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Biological treatment and its influence on the course of latent viral infections in patients with psoriasis
Laurin, Josef ; Šmahelová, Jana (advisor) ; Janovec, Václav (referee)
There are more than 80 identified autoimmune diseases. One of the most prevalent ones is psoriasis. Its prevalence is around 2-5 % worldwide. The treatment of this inflammatory skin disease can be divided as follows: in cases of low severity, topical therapies are used for local treatment and in the cases of insufficient effect, stronger therapies are used. Phototherapy is used for moderate severity, and systemic therapy is used in moderate to severe disease. Systemic agents include cytostatic methotrexate, immunosuppressant cyclosporin, or retinoids (vitamin A analogues). However, even systemic therapies may not yield the desired effects or may have adverse effects on the overall condition of the patient. In those cases, biological therapy comes to use. Biological therapy is usually conducted using antibodies and fusion proteins, which are made using recombinant technologies. Tumour necrosis factor α (TNF-α) and interleukin 12, 17 and 23 (IL-12, IL-17 and IL-23) inhibitors are the most commonly used in the treatment of psoriasis. During the inhibition of the immune system, it has been confirmed that a reactivation of viral infections can occur. These reactivations may subsequently lead to the development of various diseases caused by latent viral infections.
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Properties and functions of agnoprotein of polyomaviruses
Zosinčuková, Tereza ; Forstová, Jitka (advisor) ; Vinšová, Barbora (referee)
Polyomaviridae family includes small DNA viruses with simple structure and a small genome encoding only a few proteins. These proteins include large T and small T antigens, as well as 2 to 3 structural proteins known as VP1, VP2 and VP3. In addition, some members of the Polyomaviridae family encode in their genome a small non-structural protein called agnoprotein. Among human polyomaviruses, agnoprotein is present in BK polyomavirus and JC polyomavirus. These viruses are the causative agents of some serious diseases in immunocompromised humans and therefore, they are the subject of intensive research. Simian vacuolating virus 40 is another example of a virus which encodes the agnoprotein. Agnoprotein is capable to manipulate its host cell, disrupt vesicle transport and is also crucial for viral replication and transcription. It appears to play an important role in the morphogenesis of virions and/or in their release from the cell. This paper comprehensively summarizes the latest insights into the properties and functions of the agnoprotein BK polyomavirus, JC polyomavirus and SV40 virus, focusing on the production of this protein during infection, its structure, posttranslational modifications, cell localization, interaction partners and the overall importance of this enigmatic protein for the...
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Targeting of viral nanoparticles to cancer specific receptors
Žáčková Suchanová, Jiřina
The aim of this thesis is to reveal the potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as possible nanocarriers for directed delivery of therapeutic or diagnostic compounds to specific cells or tissues. We have chosen mouse polyomavirus VLPs because they do not contain viral DNA and are considered safe for utilization in bio-applications. In our research, we used a chemical approach for retargeting of MPyV based VLPs from their natural receptor to cancer cells. The chemical modification of the capsid surface exposed lysines by an aldehyde-containing reagent enabled conjugation of VLPs to selected molecules: transferrin and inhibitor of glutamate carboxypeptidase II (GCPII). Transferrin, as a transporter of iron to metabolically active cells, targeted VLPs to numerous types of cancer cells overexpressing the transferrin receptor. On the other hand, GCPII serves as a transmembrane marker specific for prostate cancer cells and conjugation of its inhibitor to VLPs resulted in successful recognition of these cells. Electron microscopy was used for visualization of modified VLPs and flow cytometry together with confocal microscopy for investigation of cell specific interactions and VLP uptake. Furthermore, we explored the influence of serum proteins on VLPs. The abundance of...
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Targeting of viral nanoparticles to cancer specific receptors
Žáčková Suchanová, Jiřina ; Španielová, Hana (advisor) ; Němečková, Šárka (referee) ; Ulbrich, Pavel (referee)
The aim of this thesis is to reveal the potential of mouse polyomavirus (MPyV) based virus-like particles (VLPs) as possible nanocarriers for directed delivery of therapeutic or diagnostic compounds to specific cells or tissues. We have chosen mouse polyomavirus VLPs because they do not contain viral DNA and are considered safe for utilization in bio-applications. In our research, we used a chemical approach for retargeting of MPyV based VLPs from their natural receptor to cancer cells. The chemical modification of the capsid surface exposed lysines by an aldehyde-containing reagent enabled conjugation of VLPs to selected molecules: transferrin and inhibitor of glutamate carboxypeptidase II (GCPII). Transferrin, as a transporter of iron to metabolically active cells, targeted VLPs to numerous types of cancer cells overexpressing the transferrin receptor. On the other hand, GCPII serves as a transmembrane marker specific for prostate cancer cells and conjugation of its inhibitor to VLPs resulted in successful recognition of these cells. Electron microscopy was used for visualization of modified VLPs and flow cytometry together with confocal microscopy for investigation of cell specific interactions and VLP uptake. Furthermore, we explored the influence of serum proteins on VLPs. The abundance of...
