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High-dimensional single cell cytometry approach for immune system analysis
Koladiya, Abhishek ; Němečková, Šárka (advisor) ; Cosma, Antonio (referee) ; Souček, Karel (referee)
Technological advancement allowed for the advent of single-cell technologies capable of measuring a large number of cellular features simultaneously. These technologies have been subsequently used to shed light on the heterogeneity of cellular systems previously considered homogeneous, identifying the exclusive features of individual cells within cellular niches. Today, single-cell technologies represent an essential tool for studying the underlying immunological mechanisms correlating with disease. In this context, cytometry is one of the diverse high-throughput methods capable of examining more than 50 features per cell. However, utilising cytometry at its full potential requires the development of optimized assays. Additionally, the resulting high-dimensional data represent a challenge for existing computational techniques. This thesis attempts to address these challenges. The first part of the thesis is focused on developing a non-linear embedding algorithm for rapid analysis of cytometry datasets called EmbedSOM. The comparison of EmbedSOM with other state-of-the-art algorithms suggested the superiority of EmbedSOM with faster runtime. This is critical for the analysis of large datasets with millions of cells. Furthermore, EmbedSOM has additional functionality such as landmark guided...
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Mouse polyomavirus:The way of virus translocation to the cell nucleus and sensing of viral genomes by sensors of innate immunity
Soldatova, Irina ; Forstová, Jitka (advisor) ; Němečková, Šárka (referee) ; Pichová, Iva (referee)
To understand molecular mechanisms of individual steps of virus infection is a prerequisite for successful design of specific and effective antiviral drugs. Polyomaviruses, replicating in the cell nucleus, travel from plasma membrane to the endoplasmic reticulum (ER) in endosomes. However, it is not clear how they deliver their DNA genomes from ER to the nucleus. In this thesis, we found that partially disassembled virions of the Murine polyomavirus (MPyV) interact with importin β1 at around 6 hours post infection. Mutational disruption of the nuclear localization signal (NLS) of the major capsid protein, VP1, and/or common NLS sequence of the minor capsid proteins VP2 and VP3 did not affect the structure and composition of virions, but it resulted in decreased viral infectivity (up to 80%). Virions are thus released from ER to cytosol and translocate to the nucleus via nucleopores. Mutation analyses of NLSs of individual capsid proteins showed that MPyV virions can utilize VP1 and VP2/VP3 NLSs in concert. However, one functional NLS, either that of VP1 or VP2/3 seems to be sufficient for the delivery of VP1-VP2/3 complexes into the nucleus, although none of these proteins is delivered into the nucleus separately. Thus, the conformation of NLS regions given by the presence of all three capsid...
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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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Specific antiviral immunity in immunocompromised patients, not only after bone marrow transplantation
Pelák, Ondřej ; Kalina, Tomáš (advisor) ; Lysák, Daniel (referee) ; Němečková, Šárka (referee)
-3- SUMMARY Viral reactivations after hematopoietic cell transplantation contribute to significant morbidity and mortality. Timely immune reconstitution of functional T cell immunity is crucial in controlling these viral reactivations. In this thesis we were able to identify several functional T cell populations, which are responsible for fast resolution of viral reactivation. Appearance of some of these populations may be even used for prediction of the occurrence of viral reactivation. On the other hand, the administration of corticoids due to the treatment of graft versus host disease contributes as significant negative predictor to viral reactivation incidence. We are also offering an option of adoptively transferred virus specific T cells for patients suffering from prolonged virus complications, through identification of suitable donors by two different methods. Viral reactivations cause complication also in patients who underwent the solid organ transplantation. In this thesis we have found the connection between allo and virus specific T cells. We have successfully identified several cross-reactive T cell clones which have responded to both allo and viral stimulation. Further it seems that these clones may play an important role in rejection of transplanted kidney if there is also present an ongoing...
