|
|
The role of small non-coding RNAs in the regulation of plant reproductive development
Hromadová, Michaela ; Honys, David (advisor) ; Rothová, Olga (referee)
Small RNAs (sRNA) are broadly defined as regulatory molecules of 21-24 nucleotides in length which belong to the class of noncoding RNAs. They usually originate in response to the presence of double-stranded RNA in the cells and facilitate transcriptional and posttranscriptional gene silencing of complementary sequences. Their role lies not only in defence against exogenous nucleic acids, but primarily in the regulation of endogenous genes. Typical target molecules of plant sRNA are transposable elements and genes encoding the transcription factors involved in the control of key developmental transitions like the initiation of the reproductive phase and the regulation of its progress. The aim of this thesis is a summary of functional roles of individual small RNA in plant reproductive development, with focus to the female and male gametophytes and in the protection against the transition of transposable elements to following generations. Both endogenous and exogenous sRNA are amply utilized in plants, because they provide an immediate and direct response of the organism to rapidly changing conditions, and thus undoubtedly belong to the key factors which contribute to their phenotypic plasticity.
|
|
|
Use of RNAi and CRISPR systems in genetic modifications of parasitic protists
Kaiserová, Veronika ; Votýpka, Jan (advisor) ; Stojanovová, Darja (referee)
In organisms, RNA interference serves as a defence mechanism against foreign nucleic acids. RNAi has a negative effect on translation, via the binding of small non-coding molecules to the complementary region of mRNA, resulting in its degradation. CRISPR, a new method of genetic engineering, is based upon modulating genetic expression via creating double-stranded breaks in target DNA, aided by a ribonucleoprotein complex, consisting of the prokaryotic endonuclease Cas9 and sgRNA. Both of the aforementioned methods can be utilised in functional analysis of proteins and the characterisation of metabolic pathways in organisms of interest. This work summarises the current state of knowledge regarding RNAi and CRISPR and their use in genome editing of parasitic protists.
|
|
|
Spectroscopic Study of miRNA Structure and Interactions
Homolka, Radim ; Štěpánek, Josef (advisor) ; Maloň, Petr (referee)
MiRNA are short RNA molecules that mediate post-transcriptional regulation of gene expression by binding to a target mRNA. In this thesis, some structural properties and thermodynamic parameters (melting temperature, enthalpy ∆H and entropy ∆S changes connected with the transition between particular conformations) of oligonucleotides simulating complex of miRNA and mRNA, were characterized by using spectroscopic methods and a gel electrophoresis. Temperature dependences of UV and Raman spectra were measured, the results treated by means of a factor analysis, and the abovementioned thermodynamic parameters determined by a fit to a feasible model. The outputs have demonstrated the capability of the used methods to determine thermodynamic and structural properties of non-canonical RNA complexes. Powered by TCPDF (www.tcpdf.org)
|
|
|
Molecular aspects of musculoskeletal diseases and the role of small regulatory RNAs
Pleštilová, Lenka ; Vencovský, Jiří (advisor) ; Šedivá, Anna (referee) ; Hrnčíř, Zbyněk (referee)
Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...
|
|
|
Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Flachsová, Monika
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
|
|
|
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...
|
|
|
Optimization of miRNA analysis in fine-needle biopsy samples of pancreatic cancer tissue.
Čuperková, Romana ; Benešová, Lucie (advisor) ; Kuthan, Martin (referee)
Pancreatic cancer (PC) is extremely severe malignant disease with a five-year survival of less than 5%. Currently there is no reliable tool for the diagnosis of PC in its early stages. At the time of clinical symptoms most patients are in an advanced stage of the disease and the treatment does not usually have a significant effect. For these reasons emphasis is gradually shifting to the search for the suitable molecular markers for improvement of the diagnosis and assessment of the survival prognosis with respect to a possibility of surgical treatment. MiRNA represent one of the most promising markers, although, their examination in pancreatic tissue is a complicated process. One of the reasons is the very small amount of the source material coming from a fine needle biopsy. A second cause of problems is the subtle character of the pancreatic tissue resulting in significantly lower yields of molecular genetic analysis when compared to other epithelial tissues. An additional negative factor is heterogeneity of the tissue resulting in disproportionate representation of tumor cells within the sample. A suitable choice of procedures for isolation of nucleic acids (NA) and subsequent analysis including quantification of tumor cells is critical for accurate evaluation of the miRNA levels. This work is...
|
|
|
Forensic analysis of epigenetic factors and mRNA
Andreasová, Karolina ; Šimková, Halina (advisor) ; Zachová, Markéta (referee)
In last years, forensic genetics focused on a research of epigenetic factors and mRNA. It turned out that their analysis provides valuable information that can be obtained from a small amount of a biological trace and which included e.g. estimation of circumstances of death, age estimation, discrimination within monozygotic twins, identification of tissues and body fluids, sample authentication, determination of paternal allele, etc. In this thesis, it is discussed methods of epigenetic factors and mRNA analysis and their potential application in future forensic practice.
|
|
|
Cell death-regulating micro RNAs and their role in the development and pathological processes.
Běhounek, Matěj ; Anděra, Ladislav (advisor) ; Seifertová, Eva (referee)
MicroRNAs are small protein non-coding, ~ 22 nucleotides long dsRNAs. Their main task is suppression of gene expression via removal/destabilization of mRNA or its targeting to degradation. These small molecules play an important role in the regulation of many cellular processes and have been found to affect expression of more than 30% of human genes. Among the processes affected or regulated by miRNAa belongs also programmed cell death. Although this work is mainly focused on the analysis and characterization a role of distinct miRNAs in the regulation of apoptotic cell death, miRNAs can also participate in the regulation of autophagic cell death or programmed necrosis. MiRNA can enhance cellular sensitivity to apoptosis by suppressing the expression of death receptor genes, but can also drive cells to apoptosis by regulating expression of anti-apoptotic protein Bcl-2. In many different organisms were already discovered and described thausends of micro RNAs anddozens of them participate in the regulation of cell death. Poor or impaired function of miRNAs and related disturbance in apoptotic signaling could lead to a number of pathological processes as tumorigenesis or disturbances in tissue development and homeostasis. . Understanding how miRNA functions in cell death and possible practical...
|
|
|
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.
|
guest ::
