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Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
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Pulse proteolysis in evaluation of conformational stability of cytochromes b5
Maroušková, Růžena ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Mixed-function oxidases play a major role in the metabolism of xenobiotics. The main component of this system is the cytochrome P450, it oxidizes substrates coming into our body to more polar products. Another component of mixed-function system - the cytochrome b5 (cyt b5) is able to modulate the function of cytochrome P450, the mechanism of this modulation is yet unknown. However, it is believed that it could be mediated via transfer of electron or allosteric modulation of cytochrome P450 caused by interaction with cyt b5. The aim of this thesis was to find and prepare analogs of cyt b5, which are unable to transfer electrons to cytochrome P450 and simultaneously are structurally very similar to native cyt b5. The conformational stability of cyt b5 and its analogs was monitored using pulse proteolysis. This method employs proteases to cleave the evaluated protein at varying concentration of a denaturant. For soluble proteins, urea is typically used as denaturant in combination with thermolysin as protease. While for membrane proteins, sodium dodecyl sulfate (SDS) is usually used as denaturant together with subtilisin as protease. The aim of this thesis was to use these methods to compare a conformational stability of the native human cyt b5 with apo-cyt b5 and analogs of the cyt b5 reconstituted...
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Optimization of DNA amplification isolated from buccal swabs for sequencing purposes
ROŠTÍKOVÁ, Kristýna
The topic of my bachelor thesis was the issue of taking primary DNA samples from the buccal mucosa, determination of the ideal length and optimal conditions and subsequent analysis of these samples. In the theoretical part, I focused on the preanalytical phase of the laboratory examination. It was about, the methods of sampling and the type of material that is most often used used for DNA isolation in the molecular biological laboratory. Subsequently, I focused on the methods themselves, which are used for DNA analysis. These included DNA isolation, DNA amplification methods, nucleic acid electrophoresis and DNA sequencing. In the practical part, I processed 42 samples. First, I isolated DNA from the samples and measured its concentration and purity. Then I performed electrophoresis in these samples and based on its results I decided which samples I would process further by PCR. Subsequently, I determined by electrophoresis whether DNA was amplified in the samples. Finally, I selected samples that I sent for sequencing. The aim of my bachelor thesis was to determine the optimal length and storage conditions of samples from the buccal mucosa for further processing. Then I evaluated how these parameters affect the quality, purity and quantity of isolated DNA as well as the course of the polymerase chain reaction. According to the obtained results, the best option is to store the samples in a refrigerator temperature and perform their analysis in the shortest possible time, or to store samples in a refrigerator temperature and process them within weeks to three months.
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Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
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Health risk and profit of genetically modified golden rice
KUČEROVÁ, Kateřina
People in developing countries very often suffer from severe vitamin A deficiency due to an insufficient and unbalanced diet. This vitamin is necessary, among other things, for the production of the visual pigment rhodopsin. According to the World Health Organization, up to half a million children go blind each year because of this deficit. Its deficiency also weakens the immune system and thus significantly increases the risk of death from various infectious diseases. The best solution to this deficiency would be if the vitamin was contained directly in the only food that these people get - in husked rice. There is already a special, genetically modified rice, into which has been introduced by a complicated and extensive modification of its DNA the entire metabolic pathway, which ensures the production of betacarotene in this crop. In the theoretical part I deal with the golden rice project and the origin of this genetically modified crop by the method of transgenesis, using bacteria of the genus Agrobacterium tumefaciens. Plasmids of this bacterium are able to incorporate parts of their genetic information into the target organism. Thanks to restriction enzymes, we can insert genes selected by us into plasmids, which we then introduce into a specific plant. But are these products safe? Opinions on these crops are very diverse, but one thing interests everyone: how to safely identify a genetically engineered plant? In the laboratory, these experiments can best be mastered on model material. Therefore, in the experimental part of my bachelor thesis I deal with the transgenesis of the model plant Nicotiana tabacum using a selected strain of Agrobacterium tumefaciens. These bacterial strains were provided to me from the private collections of the Institute of Molecular Plant Biology in České Budějovice. The aim of my work in the laboratory was to master the practical methodology of preparation of genetically modified plant and subsequent verification of the presence of introduced genes in the examined samples of the model organism. Specifically, genes for antibiotic resistance. DNA isolation, PCR amplification and electrophoretic assays were used for this purpose. The signal gene used in agroinfection was verified too. Also, it was monitored how many copies of the transgene were integrated into the research material during agroinfection.
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Human infectious diseases and GMO
KOBLIHOVÁ, Tereza
This bachelor thesis is focused on the detection of the transgenes in DNA of the genetically modified plants, especially on the detection of the transgene nptII. Moreover, it will discuss the determinations of the fission ratio (the amount of copies) of the injected transgenes into the next generation. The theoretical part of this bachelor thesis includes information about bacteria because some of their kinds are the reasons of human infection diseases, however, couple of them are used in genetic engineering for plant transformation at the same time. The diseases which are caused by bacteria are cured by antibiotics. Antibiotics are antimicrobic substances which are used to stop the growth of or exterminate these microorganisms. The theoretical part is especially focused on findings related to antibiotics and gene manipulated organisms (GMO). They are related to the concerns about using the selective gene nptII in the construct of these organisms, because this gene codes enzyme neomycin phosphotransferase II, which deactivates the effects of the Antibiotic called kanamycin. Organisms, where this gene is included are getting resistant to these antibiotics. The aim of the methodical part was the cultivation of own genetic modified plants carrying artificially injected genes for the resistance to kanamycin (nptII) and the signal gene GUS. Furthermore, we were looking for the subsequent determination of presence of the gene ntpII in the samples from these plants. The first necessary step to the determination of the gene nptII in the transgenic plants was multiplying of the DNA section containing this gene. That was accomplished by Polymerase Chain Reaction (PCR). After PCR was adapted, the detection of nptII gene in samples was made by gel electrophoresis. A histochemical test was used for the detection of the presence of the gene nptII in the plant samples and the confirmation of the transformation. As a model organism, the Nicotiana tabacum was chosen, which was transformed by Agrobacterium tumefaciens.
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Analysis and identification of proteins in organ dysfunction using proteomic methods
Tůma, Zdeněk ; Matějovič, Martin (advisor) ; Lopot, František (referee) ; Hernychová, Lenka (referee)
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...
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Structural study of the ASK1:thioredoxin complex.
Pšenáková, Katarína
5 ABSTRACT The mitogen-activated protein kinase (MAPK) cascade is an essential member of the cell defense system against stressors. The capability and efficiency of the cell reactions to different stress signals depend on signal transduction pathway, where signals from MAPK kinase kinase (MAP3K) are transferred through phosphorylation to downstream MAPK kinase (MAP2K) and finally to MAPK. Apoptosis signal-regulating kinase 1 (ASK1) is a member of a MAP3K family and its activation and inhibition has a significant participation in a regulation of cell response to stress stimuli. The regulation of ASK1 has a strong influence in pathogenesis of several diseases, the excessive activation of human ASK1 or failure in the control of its function are associated with cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, infectious diseases, tumorigenesis, asthma, diabetes and ageing. The activity of ASK1 is regulated by its interaction with several proteins, the attention is focused on two physiological inhibitors, mammalian thioredoxin (TRX) and the 14-3-3 protein. ASK1 in its inactive form is inhibited by bonds formation with TRX and 14-3-3, however the explicit mechanism of this interaction is unclear due to the absence of structural data. This work is a part of an extensive research about...
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The cancer cell proteome and its changes after anti-cancer drug treatment
Tylečková, Jiřina
Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...
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