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Biological characteristics of polysccharide based contrast agents for cancer diagnostics
Křivánková, Markéta ; Jirák, Daniel (advisor) ; Shapoval, Oleksandr (referee) ; Vannucci, Luca Ernesto (referee)
Despite all the progress made in the treatment of cancer in recent years, it is still necessary to continue with the research of more effective and specific drugs. In recent years, there has been a growing interest in personalized medicine and its application through drug delivery systems, which could help increase the specificity of cancer treatment and subsequently its effectiveness. Drug delivery systems can use different platforms for their design, whether they are liposomes, micelles, nano crystals or others. A very interesting platform for the construction of drug delivery systems are polysaccharides, which were, as carriers of contrast agents in order to effectively display tumours, characterized in this doctoral thesis. But polysaccharides are interesting for more reasons. Both by its availability, and by its biocompatibility and non-toxic character. In this doctoral thesis we deal with two types of polysaccharides conjugates with linked contrast agents for magnetic resonance and fluorescent imaging. The first type of polysaccharide is glycogen, the second is mannan. Both constructs - glycogen and mannan based, were synthesized in a version with and without polymethyloxazolin, which should prolong their circulation in the organism. Both types of polysaccharide conjugates used passive...
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Alternative methods for visualization of pancreatic islets.
Gálisová, Andrea ; Jirák, Daniel (advisor) ; Krššák, Martin (referee) ; Kratochvílová, Simona (referee)
Transplantation of pancreatic islets (PIs) represents an alternative treatment for type 1 diabetes mellitus. Post-transplant monitoring of islets by a reliable imaging method may contribute to the improvement of the transplantation outcome. In this thesis, novel visualization approaches for PIs were tested using magnetic resonance (MR) and optical imaging on phantoms and experimental animals, including Chemical Exchange Saturation Transfer (CEST) MR, fluorine (19 F) MR, bioluminescence and fluorescence imaging. MR imaging based on frequency-selective method CEST was performed on islets labeled with Eu-/Yb-based chelates. Labeled islets possessed low MR signal in phantoms, what would have been unsatisfactory for in vivo applications. Moreover, viability and function of labeled islets was impaired reflecting limited applicability of these agents for islet labeling and visualization. Genetically modified bioluminescent islets showed suitable properties for longitudinal tracking of their post-transplant fate at an artificial transplant site - subcutaneously implanted polymeric scaffolds. Using multimodal imaging (MR and bioluminescence), the optimal timing for transplantation of islets into the scaffolds was assessed in diabetic rats. Islets transplanted into scaffolds using the optimized timing scheme...
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Metabolism of new polysacharidic nanomaterials for biomedicinal applications
Jirátová, Markéta ; Hrubý, Martin (advisor) ; Jirák, Daniel (referee)
Cancer is one of the leading cause of death in modern world, so there is an emerging demand for better diagnostic tools and more specific less toxique therapeutics. Nanoparticles offers characteristics that could fullfill such perspectives. They can easily target tumor by ehanced permeation and retention effect (EPR). Nanoparticles can combine more than one imaging properties, so we can say that they are multimodal, some of them could combine diagnostic and therapeutic molecules in one nanoparticle, which is now highly popular topic of nanoparticles for theranostics . The aim of this thesis was to characterize new multimodal glycogen-based nanoparticle. Glycogen is an ideal structure for nanoparticle design. Glycogen is part of natural dendrimers group which are easily to modify. Glycogen's size is suitable for EPR effect. We have evaluated biological characteristics of five different types of modified glycogen. The in vitro experiments were carried on HepG2 cells. We have set time curve of cellular uptake of this glycogen probes, evaluated cytoplasmatic localization and for the first time we have carried MTT assay. Biodistribution studies on CD1-Nude mice were performed by using non-invasive method for measuring in vivo fluorescence. In conlusion we've provided some of the biological characteristics of new...
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Surface-modified nanoparticles in the treatment of serious CNS diseases
Mareková, Dana ; Jendelová, Pavla (advisor) ; Králová, Věra (referee) ; Jirák, Daniel (referee)
Glioblastoma multiforme (GBM) is a primary adult brain tumour with an unfavourable prognosis. Standard therapies have a number of side effects, posing a challenge and leading to attempts at new therapeutic approaches. One of these is the use of theranostic nanoparticles with modified surfaces. We have established primary lines from GBM patient samples and characterized them not only in 2D and 3D cultures but also after passaging in the brains of immunodeficient mice. We tested our modified superparamagnetic nanoparticles carrying the drug doxorubicin and RGDS peptides targeting tumor cells on these lines. The γ- Fe2O3@P(HP-MAH)-RGDS-Dox nanoparticles not only reduced cell proliferation in vitro but also incorporated into the tumor and inhibited tumor growth in vivo. We also developed magnetic nanoparticles containing Mn and Zn, which can replace commercially available iron oxide-based contrast agents with their magnetic properties and can be used to label cells for in vivo magnetic resonance imaging. Both in vitro and in vivo tests of the new nanoparticles have revealed that they can be used as a suitable and effective tool for cell tracking using magnetic resonance imaging even where the use of current contrast agents is contraindicated.
