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Regulation of STING function during murine polyomavirus infection
Šnejdarová, Aneta ; Horníková, Lenka (advisor) ; Pimková Polidarová, Markéta (referee)
Stimulator of Interferon Genes (STING) is the adapter protein of an innate immunity signalling pathway, involved in detection of double-stranded DNA (dsDNA) in the cell cytoplasm, which leads to the expression of pro-inflammatory genes, including the production of type I interferon. Eventhough during the infection with a dsDNA virus, murine polyomavirus (MPyV), the STING protein is activated, the resulting interferon production is moderate. Therefore, it can be assumed that the function of the STING protein is regulated in MPyV-infected cells. The aim of this thesis was to investigate three mechanisms by which the regulation can occur, namely through protein interaction partners, post- translational modifications, or changes in the subcellular localization of the STING protein. A cell-line of mouse fibroblasts stably expressing the STING protein fused with the HA-tag was established to facilitate the research. Furthermore, two plasmids were prepared, that encode the STING protein fused with the green fluorescent protein, facilitating the monitoring of the localization of the protein in the cell, or with a composite tag containing an in vivo biotinylated BioEaseTM -tag enabling effective isolation of the STING protein. The results of colocalization observations and coimmunoprecipitation suggest that...
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The importance of cell-free HPV DNA detection
Milt, Petr ; Saláková, Martina (advisor) ; Horníková, Lenka (referee)
Human papillomaviruses (HPV) are small, nonenveloped DNA viruses that are abundant in the population. They are sexually transmitted or spread by close contact with mucosa and skin. Papillomaviruses can cause lesions and warts on the skin and mucosa. In addition, high-risk HPV types, especially HPV 16 and 18, are associated with squamous cell carcinomas such as cervical cancer, oropharyngeal cancer and carcinomas of the vulva, anus, penis and vagina. Early detection and the right evaluation of the risk of recurrence are crucial for effective treatment. Cell-free DNA released from cells into body fluids has potential in cancer diagnosis. Cell-free circulating HPV DNA, in the blood of patients with HPV-associated cancers is a promising and highly sensitive biomarker, useful for monitoring treatment efficiency, early detection of the disease and estimation of recurrence risk. Key words: HPV, carcinogenesis, cfDNA, cfHPV DNA, significance of detection, cervical cancer, oropharyngeal cancer
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Oral microbiome and carcinogenesis
Šimáčková, Šárka ; Šmahelová, Jana (advisor) ; Horníková, Lenka (referee)
Microorganisms that colonize human body participate to well functioning of human organism and they are very important for human health. The composition of these communities (microbiomes) is specific for everyone and changes in the composition may participate to induce and progression of disease. These diseases also include carcinogenesis and tumors. Main goal of this work are specially tumors in head and neck area which risk factors are smoking, alcohol consumption and human papillomaviruses infection. Depending on these factors is influenced the composition of microbiome of oral cavity. Many studies show differences between oral microbiomes of patients with head and neck cancer and healthy people as controls. Keywords: microbiome, oral cavity, head and neck cancer, papillomavirus
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Analysis of serological cross-reactivity between antibodies against BK polyomavirus variants
Caisová, Helena ; Horníková, Lenka (advisor) ; Roubalová, Kateřina (referee)
BK polyomavirus (BKPyV), which asymptomatically infects about 80 % of the human population, can reactivate in immunosuppressed patients after kidney transplantation and cause nephropathy. In the European population, there are predominantly two BKPyV sub- types, BKPyV-I and BKPyV-IV, which behave as different serotypes. Recipients who receive a kidney from a donor positive for a different subtype are at a greater risk of graft rejection. So far, it is impossible to distinguish between these two subtypes using a serological ELISA test as serum antibodies against these two subtypes cross-react. This work aimed to iden- tify epitopes on the major antigenic protein VP1, which are responsible for the binding of cross-reacting and/or subtype-specific antibodies. The identification of such epitopes could further lead to improvement in pre-transplant diagnostics and better management of pati- ents after transplantation. Therefore, two types of antigens composed of the VP1 protein of the BKPyV-IV subtype with introduced mutations characteristic of BKPyV-I in the DE and EF loops of VP1 were prepared and tested using an antigen competition assay with a panel of human sera. The results showed that the DE loop region is an immunogenic epitope that binds specific antibodies for BKPyV subtype I. Conversely, a mutation in...
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Tunneling nanotubes and life cycles of viruses
Lišková, Jitka ; Horníková, Lenka (advisor) ; Španielová, Hana (referee)
Tunneling nanotubes are a specific type of an intercellular junctions. These structures are thin long protrusions of cellular membrane containing mainly F-actin and form connections between the cells. These structures facilitate intercellular transport and the cargo transported by tunneling nanotubes is very variable - from cellular organelles to ions and proteins. Moreover, intracellular pathogens, like bacteria and viruses can use these structures to spread in the organism. Transport of the whole virions or viral proteins mediated by tunneling nanotubes was described for several families of enveloped viruses, e.g. Retroviruses, Herpesviruses or Ortomyxoviruses. Viruses from these families use nanotubes to spread their own viral progeny to uninfected cells and this type of transport allows them to escape from control of the host`s immune system.
