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The Persistence of Human Polyomaviruses
Blažková, Kristýna ; Drda Morávková, Alena (advisor) ; Kuthan, Martin (referee)
Despite years of research, even the most scrutinized Polyomaviruses - BK and JC - have not yet been thoroughly understood. With a number of new Polyomaviruses - KIV, WUV, MCV, HPyV6, HPyV7, TSV and HPyV9 described in the past few years, the need to understand how Polyomaviruses operate in their hosts has become even more urgent. The probable route of transmission appears to be either respiratory or faecal-oral. The initial infection occurs most likely in the early childhood or early-adolescence and is followed by a life-long persistence. The seroprevalence of Human Polyomaviruses among healthy adult population is high: BKV (81-97 %), JCV (35-69 %), KIV (55 %), WUV (69 %), MCV (25-46 %) and TSV (70-80 %). Human Polyomaviruses can cause fatal diseases in immunocompromised patients. The site of persistence in humans probably varies depending on the specific Polyomavirus. BK and JC are known to persist in kidneys and the urinary tract. Human Polyomaviruses have been detected in the lymphatic tissues, blood, respiratory, urinary, and gastrointestinal systems. It is not clear, however, if they persist in all of these sites. Mechanisms which Polyomaviruses use to establish and maintain persistent infection could include the viral miRNA and viral agnoprotein, which would result in a modulation of viral...
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The expression of miRNA in HPV-associated and HPV-independent head and neck tumors
Vojtěchová, Zuzana ; Tachezy, Ruth (advisor) ; Hejnar, Jiří (referee) ; Slabý, Ondřej (referee)
Head and neck cancers represent a group of tumors with two different etiologies. The first type is associated with the viral HPV infection, the second one is virus-independent and it is associated with smoking and alcohol consumption as two main risk factors. Numerous studies show that HPV-positive tumors are more frequent in younger patients, as well as that the prognosis and overall survival of these patients is remarkably better. Therefore, the modification of the treatment is considered. For this, however, specific, sensitive and clinically relevant biomarkers for accurate identification of tumor etiology is needed. Suitable candidates for such biomarkers are miRNAs, small non-coding regulatory molecules stable in archived samples, that have been shown as differentially expressed in human cancers and the expression pattern seems specific for tumors of different origin. The submitted thesis focuses on miRNA profiling in HPV-positive and HPV-negative tonsillar tumors and cervical carcinomas with the aim to find out the differences between regulation of important carcinogenetic pathways of tumors of viral and non-viral etiology. Our data have shown very large heterogeneity of the miRNA expression profiles of these tumors. Despite the well characterized and uniform samples collection, we have found...
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Analysis of miRNAs in HPV-associated carcinomas
Pagáčová, Lucie ; Tachezy, Ruth (advisor) ; Vopálenský, Václav (referee)
Papillomaviruses are small DNA viruses that are associated with the induction of epithelial tumors. HPV is an important infectious agent causing almost 100 % of cervical tumors but it can also cause tumors in other anogenital and head and neck locations in both men and women. Active HPV infection induces changes in miRNA expression that contribute to the tumor formation and progression. It is already known that papillomaviruses do not encode their own viral miRNAs but they affect the expression of cellular miRNAs. In my thesis I have in selected epithelial tumors (vulva, cervix, anus and tonsils) determined their etiology and analyzed the presence of miRNAs in tissues by next generation sequencing. From these data I determined the expression profiles of deregulated miRNAs in tumors relation to healthy tissues of corresponding location. Even though, sufficient number of samples was analyzed, it was not possible to detect HPV-core miRNA common to all analyzed HPV-induced tumors due to the absence of statistically relevant differentially expressed miRNAs in HPV positive vulvar tumors. Among the tumors of the other sites I found an overlap in three miRNAs. One of these miRNAs (miR-139-5p) and another one (miR-9-5p) which I have selected based on the study of other published data, were used for...
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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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The role of microRNAs in chronic lymphocytic leukemia
Moravec, Martin ; Hájek, Miroslav (advisor) ; Macůrková, Marie (referee)
2 Abstract MicroRNAs (miRs, miRNAs) are recently discovered molecules (19-25 nucleotides long) that regulate gene expression at post-transcriptional level by either blocking protein synthesis or mRNA degradation. As a part of gene silencing mechanism, miRNAs are involved in cellular processes, such as apoptosis, cell proliferation, development and viral defence. miRNAs have been intensely studied in connection to disease pathogenesis. Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries affecting mostly elderly people. In my work I focus on explanation of miRNA functions and their contributions to chronic lymphocytic leukemia (CLL). I describe previously published data about miRNA-15, miRNA-16, miRNA-143, miRNA-145 and miRNA-155 in connection to this disease. Based on recent reports, I also discuss the potential role of miRNA-326 in CLL pathogenesis.