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Raccoon polyomavirus: example or exception of polyomavirus driven oncogenesis?
Schreiberová, Lucie ; Španielová, Hana (advisor) ; Hirsch, Ivan (referee)
Polyomaviruses (PyV) are widespread through human and animal populations and typically associated with asymptomatic persistent infection. Rarely, natural PyV infections can lead to oncogenic transformation. Virus genome is usually integrated into the host DNA of tumour tissue. Over the past few years, an increased number of very aggressive brain tumours and olfactory tumours have been observed in raccoons. These tumours are associated with the newly discovered raccoon polyomavirus, which was found as an intact episome in host cells. This bachelor thesis is therefore focused on comparison of current state of knowledge on raccoon polyomavirus with previously described mechanisms of PyV tumorogenesis. Unlike for other PyVs, the fact that primary neuronal stem cell infection is most likely to occur can play a key role in raccoon polyomavirus driven oncogenesis. Tumours also exhibit unusually high expression of virus-encoded micro RNA that can be connected with tumour induction. Similary to other tumours caused by PyV, a large amount of early viral proteins with oncogenic potential is found in tumours. Revealing unknown factors responsible for the development of tumours caused by raccoon polyomavirus may help in understanding of mechanisms of oncogenesis. Key words Polyomavirus, oncogenesis, raccoon...
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Histone modifications and methylation of polyomaviral genomes during the infection
Mrkáček, Michal ; Forstová, Jitka (advisor) ; Šmahelová, Jana (referee)
Similarly to other viruses, polyomaviruses require for their successful replication enzymes and other proteins encoded by their host cells. Additionally, because of their relatively small genome with only a few genes, polyomaviruses utilize for their efficient replication cellular regulation mechanisms. One of these regulations are posttranslational modifications of histones, which form nucleosomes together with viral DNA. The spectrum of these modifications is very wide, but in case of polyomaviruses, almost only ones studied are histone acetylations and methylations. Second possible regulation is a methylation of cytosine in CpG dinucleotides, which is associated with repression of gene expression. Current knowledge however suggest that polyomaviruses do not utilise this kind of modification. Moreover, because of a relatively small amount of CpG dinucleotides present in their genomes, they seem to avoid it. The goal of this work is to describe the individual types of these modifications and show their possible importance in the infectious cycle of polyomaviruses. Key words: polyomavirus, epigenetics, histone modification, DNA methylation, CpG dinucleotides
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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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Vesicular trafficking from acidic compartments to the endoplasmic reticulum
Polidarová, Markéta ; Forstová, Jitka (advisor) ; Plocek, Vítězslav (referee)
The cell uses retrograde transport from endosomes to Golgi apparatus and further to the endoplasmic reticulum to recycle its receptors and other proteins. There are several pathways starting on different types of endosomes aimed to the trans-Golgi network and from it further to the endoplasmic reticulum. From the early and maturing endosomes the proteins are transported using the retromer complex. Rab9 GTPase is essential for transport from the late endosomes. Rab6 and Rab11 play major role in the transport form the recycling endosomes. There are two pathways going through the Golgi apparatus. The first one is mediated by COPI vesicles which are regulated by Arf1 GTPase and the pathway is sensitive to brefeldin A. The second pathway is regulated by Rab6 GTPase. Except for endogenous proteins the retrograde transport is used by protein toxins and small unenveloped DNA viruses as well. Rab6 pathway from the recycling endosomes and through the Golgi apparatus is characteristic for Shiga toxin. The retrograde transport of ricin starts on the early endosomes and is less clear. Scientists only started uncovering the transport of small unenveloped DNA viruses.
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Preparation of expression vectors and virus mutants for studies of the minor structural proteins of polyomaviruses.
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šroller, Vojtěch (referee)
Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...
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Experimental system for the mouse polyomavirus life cycle study
Pergner, Jiří ; Mašek, Tomáš (referee) ; Španielová, Hana (advisor)
Experimental system for the mouse polyomavirus life cycle study Abstract: Murine polyomavirus (MPyV) is the prototype of the Polyomaviridae family. This family includes also some important human pathogens (BKV, JCV, Merkel cell polyomavirus). Due to their specific properties viruses within this family may serve as versatile vectors for gene therapy or recombinant vaccine production. New methodological approaches may help to understand some yet unknown facts about MPyV life cycle. Clarification of some processes during murine polyomavirus life cycle may be also important to fully exploit polyomaviruses for therapeutic purposes. The aim of this diploma thesis was to preparare two innovative experimental systems that extend possibilities of studying the life cycle of MPyV. The first part of the diploma thesis focusses on construction of recombinant MPyV which expresses yellow fluorescent protein (EYFP) in the early stages of infection. Such virus can be very useful for studying the infection spreading by live- cell imaging and Fluorescence-Activated Cell Sorting (FACS) and can be employed for co- localization studies of YFP-tagged LT antigen with certain cellular proteins. Second part of the diploma thesis describes preparation of a hybrid cell line prepared by fusion of mouse and monkey cells. This new cell...
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