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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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Modifikace ferritinových nanoklecí cílícími modalitami
Kurcová, Jana
In last two decades, application of protein nanoparticles has emerged as an excellent candidate in a field of biomedical research, especially as a tool for targeted drug delivery. Nanocarriers can ensure specific drug delivery into the damaged cells without the risk of premature release of the drug hence damage to normal cells. It is possible to modify the surface of nanocarriers with targeting modalities and use active targeting to improve cell specificity and internalization of nanocarriers. Therefore, this thesis is focused on specific surface modifications of nanocarriers with respect to active targeted drug delivery. Herein, a new method of covalent binding of the hexameric HWR (HWRGWV) peptide to the surface of fluorescently labeled apoferritin cages (APO-Cy7) using a zero-length crosslinker N,N’ dicyclohexylcarbodiimide (DCC), is presented. HWR peptide can specifically bind Fc region of antibodies enabling its application for site directed conjugation with various targeting antibodies. The optimal ratio of HWR to APO for maximum reaction effect was 1.26 mg HWR to 1 mg APO-Cy7. Furthermore, the stability of as-prepared fluorescent nanocarrier was confirmed after the binding with peptide. Fluorescence of nanocarrier increased in the emission maximum (λ = 773 nm) from 1943 ± 209 a.u. to 2594 ± 62 a.u. due to the transfer of electrons from the tryptophan residues of the peptide to Cy7. The size of the nanocarrier was 12.71 ± 0.49 nm with a surface ζ-potential of -14.37 ± 3.15 mV. The correct binding orientation of the peptide was confirmed using polyclonal IgG antibodies. After incubation and following purification, the presence of antibodies was proven spectrophotometrically, via SDS-PAGE and by changing the size of the nanocarrier. Thus, the presented work demonstrates a versatile platform suitable for a surface modification with targeting antibodies for utilization in active anticancer nanomedicine.
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Cílený transport bioaktivních molekul pomocí ferritinových nanoklecí
Rogotovskaya, Alexandra
Abstract This bachelor's thesis deals with the topic of targeted transport of bioactive molecules using ferritin nanocages. Nanoparticles are widely tested for use in contemporary medicine, not only for their use in cancer management. The use of these materials is preferred due to their ability to decrease unwanted and toxic effects of conventional chemotherapy. The use of nanoparticles ensures targeted transport, which results in reduction of the toxic effect of drugs. Among the most promising nanomedicinal vehicles belong ferritins. Ferritin is a globular protein and the main natural intracellular iron storage molecule in almost all living organisms. Ferritin specifically recognizes transferrin receptor 1 (TfR1), which is usually highly expressed on various types of tumor cells. Ferritin exhibits unique ability of reversible self-assembly. Furthermore, the surface of ferritins can be chemically or genetically modified to bear variety of cancer targeting ligands. In addition, due to its origin, ferritins are biocompatible and do not induce unwanted immune reactions. Thus, ferritins are promising delivery vehicles for targeted cancer therapy.
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Erythrocytapheresis in the Treatment of Patients with Hereditary Haemochromatosis
Řeháček, Vít ; Bláha, Milan (advisor) ; Gašová, Zdenka (referee) ; Penka, Miroslav (referee)
BACKGROUND: Hereditary hemochromatosis (HH) is one of the most common hereditary diseases of the Euro-American population. The disease is caused by increased absorption of iron from the intestine, regardless of its current stores in the body. Excess iron is stored in tissues and organs and causes their significant damage. HH treatment consists of regular blood withdrawal, activation of erythropoesis causes the utilization of excess iron and normalization of its reserves. The standard method of blood withdrawal is venipuncture, an alternative method with the possibility of collecting larger red cell volume in one procedure is erythrocytapheresis. The aim of the research was to create a group of patients with newly diagnosed HH, apply erythrocytapheresis, verify their effectiveness, optimize and standardize treatment to reduce ferritin levels in the induction phase of treatment below 50 μg/l and subsequently keep ferritin levels below 100 μg / l in the maintenance phase of treatment. METHODS: The group of patient was created in cooperation with hepatology departments of the Hradec Králové region, by testing related patients with HH and by screening blood donors. For erythrocytapheresis, a Haemonetics MCS+ separator (Haemonetics Corp., Braintree, MA, USA) was used. The SW of MCS+ can modify red cell...