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Novel hepatitis C virus proteins
Zeman, Jakub ; Vopálenský, Václav (advisor) ; Horníková, Lenka (referee)
The hepatitis C virus (HCV) is a major etiological agent of chronic liver diseases. More than 170 million people worldwide are chronically infected, and more than 100 thousand of them develop hepatocellular carcinoma a year. HCV is an enveloped, positive-sense single-stranded RNA virus (+ssRNA virus) of the family Flaviviridae. Its genome is translated to produce a single polyprotein precursor that is further processed by cellular and viral proteases to form 10 viral proteins. Moreover, there is another protein encoded in an alternative reading frame. Two alternative translation mechanisms have been proposed for expression of this alternative reading frame protein (ARFP): a frameshift mechanism and translation initiating from internal start codons. Despite ten years of research its role in vivo is not yet explained. It appears that secondary structures in the core encoding region of HCV genome but not ARFP expression are required for robust viral translation and replication. The results of recent studies suggest that mutations distorting these structures may result not only in slowing down the viral cycle but also in a brand new and utterly unusual serological profile in patients as well as an increased level of expression of ARFP.
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The role of caspase 2 in apoptosis induction in tumor cells.
Schmiedlová, Martina ; Kovář, Jan (advisor) ; Horníková, Lenka (referee)
Within the cell, caspase-2 probably fulfills several functions. Caspase-2 can be involved in apoptosis induction, DNA repair as well as cell cycle regulation. Caspase-2 has the character of initiator and also executioner caspase. A stimulus for caspase 2 activation can be oxidative stress or DNA damage. Caspase-2 is activated by cleavadge during an interaction with protein complexes. One of protein complexes,i.e. PIDDosome, is made of protein PIDD, RAIDD and pro-caspase-2. Withine the PIDDosome, caspase-2 is activated. Activated caspase-2 occures in a short S form and in long L form. L form of caspase-2 has proapoptotic effects and S form of caspase-2 has antiapoptotic effects. Caspase-2S has been only detected on mRNA level but not on protein level. The main role of caspase-2L is apoptosis induction in normal and tumor cells. Caspase-2 in tumour cells is activated by extrinstic as well as intristic apoptotic pathway. Apoptosis induction by caspase-2 is for example studied in connection with breast cancer treatment with taxanes. Caspase-2 ability of apoptosis induction in cancer cells independently of p53 protein is employed in cancer treatment including overcoming the resistance to apoptosis induction which is based on loosing p53 activity. Caspase-2 is involved in apoptosis induction by different...
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Endoplasmic reticulum stress
Červenka, Jakub ; Schierová, Michaela (advisor) ; Horníková, Lenka (referee)
The accumulation of unfolded or misfolded proteins in endoplasmic reticulum (ER) leads to ER stress and the activation of unfolded protein response (UPR). Recent studies show that ER stress or UPR are associated with many diseases such as diabetes, hepatitis type C, prion disease, different kinds of tumors or Alzheimer's, Parkinson's and Huntington's disease and also with physiological processes like cell differentiation. When UPR is activated in yeast Saccharomyces cerevisiae, Ire1 protein oligomerizes, transautophosphorylates and activates itself. After this, Ire1 cleaves HAC1 mRNA to remove an intron. The spliced form of HAC1 mRNA is translated into the Hac1 transcription factor, which induces transcription of genes for chaperones of lumen ER, proteins involved in ERAD, synthesis of lipids etc. The cell uses this to reestablish homeostasis in ER. In mammals, the UPR is more complex and except Ire1 dependent pathway, it comprises Perk and Atf6 pathways, which are missing in yeast. Nevertheless, Perk is activated and regulated by the similar mechanism as Ire1 in S. cerevisiae. In consideration of broad spectrum of methods for genetic manipulation, rapid growth and well annotated genome, the yeast S. cerevisiae is a useful model for study of general mechanisms of UPR in mammals.
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Minor Structural Proteins of Polyomaviruses: Attributes and Interactions with Cellular Structures
Vinšová, Barbora ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Even though polyomaviruses have been intensively studied for more than 60 years, the role of minor structural proteins VP2 and VP3 in some important steps of viral life cycle has still not been fully elucidated, explicitly their role in viral genome delivery to the cell nucleus and their involvement in late phases of viral life cycle. This diploma thesis focuses on the study of minor proteins of Mouse polyomavirus (MPyV) and Human polyomavirus BK (BKV). Four rabbit polyclonal antibodies against minor proteins of polyomaviruses MPyV or BKV have been prepared within this diploma thesis. Two of these prepared antibodies target minor proteins of MPyV (α-MPyV VP2/3) or BKV virus (α-BKV VP2/3), other two prepared antibodies recognize C-terminal sequence common to minor proteins VP2 and VP3 of MPyV (α-MPyV C-termVP2/3) or BKV virus (α-BKV C-termVP2/3). In the second part of this diploma thesis we aimed to study toxicity of BKV virus minor proteins during individual production in mammalian cells. Obtained results suggest that minor proteins of BKV virus might not exhibit as high levels of cytotoxicity as minor proteins of MPyV virus. Third part of this diploma thesis is devoted to investigation of interactions of BKV and MPyV minor proteins with cellular proteins and within one another respectively....
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