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Micronuclei and their connection with intracellular innate immunity and viral infection
Knoblochová, Kateřina ; Bruštíková, Kateřina (advisor) ; Španielová, Hana (referee)
Micronuclei are tiny structures that contain nuclear DNA and a membrane derived from the nucleus. They emerge in cells that have been exposed to severe stress factors, such as viral infections, radiation, or genotoxic substances. While micronuclei have long been used as markers of genotoxic stress, the mechanism of their formation and internal processes are not yet fully understood. DNA enclosed inside micronuclei is restructured in an atypical manner, which may induce mutations and accelerate oncogenic transformation of the cell. Due to these processes micronuclei can also act as reservoirs of immunostimulatory nucleic acids, which may potentially be detected by molecular sensors. Therefore, studying micronuclei is significant in relation to the activation of signaling pathways that are part of the innate intracellular immunity. This work summarizes the current knowledge about micronuclei and their connection to innate intracellular immunity and viral infection. Keywords: micronuclei, innate immunity, molecular sensors, chromotripsis
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The role of phosphoinositides in macropinocytosis
Hájek, Tomáš ; Doubravská, Lenka (advisor) ; Španielová, Hana (referee)
Macropinocytosis is non-selective actin-dependent type of endocytosis. It is important for the normal physiology of some cell types. However, it is used by intracellular parasites which internalise themselves into host cells in this way and also play a role in the nutritional supply in some type of cancer cells. During macropinocytosis a self-organized subdomain of plasma membrane is separated by a diffusion barrier named macropinocytic cup. RAC1-driven actin polymerization is required for membrane protrusion at the cup periphery, where a narrow ring of the actin nucleating factors is present. In contrast, actin dissociation occurs at the base of the cup due to RAS-controlled formation of phosphatidylinositol trisphosphates (PIP3). During cup closure sequential breakdown of PIP3 to phosphatidylinositol and acquisition of the endosomal identity of the newly formed vesicle is necessary. As a result of tubulation in the early stages of macropinocytosome maturation the vesicle decreases in diameter and stabilizes. At late stages the macropinocytic vesicle may fuse with the lysosome, allowing internalized material to enter this degradative organelle. Throughout the process specific types of phosphatidylinositols are part of the membrane providing signal transduction and membrane identity. These phospholipids...
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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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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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Molecular genetic characterization of the rare tumours of the urogenital tract.
Šteiner, Petr ; Vaněček, Tomáš (advisor) ; Španielová, Hana (referee)
The aim of this study was molecular characterization of four types of renal tumours (papillary renal cell carcinoma [PRCC], tubulocystic renal carcinoma [TCRC], pseudorossette forming renal carcinoma [PRRC] and unclassified renal carcinomas [URC]) and two types of rare tumours of the testes (Adult type of granulosa cell tumours [ATGCTs] and Incompletely differentiated sex cord stromal tumours [ISCSTs]). In case of TCRC the activity of signalling pathways involved in angiogenesis was studied. The aim was to determine the suitability of antiangiogenic agents for treatment of TCRC. Next, the methylation profile of 24 tumor suppressor genes was studied in TCRC and PRCC in order to analyze their similarity. Eventual differences could be helpful tool in differential diagnostics. Also, spectrum of chromosomal aberrations was analyzed by array-CGH in one case of PRRC and two cases of URC. Any unique aberration found would be useful in differential diagnostics of these tumors. Last, but not least, the specificity of mutation c.402C>G of FOXL2 gene for ovarian ATGCTs was verified by studying its occurrence in testicular ATGCTs and ISCSTs. Analysis of mRNA levels did not reveal any enhanced activity of the studied signalling pathways. Cluster analysis of methylation profiles showed close relationship between PRCC a...
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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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Stratification risk of disease progression in patients with abnormal cervical cytologic finding by means of molecular genetic analysis of selected biological factors
Gomolčáková, Barbora ; Kašpírková, Jana (advisor) ; Španielová, Hana (referee)
The aim of this thesis was to track the impact of selected herpesviruses, polyomaviruses, Chlamydia trachomatis and methylation of tumor supressor genes at the development and progression of high grade- lesion in HPV - positive patients by means of molecular-genetic techniques. Confirmation of these markers presence in women with severe lesions of cervix would help to raise necessary specificity of molecular genetics HPV testing and recommend it as a primary screening test for cervical carcinoma prevention. HPV testing could thus replace currently prevailing cytology which has relatively low sensitivity and therefore the number of false negative results. The analyzed samples consisted of cytological cervical smears of 51 HPV positive women, with histologically confirmed presence of severe lesions, collected in liquid medium. Samplings from 51 women without infection were used as a control. The possible effect on disease progress was confirmed only in the case of gene promoters' methylation whose presence was detected in up to 26 patients. It is, however, very unlikely that cancer would develop in all these women. This marker could thus help to stratify patients at risk but only to some extent. Although the individual effect of remaining markers has not been established in the carcinogenesis of cervical...
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Role of the PDZ Domain-Binding Motif of the Oncoprotein E6 in the Pathogenesis of High Risk Papillomaviruses
Faflíková, Tereza ; Španielová, Hana (advisor) ; Plocek, Vítězslav (referee)
The infection by high-risk human papillomaviruses is involved in causing cancers. The extreme carboxy terminus of high-risk α-HPV E6 protein contains a PDZ domain-binding motif. The E6 protein of high-risk HPV binds by PDZ domain-binding motif to cellular proteins containing PDZ domain, which participate in the maintenance of the cell polarity, in the stabilization of cell-cell junctions or in the regulation of cellular signaling pathways, e.g. hDlg (human homologue of Discs large protein), hScrib (human homologue of Drosophila tumor suppressor Scribble). Binding of E6 with cellular proteins prevents the induction of apoptosis and influences the enhancement of growth rate of infected cells, degradation of tight-junctions, regulation of cell polarity and vesicular transport. The interaction between E6 and cellular proteins increases the stability of E6 protein and protects E6 from proteasomal degradation. PDZ domain-binding motif of E6 high-risk HPV contributes to the development of malignant tumors.
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Preparation and characterization of modified viral particles derived from mouse polyomavirus for the transport of genes to increase the efficiency of transduction
Škvára, Petr ; Španielová, Hana (advisor) ; Sýkora, Michal (referee)
Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...
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