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Alternative methods for visualization of pancreatic islets.
Gálisová, Andrea ; Jirák, Daniel (advisor) ; Krššák, Martin (referee) ; Kratochvílová, Simona (referee)
Transplantation of pancreatic islets (PIs) represents an alternative treatment for type 1 diabetes mellitus. Post-transplant monitoring of islets by a reliable imaging method may contribute to the improvement of the transplantation outcome. In this thesis, novel visualization approaches for PIs were tested using magnetic resonance (MR) and optical imaging on phantoms and experimental animals, including Chemical Exchange Saturation Transfer (CEST) MR, fluorine (19 F) MR, bioluminescence and fluorescence imaging. MR imaging based on frequency-selective method CEST was performed on islets labeled with Eu-/Yb-based chelates. Labeled islets possessed low MR signal in phantoms, what would have been unsatisfactory for in vivo applications. Moreover, viability and function of labeled islets was impaired reflecting limited applicability of these agents for islet labeling and visualization. Genetically modified bioluminescent islets showed suitable properties for longitudinal tracking of their post-transplant fate at an artificial transplant site - subcutaneously implanted polymeric scaffolds. Using multimodal imaging (MR and bioluminescence), the optimal timing for transplantation of islets into the scaffolds was assessed in diabetic rats. Islets transplanted into scaffolds using the optimized timing scheme...
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Preparation and characterization of synthetic mRNA coding for pancreatic transcription factors
Loukotová, Šárka ; Hodek, Petr (advisor) ; Jirák, Daniel (referee)
Diabetes mellitus type I is severe autoimmune disease which is caused by destruction of insulin-producing β-cells in pancreas. Diabetic patients are dependent on external usage of insulin during their whole life. Nowadays the only treatment of diabetes type I is transplantation of entire pancreas or isolated Langerhans islets. Due to the fact that this kind of treatment is very demanding and limited availability of suitable donors, the researchers are intensively working on development of new alternative ways how to produce the insulin-producing cells. One of the possible approaches on producing insulin-positive cells is transdifferentiation of pancreatic exocrine cells via transcription factors. In this diploma thesis, the transdifferentiation of exocrine cells AR42J was carried out with in vitro synthesized mRNA encoding transcription factors Pdx1, Ngn3 and MafA. The primary mRNA structure was optimized in order to prepare highly stable mRNA which is correctly translated into the protein. The main stabilizing elements in mRNA structure include 3' and 5' untranslated region derived from highly stable β-globin mRNA. In order to verify the function of synthetic mRNA the immunofluorescence staining of transcription factors has been investigated. Synthetic mRNAs encoding transcription factors Pdx1,...
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Metabolism of new polysacharidic nanomaterials for biomedicinal applications
Jirátová, Markéta ; Hrubý, Martin (advisor) ; Jirák, Daniel (referee)
Cancer is one of the leading cause of death in modern world, so there is an emerging demand for better diagnostic tools and more specific less toxique therapeutics. Nanoparticles offers characteristics that could fullfill such perspectives. They can easily target tumor by ehanced permeation and retention effect (EPR). Nanoparticles can combine more than one imaging properties, so we can say that they are multimodal, some of them could combine diagnostic and therapeutic molecules in one nanoparticle, which is now highly popular topic of nanoparticles for theranostics . The aim of this thesis was to characterize new multimodal glycogen-based nanoparticle. Glycogen is an ideal structure for nanoparticle design. Glycogen is part of natural dendrimers group which are easily to modify. Glycogen's size is suitable for EPR effect. We have evaluated biological characteristics of five different types of modified glycogen. The in vitro experiments were carried on HepG2 cells. We have set time curve of cellular uptake of this glycogen probes, evaluated cytoplasmatic localization and for the first time we have carried MTT assay. Biodistribution studies on CD1-Nude mice were performed by using non-invasive method for measuring in vivo fluorescence. In conlusion we've provided some of the biological characteristics of new...
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Cardiovascular involvement in patients with Fabry disease
Jirák, Daniel ; Hájek, Milan (advisor) ; Vymazal, Josef (referee) ; Žižka, Jan (referee)
The evaluation of clinical magnetic resonance (MR) images is influenced by errors (subjective evaluation, noise, etc.). It is possible to eliminate these errors by using certain mathematical and statistical methods. One of these methods is texture analysis (TA), which can describe images quantitatively by computed texture parameters. The aim of this thesis was to analyze MR images of various subjects (phantoms, apples, calf muscles, livers) by TA and to evaluate its possible use in clinical practice. The main tasks of the thesis were: 1) to develop a new phantom suitable for TA and MR imaging that is biochemically and mechanically stable 2) to optimize an algorithm for TA parameter selection and classification 3) to apply TA in evaluating MR images. The first methodical part of the thesis deals with the optimization of texture parameter selection and texture classification. For this purpose a new type of phantom was developed. This PSAG phantom, with a nodular structure from polystyrene spheres (PS) placed into agar (AG), is suitable for MR imaging and for TA because it produces a strong MR signal and because its T1 and T2 relaxation times are in the range of biological tissue and can be easily modified by employing various densities of PS spheres or by the addition of a contrast agent. Measurements of PSAG...
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