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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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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...
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Circulating miRNAs as biomarkers in the diagnosis and treatment of rheumatic diseases
Prajzlerová, Klára ; Filková, Mária (advisor) ; Hrnčíř, Zbyněk (referee) ; Procházková, Leona (referee)
Background: MicroRNAs (miRNAs) are small non-coding single-stranded RNAs involved in the posttranscriptional inhibition of gene expression and thereby regulating all cellular functions. Their dysregulation contributes to the pathophysiology of many diseases, including rheumatic diseases. MiRNAs can also be found extracellularly in body fluids and represent promising diagnostic and prognostic biomarkers. Our study aimed to investigate miRNAs as biomarkers of stage and activity and predictors of therapeutic response of two most common inflammatory rheumatic diseases: spondyloarthritis (SpA) and rheumatic arthritis (RA). Results: We found several circulating miRNAs differentially expressed in SpA patients reflecting the severity of axial involvement and/or disease activity. The decrease in circulating miR-145 in plasma of patients with ankylosing spondylitis 3 months of anti-TNF therapy predicted a good therapeutic response and low disease activity after a year of therapy. Circulating and intracellular expression of miR-125b in peripheral blood mononuclear cells (PBMC) was lower in treatment-naïve patients with early RA than in healthy controls. Baseline expression of miR-125 in PBMC predicted a (non)adequate therapeutic response. We also found the increased expression of miR-451 in PBMC in...
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Novel bile acid derivatives as a promising therapeutic approach for liver and metabolic disorders
Štefela, Alžbeta ; Pávek, Petr (advisor) ; Vítek, Libor (referee) ; Juřica, Jan (referee)
IN ENGLISH LANGUAGE Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacology and Toxicology Candidate: Mgr. Alžbeta Štefela Supervisor: Prof. PharmDr. Petr Pávek, PhD. Title of the doctoral thesis: Novel bile acid derivatives as promising therapeutic approach Bile acids (BAs) are amphipathic steroidal molecules that are traditionally known to facilitate intestinal digestion and absorption of lipids and fat-soluble substances. On top, the recent findings have revealed that they represent important signaling agents involved in the orchestration of lipid, glucose and energy metabolism and immune response. BAs exhibit these roles by activating intracellular nuclear receptors such as farnesoid X (FXR), pregnane X (PXR) vitamin D receptors. Furthermore, BAs act as endocrine signaling molecules and activate numerous biological cascades via a membrane G-protein-coupled receptor, termed TGR5. Therefore, the extensive modulation of BA scaffold underwent to identify compounds with specific targeting of above-mentioned receptors as a promising therapeutic approach for the treatment of various liver and metabolic disorders including cholestasis, biliary cirrhosis, nonalcoholic steatohepatitis or diabetes. The principal aim of this doctoral thesis was to investigate the structure...
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MicroRNA pathway activity in mammalian oocytes
Kataruka, Shubhangini ; Svoboda, Petr (advisor) ; Simard, Martin (referee) ; Cochella, Luisa (referee)
(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events where a differentiated oocyte gives rise to a totipotent zygote. During the growth phase an oocyte prepares for fertilization and progression to zygotic genome activation. It does so by transcribing and storing the necessary mRNAs till a fully-grown oocyte attains transcriptional quiescence. Therefore, transcriptome regulation in a fully-grown oocyte is of utmost importance. Study of post-transcriptional regulatory pathways revealed that the small-RNA mediated regulatory pathways exist in a unique conformation in mouse oocytes. Endogenous RNAi pathway is essential for mouse female germline while miRNA pathway which is ubiquitously present in most cell types is dispensable for oocyte maturation and fertilization. My PhD project was aimed at understanding the constraints of the miRNA pathway in the oocyte which makes it non-functional. As a fully-grown oocyte is a huge cell with a proportionally large maternal transcriptome we analysed the miRNA: mRNA stoichiometry changes that occur from growing to the fully-grown mouse oocyte. Inability of miRNAs to accumulate during oocyte growth phase leads to their dilution in fully-grown oocyte rendering them inactive. Low miRNA concentrations were also observed in rat,...
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