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Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2
Font Haro, Albert ; Hirsch, Ivan (advisor) ; Němečková, Šárka (referee) ; Saláková, Martina (referee)
Albert Font Haro ABSTRACT Modulation of plasmacytoid dendritic cell function: role of immunoreceptors TIM-3 and BDCA-2 Plasmacytoid dendritic cells (pDCs) are key players in the antiviral response as well as in linking innate and adaptive immune response. They express endosomal toll-like receptors 7 and 9, which can detect ssRNA and unmethylated CpG DNA, respectively. Due to the constitutive expression of the transcription factor IRF7, pDCs are able to rapidly produce massive quantities of type I (α, β, ω) and type III (1, 2, 3, 4) interferons (IFN-I and IFN-III) as well as pro- inflammatory cytokines such as IL-1, IL-6 and TNF-α. After maturation, they also function as antigen-presenting cells. Despite intense research, the mechanisms of IFN and pro-inflammatory cytokines production and regulation are still poorly understood. Using the pDC cell line GEN2.2 and also primary human pDCs, we shed light on the role of kinases MEK and SYK in IFN-I production and regulation. We found that SYK is not only involved in the regulatory receptor (RR)-mediated BCR-like pathway that represents the negative regulation of IFN-I and IFN-III secretion but also in the positive TLR7/9-mediated signal transduction pathway that leads to IFN-I production, representing the immunogenic function. We also found that MEK plays a...
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The effect of gut microbiota composition on condition in birds
Černá, Kateřina ; Vinkler, Michal (advisor) ; Saláková, Martina (referee)
The aim of this thesis is to summarize present knowledge concerning the effects of gut microbiota composition on condition in birds. The effect of gut microbiota on various aspects of animal physiology is currently intensively studied and the understanding of its biomedical importance is a topical theme. However, the majority of the current research is focused on humans and other mammals, and in birds on agriculturally significant species. Therefore, this thesis focuses also on the applicability of results of agriculturally orientated research on free-living birds' study. The results of agricultural research show a high interspecies and intraspecies variability in the effects of certain bacteria on the condition of the host. That should be taken into account when designing experiments in wild-living species. This thesis summarizes current knowledge concerning the effects of bacteria on condition of birds that was gained in correlational and manipulative studies. Manipulative studies include those in which probiotics or antibiotics were given to birds. The main modes of action of probiotic bacteria are also summarized here. One of the aims of this thesis was also to find out the possibilities of using detection of certain bacterial species as a non-invasive method to assess birds' condition. This thesis...
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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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The role of histone deacetylase 6 in murine polyomavirus replication cycle
Vlachová, Štěpánka ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
The replication cycle of polyomaviruses is, consistently with other viruses, fully dependent on host cells. Not only the cellular replicational and translational mechanisms are important for viruses, but also the virus infection is affected by other cellular proteins. This work is focused on the role of major cytoplasmic deacetylase, histone deacetylase 6 (HDAC6) in replication cycle of murine polyomavirus (MPyV). We showed that the presence of fully functional HDAC6 is essential for successful and productive infection. We found that HDAC6 affects not only early phase, but also late phase of infection. Cells with inhibited, or absent HDAC6 are infected with decreased effectivity and moreover lower amount of infectious viral particles is produced. On the other side, using cells with partially functional HDAC6, either in its deacetylase activity or in ubiquitin-binding activity, leads to increased ability of MPyV to infect those cells. Analysis of levels of early LT antigen and late structural protein VP1 in the infected cells showed, that viral proteins are affected by HDAC6. Our data suggest, that in the replication cycle of MPyV mainly the ubiquitin-binding domain of HDAC6 is required and the role of this domain in protein metabolism and degradation. In the second part of diploma project, we...
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The role of proteins acetylation in life cycle of Polyomaviruses
Dostalík, Pavel ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...