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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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Comparison of surface ferritin with levels of CRP and erythrocyte sedimentation rate as markers of acute-phase inflammation.
STRUČKOVÁ, Daniela
The aim of the study is to compare the levels of ferritin, CRP and the rate of erythrocyte sedimentation. Ferritin and CRP belong to the acute phase proteins that are formed in increased levels in inflammatory conditions and certain diseases. CRP is one of the most important markers of acute phase inflammation for high sensitivity, it is increasing hours after the onset of the acute phase reaction and rises more than a hundred times during the first days. CRP was measured on a Beckman Coulter AU 680 analyzer using the reaction of antigen-antibody and photometric measurements. The ferritin measurement on the Architect analyzer is a two-stage immunoassay of the immunological chemiluminescent microparticle using. The resulting reaction is measured in the optical system. Examination of the rate of erythrocyte sedimentation is frequent, although not specific to a particular disease. However, it can be used in diagnosing or monitoring of the effect therapy efficiency. The principle of measuring on Alifax Test analyzer is monitoring the aggregate capacity of red cells through optical density. These three parameters were measured by 1000 patients, the ratios of all measured values were calculated and compared with each other by means of linear regression graphs and the calculated correlation coefficients. The correlation coefficients have shown a low degree of dependence between ferritin and CRP and between CRP and age. The medium degree of dependence was proofed between ferritin and ESR parameters, between CRP and ESR and between age and ferritin, and age and ESR. All samplies were performed lege artis according to the instructions of the standard operating procedure of the company synlab czech, s.r.o. After collecting, these samples were measured in the laboratory synlab czech, s.r.o., Vrbenská 197/23, České Budějovice.
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New ferritin nanoparticles for specific targeting of experimental melanoma in mice: in vitro and in vivo tests.
Rajsiglová, Lenka ; Vannucci, Luca Ernesto (advisor) ; Šírová, Milada (referee)
Cancer diseases represent second most frequent cause of death after cardiovascular diseases in Europe. Nowadays used medical treatments like chemotherapy and radiotherapy are nonspecific and cause huge side effects. Various systems to deliver therapy directly inside the tumour microenvironment and reduce side effects are under development. Protein nanoparticles seem to be very promising strategy to achieve that goal. Our group in cooperation with CNR in Rome tested nanoparticles based on heavy chain of human ferritine. These constructs, modified to expose the tumor targeting molecule, were able to be specifically internalised by B16F10 melanoma cells in vitro. They also specifically target and localise at the sites of primary melanoma and lung metastases of different size in mouse in vivo model. These nanoparticles can carry either therapeutic or diagnostic molecules. Thus they represent a suitable candidate for further studies for potential use in clinical praxis as a diagnostic and/or therapeutic agents (theranostics). Powered by TCPDF (www.tcpdf.org)
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Anaemia focusing on microcytic anaemia
REMTOVÁ, Eliška
The theme of my bachelor thesis is focused on the anemia, especially microcytic anemia. The introduction describes only the general facts about this disease and further focuses only on the microcytic anemia. Every single type is described in the separate theoretical part. Iron deficiency anemia or anemia caused by the lack of iron is the most common type at all. It occurs in up to one third of the population in developing and also developed countries all over the world. I describe the second most common chronic disease anemia as well and then rarer thalassemia at the end of this paper. The practical part is devoted to analyze all of the collected data. At first the results of blood counts of the patients, who were examined by the Department of Clinical Hematology at the Ceske Budejovice Inc. hospital, are evaluated. The evaluation is performed on an automated hematology analyzer Unicel DxH 800 Beckman Coulter. I had examined the frequency of an anemic and subsequently microcytic patients from a total of 7,664 tested patients. The resulting number with this anemia was 28%. I had also focused on the evaluation of iron, transferrin and ferritin parameters which are determined on the analyzer ADVIA. I gained the necessary data at the Department of Clinical Chemistry of Ceske Budejovice Inc. hospital. The most important goal was to determine how many of microcytic patients were tested for iron deficiency. The resulting number was only 18% examined patients in this way. Then I had looked up how many of these patients were subsequently examined to transferrin and ferritin. There were many patients who were completely missing the results of these tests, so it was not possible to determine the exact number. Based on all these detected parameters I could evaluate the patients, who suffered the anemia and which type they suffered. The anemia of chronic disease was the most often type and it was found in 24 patients. The iron deficiency anemia suffered only 11 of the examined patients.
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