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Molekulárně-biologická diagnostika lidských polyomavirů
Ryšavá, Markéta
Most of the human population encounters human polyomaviruses during childhood, when the first infection is asymptomatic or with mild symptoms. However, these viruses persist in the human body and most often, they reactivate during immunosuppression. Together with JC virus reactivation, the progressive multifocal encephalopathy is associated. Hemorrhagic cystitis is associated with BK virus, resulting in the loss of allograft during kidney transplantation. Accurate diagnostics can detect viruses in a timely manner and mitigate tissue damage. This diploma thesis deals with biotechnologies, which can be used in virus detection with a focus on real time PCR, which is the gold standard in virus diagnostics. The literary research summarizes basic information about polyomaviruses with a focus on human BKV and JCV polyomaviruses. It summarizes the biotechnological methods used for detection of polyomaviruses, both in routine diagnosis and in alternative approaches involving biosensors and CRISPR. The experimental part of the work includes the design of a detection system for polyomaviruses from in silico genome analysis, through the design of potential primers, to theoretical specificity analysis. The practical part of the work compares the efficiency and sensitivity of amplification of two reaction mixtures, where one is intended for simple systems and the other for multiplexes. It also includes testing of selected additives of PCR and determining the sensitivity and validity parameters of the reaction mixture test selected for PCR system development. The results showed that detection using a multiplex reaction mixture is sufficiently sensitive, as it meets the conditions and requi-rements of clinical recommendations and at the same time shows very good values of sensitivity and specificity of the test comparable to published technologies. This reaction mixture appears to be relevant to the use of the development and optimization of a PCR system for the detection of polyomaviruses.
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Virom of lower urinary tracts
Cirbusová, Adéla ; Saláková, Martina (advisor) ; Španielová, Hana (referee)
The human urinary tract was considered to be a sterile environment for many years. However, studies over the past decade have shown that urine harbours rich microbial community which includes also viruses. Nevertheless, there is only very little known about urinary virome so far. Optimised Next Generation Sequencing (NGS) protocol was used to describe the urinary virome of three individuals. However, characterization of the virome from urine samples using NGS proved to be quite challenging, mainly due to observed viral genomes fragmentation. Despite this problem, it was possible to identify human endogenous retroviruses in all individuals and also JC polyomavirus in two of them. Quantitative PCR was further used to characterize part of the urinary virome represented by human DNA viruses. Possible differences in prevalence and viral load of human DNA viruses were observed in individuals with and without bladder carcinoma (bc). Urine of these patients was obtained from different sites of the urinary tract to further establish, if there is a difference in these samples. Torque Teno virus and JC polyomavirus were found as the most common viruses. Torque Teno virus was detected in 75 % patients with and 60 % patients without bc, JC polyomavirus in 43,8 % patients with and 50 % patients without bc. BK...
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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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Experimental system for the mouse polyomavirus life cycle study
Pergner, Jiří ; Španielová, Hana (advisor) ; Mašek, Tomáš (referee)
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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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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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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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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The noncoding control region of human polyomaviruses
Pešek, David ; Saláková, Martina (advisor) ; Váňová, Jana (referee)
Genome of human polyomavirus consists of circular dsDNA around 5000 base and can be divided into three functional regions - the early viral gene region (EVGR), that encodes the regulatory T antigen and miRNAs, noncoding control region (NCCR) harboring the minimal cis- acting elements involved in viral replication and the late viral gene region (LVGR), that encodes the structural capsid proteins. Noncoding control region contains the origin of viral replication that overlaps the promoters that control expresion of early and late gene region. Noncoding control region sequences include a large number of various binding sites for cellular transcription factors involved in regulation expression from LVGR and EVGR. This thesis describes the organization of the most variable region of the PyV genome, NCCR, in chosen polyomaviruses SV40, BKPyV and JCPyV. This region often undergoes rearrangements, deletion and point mutations that affects exression of human polyomavirus. Key words: polyomavirus, noncoding control region, BKPyV virus, JCPyV virus, SV40, large T antigen, transcriptional factor
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Effect of polyhistidine modification of viral particles on their intracellular localization and gene delivery to the nucleus
Číhařová, Barbora ; Španielová, Hana (advisor) ; Grantz Šašková, Klára (referee)
Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle's internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications - LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine...
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