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Study of disassembly/reassembly mechanisms of ferritin protein cages and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger, Zbyněk (advisor)
Diploma thesis deals with the study of dissociation and reassociation of ferritin protein cages and their use in nanomedicine. Most studies that are focused on targeted transport of pharmaceuticals using ferritin cages work with horse spleen ferritin. It is, however, its origin, which leads to increasingly frequent questions about possible immunogenicity in the patient's organism, which also provides the main motivation to test the possibility of encapsulation of low-molecular drugs into ferritins originating from alternative organisms. In the practical part the method for the study of dissociation was experimentally designed. Native polyacrylamide gel electrophoresis was used to study dissociation of equine ferritin composed of different subunit, human ferritin, and archeal Pyrococcus furiosus ferritin. The obtained subunit dissociation results were used to encapsulate the low molecular chemotherapeutic drug doxorubicin and for further characterization of the ferritin-doxorubicin complex. The efficacy of the designed nanoformulations has been verified in the treatment of malignant breast cancer. Human ferritin proves to be the optimal one. Its composition of heavy subunits corresponds to a lower protein stability, thus a more efficient opening of the structure and consequent encapsulation of the cytostatics occurs. With its 60% encapsulation efficiency of doxorubicin, low polydispersity index, effective cytotoxicity of ferritin-doxorubicin complex and minimal risk of immune response to the patient's organism, human ferritin achieves better results than commonly used horse spleen ferritin.
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Study of expression of transferrin receptors (TfR1) and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger,, Zbyněk (advisor)
Bachelor thesis deals with the expression of the transferrin receptor (TfR1) and its use in nanomedicine. During the last decade, nanotechnology emerged as one of the central milestones in connecting all scientific and technological disciplines. Nanomedicine already demonstrated efficacy not only in animal models of cancer but also in clinical practice. The theoretical part is not only aimed at cancer of the human population, but also at the possibilities of targeted drug delivery into the tumor tissue, which greatly reduces the otherwise serious side effects of conventional treatment – systemic toxicity. The practical part is focused on optimization for studying the expression of the transferrin receptor, a protein overexpressed by neoplastic cells aiming to enrich the higher metabolic needs of tumor cells. The optimal conditions were as follows: lysate of 50 000 cells applied with nonreducing nondenaturing buffer and the concentration of the primary antibody of 1.0 𝜇g/ml. Different levels of TfR1 expression were detected, depending on the type of tumor cells. The cell lines of neuroblastoma, prostate cancer (occurence in every 7th man) and breast cancer (occurence in every 8th woman) were selected for the next experiments. Via this transferrin receptor, apoferritin, which is a protein storaging iron ions in many organisms, can be internalized into cells. Artificially, the internal cavity of apoferritin may be used for encapsulation and transport of any molecules. In the case of this bachelor thesis, the apoferritin was used for delivery of doxorubicin. Doxorubicin has been used for cancer treatment for more than 30 years; however, its administered dose is limited by its high toxicity. This can be reduced by its encapsulation in a suitable vector for targeted transport to the tumor cells only. Apoferritin could serve as such suitable vector. In this thesis, the suitable usage of apoferritin as a nanocarrier for chemotherapeutic delivery was confirmed. Its molecule size of 10-12 nm allows it to employ the effect of increased permeability and retention. At the same time, this size makes it possible to avoid renal clearance. The properties of encapsulated doxorubicin are not affected by apoferritin, thus preserving its toxicity for cells with a high level of TfR1 expression (30% growth inhibition of these cells after 24 h of treatment).
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The effect of aspartate β-hydroxylase inhibition on immune reactions
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Filipp, Dominik (referee)
Aspartate β-hydroxylase (ASPH) is an enzyme that contributes to tumor progression by enhancing the proliferation, migration and invasiveness of cancer cells. Its expression is mostly negligible in normal cells, whereas it is often overexpressed in cancer cells. An inhibitory effect of ASPH on immune cells, specifically on natural killer cells, has also been demonstrated. Thus, the ASPH enzyme could be a new target for cancer immunotherapy. The aim of this thesis was to show the effect of ASPH inhibition on the immune response. Firstly, it was found that ASPH inhibition contributes to the anti-tumor effect of DNA vaccination. The reduction of tumors induced by ASPH inhibition in combination with DNA vaccination was shown to be mainly caused by CD8+ T lymphocytes. Subsequently, the specific activation of T lymphocytes was confirmed by the ELISPOT assay. In case of non-specific activation of T lymphocytes, ASPH inhibition had no effect on the direct activation of CD8+ T lymphocytes. Finally, it was found that plasmacytoid dendritic cells did not contribute to CD8+ T lymphocyte activation after ASPH inhibition. Thus, the results demonstrate that inhibition of ASPH contributes to the activation of adaptive immunity induced by DNA vaccination, but the mechanism of this activation remains unknown.
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Immune reactions induced by SARS-CoV-2 infection
Krausová, Kateřina ; Šmahel, Michal (advisor) ; Šroller, Vojtěch (referee)
Coronavirus disease 2019 (COVID-19) pandemic caused by newly discovered Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe health and economic problems all over the world. The disease severity depends mainly on the host's immune response to SARS-CoV-2. This virus uses many mechanisms for escape from the host's immune system. The major evasion mechanisms include suppression of interferon production at the early phase of infection, exhaustion of natural killer cells and induction of a cytokine storm. After the innate immune response, mechanisms of adaptive immunity join the defense against the virus. Patients with severe cases have a significant reduction in the amount of both helper CD4+ T cells and cytotoxic CD8+ T cells. On the contrary, these patients have an increased level of antibodies. Even though there have been many findings about immune reactions to SARS-CoV-2 in the year after its discovery, there are still many unknowns. Vaccines, which are successful at preventing COVID-19, have been developed in a short time. However, an important remaining question for further research is the longevity of immune memory after vaccination or after suffering from COVID-19.
