracellular innate immunity and viral infection
Knoblochová, Kateřina ; Bruštíková, Kateřina (advisor) ; Španielová, Hana (referee)
Micronuclei are tiny structures that contain nuclear DNA and a membrane derived from the nucleus. They emerge in cells that have been exposed to severe stress factors, such as viral infections, radiation, or genotoxic substances. While micronuclei have long been used as markers of genotoxic stress, the mechanism of their formation and internal processes are not yet fully understood. DNA enclosed inside micronuclei is restructured in an atypical manner, which may induce mutations and accelerate oncogenic transformation of the cell. Due to these processes micronuclei can also act as reservoirs of immunostimulatory nucleic acids, which may potentially be detected by molecular sensors. Therefore, studying micronuclei is significant in relation to the activation of signaling pathways that are part of the innate intracellular immunity. This work summarizes the current knowledge about micronuclei and their connection to innate intracellular immunity and viral infection. Keywords: micronuclei, innate immunity, molecular sensors, chromotripsis
|
|
|
Role of nuclear lamins and Nup358 in BK polyomavirus infection
Išler, Lukáš ; Bruštíková, Kateřina (advisor) ; Němečková, Šárka (referee)
BK polyomavirus (BKPyV) has been causing serious health complication for several decades, especially in immunocompromised patients. The aim of this work was to investigate the progress of BKPyV replication during infection of human cells, as well as the influence of BKPyV infection on the nuclear lamina and nuclear pore proteins (NPC; Nuclear Pore Complex). During characterization and comparison of BKPyV infection in RPTEC/hTERT1 and MRC-5 cells we showed that BKPyV replicates better in RPTEC/hTERT1 cells as the productive infection results in six times higher viral titer. Using confocal microscopy we did not observe any nuclear lamina disruption nor VP1 accumulation under nuclear lamina that was previuosly observed in mouse polyomavirus infection. We verified previous observations of cytoplasmic deposits of NPC colocalizing with VP1 protein 24 hours post infection (hpi) with BKPyV and we showed that Nup358, protein of NPC, is a component of NPC deposits colocalizing with VP1. Neither transient expression from vectors encoding late region of BKPyV genome (pEF- BKV-late) or VP1 alone (pIaW), nor LT antigen expression analysis did not suggest any conection of observed phenomena to the productive infection. However, pseudoinfection of RPTEC/hTERT1 cells with VLPs derived from BKPyV induced VP1...
|
|
|
Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
|
|
|
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
|
|
|
Nuclear lamina, its function in the cell and its role in interaction with non-enveloped DNA viruses
Pápež, Samuel ; Bruštíková, Kateřina (advisor) ; Kuthan, Martin (referee)
The nuclear lamina is a protein network found in all metazoa. It is composed of intermediate filaments V, namely type A and type B lamins. It is a multifunctional cellular structure adjacent to the inner nuclear membrane, giving the nucleus its shape, ensuring its mechanical stability and participating in the regulation of many nuclear processes. The nuclear lamina also has a protective function manifested in the infection of cells with DNA viruses, whose life cycle takes place in the nucleoplasm. These include small non-enveloped DNA viruses. The nuclear lamina is an obstacle that viruses have had to learn to overcome in order to multiply. To do this, they use their own, but also cellular mechanisms, which disrupt the nuclear membrane together with the nuclear lamina and thus allow the virus nuclear entry and exit. This work summarizes the knowledge about changes in the nuclear lamina induced by viruses of the families Polyomaviridae, Parvoviridae and Circoviridae, on the way through the nuclear envelope. Keywords: Nuclear lamina, disassembly of nuclear lamina, DNA viruses, Polyomaviruses, Parvoviruses, Circoviruses
|
|
|
Vývoj chemických regulátorů drah mikroRNA a RNAi
Bruštíková, Kateřina ; Svoboda, Petr (advisor) ; Bařinka, Cyril (referee) ; Pospíšek, Martin (referee)
MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...
|
guest ::