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Helicase DHX9 and its role in viral life cycles
Kertisová, Anna ; Horníková, Lenka (advisor) ; Saláková, Martina (referee)
Helicases are proteins that provide strands-unwinding of polystranded conformations of nucleic acids. DHX9 is a representative of helicases with a DExD/H-box motif. It accepts molecules of RNA, DNA and hybrid strucutures as a substrate. Inside the nucleus of the eukaryotic cell, DHX9 participates in the formation of the replication fork or it acts in a complex of trancriptional factors and it connects other proteins with RNA polymerase II. Subsequently DHX9 regulates post-transcritional RNA processing. After a shuttle to the cytoplasm it assists in a correct course of translation of structurated mRNAs. Since DHX9 is involved in nucleic acids-related processes, its role in viral life cycles is investigated. Also it is a part of antiviral signaling pathways of the cell. DHX9 provides an advantage for RNA viruses and retroviruses in their replicative cycle in various manners. The binding of DHX9 on secondary structures of viral RNAs and their eventually remodelation, which causes promotion of viral replication, transcription and translation, is significant. In contrast to that, DNA viruses use DHX9 to regulate host protein expression and minimize its antiviral fuction. Understanding of relationship could lead to more effective aiming of the therapeutic drugs against viral infections. Key words:...
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Study of exosomes in polyomavirus infection
Hyka, Lukáš ; Šroller, Vojtěch (advisor) ; Saláková, Martina (referee)
Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...
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Analysis of antibody response during BK virus infection
Tomanová, Tereza ; Španielová, Hana (advisor) ; Saláková, Martina (referee)
BK virus is a human polyomavirus which is highly prevalent in the population. The virus is usually not very dangerous to its host, but it may cause complicati- ons in immunosuppressed patients. These complications commonly appear after kidney transplantation because BK virus persists in kidney epithelial cells. There are four subtypes of BK virus and it might be clinically important to screen for the identity of subtypes in matched pairs of donors and recipients of the kidney. This determination of the subtype specific antibodies by simple test could help to manage complications after the surgery. During previous project the ELISA test that could serologically differentiate between two main BK virus subtypes (I and IV) was designed, but its development is complicated by the fact that there is a strong cross-reactivity between the BK virus subtypes and antibodies. The modification of antigen towards better specificity might be required to succeed. Consequently, the main aim of this diploma thesis was to map important spots of major capsid protein VP1 of BK virus, particulary in EF and DE loops, which could participate in binding of antibodies. This aim was addressed by targeted mutagenesis of the gene coding VP1 protein in the region of the respective loop. Nucleotides coding two surface aminoacids...
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Experimental model systems to study small DNA viral infection
Bučková, Alžbeta ; Saláková, Martina (advisor) ; Horníková, Lenka (referee)
Merkel cell polyomavirus (MCPyV) and Human papillomavirus 16 (HPV 16) are members of small tumour DNA viruses Polyomaviridae and Papillomaviridae, which represent increasing risk for humans resulting from their oncogenic potential. After the acquisition HPV 16 and MCPyV are able to persist for long term in a form of asymptomatic infection, while the aggressive disease is mostly being cleared by the host immune system. Integration of viral genome into the host DNA causes cell transformation resulting in rare but fatal skin carcinomas and epithelial lesions of anogenital tract, head and oropharynx, that may progress into malignant tumours. Their mechanisms of immune system evasion and complete life cycles are not fully understood to this day which highlights some of the reasons why continuing research in this field is of importance. The aim of this thesis is to review model systems used to study infection of MCPyV and HPV 16 in vitro and in vivo. Key words: Papillomaviruses, polyomaviruses, virus-like particles, pseudoparticles, animal models, cell culture, human papillomavirus 16, Merkel cell polyomavirus, HPV 16, MCPyV
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The role of BK polyomavirus in human cancer
Cirbusová, Adéla ; Saláková, Martina (advisor) ; Šroller, Vojtěch (referee)
BK polyomavirus is small uncoated DNA virus which is ubiquitous in human population. In imunosupresed individuals can cause severe diseases, specifically BK polyomavirus associated nephropathy (BKVaN) and hemoragic cystitidis. Except that, it is presumed that BKV may be responsible for some cases of human cancer. Some features of BKV could support this idea. There are three encoded oncoproteins in BKV genome. BKV is oncogenic in rodents, where induces multiple types of tumors and it can transform cell lines as well. Moreover, BKV DNA was found in many types of human cancer. All these facts suggest a possible role of BKV in human cancer. Bladder carcinoma in pacients after transplatation and prostate adenocarcinoma are the most likely candidates to link with BKV participation. There is no complete evidence though. Therefore, future studies are necessary to proof or even exclude BKV as a possible cause of human cancer. key words: BKV, cancer, LTag, prostate, bladder
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