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Adoptive transfer of tumor-specific lymphocytes for cancer immunotherapy
Vávrová, Kateřina ; Bartůňková, Jiřina (advisor) ; Němečková, Šárka (referee) ; Reiniš, Milan (referee)
Prostate cancer is the second leading cause of cancer death in men in Europe and the US. In the context of previous preclinical experiments and clinical studies there are certain assumptions predicating successful application of immunotherapy in the treatment of patients with prostate cancer. Promising results have been achieved by a combination of different treatment modalities which provide a synergistic antitumor effect. One of these combinatorial options is the use of antitumor vaccines and adoptive T cell transfer. The topic of this thesis is to provide a fresh insight into the past and current trends following the long-term candidate's department program in the field of anti-tumor immunotherapy. The experimental part of this thesis revolves around our own results published in this field. The introductory chapter delivers a basic overview of cellular mechanisms of anti-tumor immunity and the role of individual immune components in these processes. Following chapters are dedicated to current immunotherapeutic approaches with emphasis on the adoptive T cell transfer and implication of this technology in the treatment of prostate cancer. The results section describes the establishment of our protocol for adoptive T cell transfer as well as the protocol for ex vivo enrichment of human T cell...
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Vliv zinku ve výživě drůbeže na senzorické vlastnosti masa
Němečková, Šárka
Experiment was conducted, in which were served different levels of zinc for the cockerels Ross 308. The aim of my study was to discover, if the level 204 mg Zn/kg of feed mixture will affect the sensory characteristics of the thigh and breast muscle after 30 days of storing. From the sensory analysis, performed by the 12 evaluators, appears, that the higher level of zinc did not have a statistically significant impact on the sensory characteristics of the thigh and breast muscle in majority parameters in my study. Exception was a statistically significant difference (p < 0,05) in aroma at the thigh muscle after correction of evaluator (Zn 24 mg was evaluated 1,03 +- 0,02 and Zn 204 mg was evaluated 0,97 +- 0,02). In the breast muscle of the experimental group was relatively the best rate the juiciness - 5 % above the average. In the thigh muscle of the experimental group was relatively the best rate also the juiciness - 10 % above the average.
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eating of the veterinary clinic
Němečková, Šárka ; Blasinski, Petr (referee) ; Počinková, Marcela (advisor)
The aim of this bachelor thesis is a processing of a project regarding heating system and warm water production for the veterinary clinic. The building has two overground floors and flat roof. A source of the heat for the building is an air-water heat pump. Heating surfaces are panel radiators. There is natural ventilation in the building mostly.
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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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Major structural protein of Polyomaviruses: Interactions with host cell structures
Mrkáček, Michal ; Horníková, Lenka (advisor) ; Němečková, Šárka (referee)
The main structural protein VP1 is the product of late polyomaviral genes and it is the largest and the most abundant protein of the whole polyomaviral capsid. Because of the low coding capacity of the polyomaviral genomes, it is considered that in addition to its structural role the VP1 protein might have some additional functions in the late phase of the infectious cycle. This diploma thesis is exactly on these additional functions. In the case of the VP1 protein of mouse polyomavirus, it was observed that the protein is capable of binding to the structure of cellular microtubules. The first objective of this work was to test whether pentamers of the VP1 protein are able of this binding without the participation of other cellular (or viral) proteins. Based on an in vitro experiment, we showed that protein VP1 binds to the structure of microtubules very inefficiently. The second objective of this work was to prepare a detection system that would allow an identification of potential interaction partners of BK polyomavirus VP1 protein. Therefore, expression plasmids producing the N and C-terminally tagged VP1 protein were prepared. These tagged proteins had the property of being biotinylated whilst being produced in the transfected cells. By using affinity chromatography, the entire protein complexes...
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New chimeric antigen receptor (CAR) for therapy of human cytomegalovirus (HCMV) infection
Kroutilová, Marie ; Němečková, Šárka (advisor) ; Forstová, Jitka (referee)
Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...
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