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Study of disassembly/reassembly mechanisms of ferritin protein cages and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger, Zbyněk (advisor)
Diploma thesis deals with the study of dissociation and reassociation of ferritin protein cages and their use in nanomedicine. Most studies that are focused on targeted transport of pharmaceuticals using ferritin cages work with horse spleen ferritin. It is, however, its origin, which leads to increasingly frequent questions about possible immunogenicity in the patient's organism, which also provides the main motivation to test the possibility of encapsulation of low-molecular drugs into ferritins originating from alternative organisms. In the practical part the method for the study of dissociation was experimentally designed. Native polyacrylamide gel electrophoresis was used to study dissociation of equine ferritin composed of different subunit, human ferritin, and archeal Pyrococcus furiosus ferritin. The obtained subunit dissociation results were used to encapsulate the low molecular chemotherapeutic drug doxorubicin and for further characterization of the ferritin-doxorubicin complex. The efficacy of the designed nanoformulations has been verified in the treatment of malignant breast cancer. Human ferritin proves to be the optimal one. Its composition of heavy subunits corresponds to a lower protein stability, thus a more efficient opening of the structure and consequent encapsulation of the cytostatics occurs. With its 60% encapsulation efficiency of doxorubicin, low polydispersity index, effective cytotoxicity of ferritin-doxorubicin complex and minimal risk of immune response to the patient's organism, human ferritin achieves better results than commonly used horse spleen ferritin.
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Four elements - the possibility of development of sensory perception in preschool children
KRAUSOVÁ, Kateřina
The theme of the thesis is a development of sensory perception of preschool children in terms of four elements. The theoretical part is focused on four elements from the perspective of ancient philosophy, and the concept of elements in other cultures. The thesis defines sensory perception in terms of importance and development of specialized sensory organs and their diagnostics for the development of school readiness. Furthermore, it introduces the concept of senses in Waldorf pedagogy and medicine. Last part is about defining and finding of the relationship between a child and nature. Practical part introduces specific suggestions for the development of sensory perception, empathy and creativity for each element individually: water, earth, fire / heat and air / wind. The aim of the thesis is to foster natural children curiosity, gradually develop and address various senses through which we can understand the world around us and within us. At the same time it mentions pro-social, aesthetic and intellectual topics as well.
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Logopaedic prevention in Plzeň region Kindergartens
KRAUSOVÁ, Kateřina
This bachelor's essay deals with a prevention of a speech therapy in kindergartens in the Pilsner region. A theoretical part describes a characteristic of the Pilsner region and its dialect. It contains a speech therapy preventation, definitions of basic terms and a primary speech therapy preventation in kindergartens. It defines terms of a speech therapy screening and a speech therapy edification. It mentions qualification demands of a speech therapy, teacher's competencies and conditions for a speech development's support. It defines a content of a speech therapy prevention. A practical part contains a research and thematic units. There were used questionaries to collect facts.
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Study of expression of transferrin receptors (TfR1) and their utilization in nanomedicine
Krausová, Kateřina ; Fohlerová, Zdenka (referee) ; Heger,, Zbyněk (advisor)
Bachelor thesis deals with the expression of the transferrin receptor (TfR1) and its use in nanomedicine. During the last decade, nanotechnology emerged as one of the central milestones in connecting all scientific and technological disciplines. Nanomedicine already demonstrated efficacy not only in animal models of cancer but also in clinical practice. The theoretical part is not only aimed at cancer of the human population, but also at the possibilities of targeted drug delivery into the tumor tissue, which greatly reduces the otherwise serious side effects of conventional treatment – systemic toxicity. The practical part is focused on optimization for studying the expression of the transferrin receptor, a protein overexpressed by neoplastic cells aiming to enrich the higher metabolic needs of tumor cells. The optimal conditions were as follows: lysate of 50 000 cells applied with nonreducing nondenaturing buffer and the concentration of the primary antibody of 1.0 𝜇g/ml. Different levels of TfR1 expression were detected, depending on the type of tumor cells. The cell lines of neuroblastoma, prostate cancer (occurence in every 7th man) and breast cancer (occurence in every 8th woman) were selected for the next experiments. Via this transferrin receptor, apoferritin, which is a protein storaging iron ions in many organisms, can be internalized into cells. Artificially, the internal cavity of apoferritin may be used for encapsulation and transport of any molecules. In the case of this bachelor thesis, the apoferritin was used for delivery of doxorubicin. Doxorubicin has been used for cancer treatment for more than 30 years; however, its administered dose is limited by its high toxicity. This can be reduced by its encapsulation in a suitable vector for targeted transport to the tumor cells only. Apoferritin could serve as such suitable vector. In this thesis, the suitable usage of apoferritin as a nanocarrier for chemotherapeutic delivery was confirmed. Its molecule size of 10-12 nm allows it to employ the effect of increased permeability and retention. At the same time, this size makes it possible to avoid renal clearance. The properties of encapsulated doxorubicin are not affected by apoferritin, thus preserving its toxicity for cells with a high level of TfR1 expression (30% growth inhibition of these cells after 24 h of treatment).
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