|
|
Utilization of Cannabis Sativa in Teaching Chemistry
Stránská, Adéla ; Holada, Karel (advisor) ; Beneš, Pavel (referee)
The diploma thesis deals with Cannabis sativa. The aim is to focus on possible utilization of Cannabis sativa in teaching chemistry. The theoretical part describes the botany of cannabis, overviews phytocannabinoids, substances contained in cannabis, and it also includes wide use of cannabis in agriculture and industry. The practical part deals with the selection and arrangement of a possible curriculum about chromatography as the most affordable technique for analysis of cannabis at school, including the formulation of school experiment methodology using the thin-layer chromatography of Cannabis sativa extract to demonstrate the presence of phytocannabinoids. The practical part also includes the scheme of an educational project on cannabis and describes the latest techniques, as well as the methodology of work with them, which are suitable for analysis of phytocannabinoids.
|
|
|
Visualization of Educational Experiments
Zvelebilová, Rosana ; Holada, Karel (advisor) ; Beneš, Pavel (referee)
Anotation: Thesis visualizes educational experiments using various visualization devices - video, drawing, table, diagram, equation description. It serves to educate chemistry teachers and tutors them a live demonstration of experiments in chemistry lessons and laboratory exercises. Keywords: Visualization, education, experiment, categorization, demonstrations, model.
|
|
|
Regulation of Cathepsin D Activity and Activation
Máša, Martin ; Mareš, Michael (advisor) ; Jonáková, Věra (referee) ; Holada, Karel (referee)
PhD Thesis ABSTRACT REGULATION OF CATHEPSIN D ACTIVITY AND ACTIVATION Martin Máša supervisor: Michael Mareš Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic PRAGUE 2009 Introduction Cathepsin D (CD) is an aspartic peptidase located in the lysosomes of all mammalian cells, its main role is catabolic degradation of proteins. More over CD is known to participate in a range of physiological processes such as apoptosis and tissue homeostasis, as well as in the regulation of angiogenesis and the production of peptidic antigens. The role of CD in pathophysiology is associated with several diseases such as Alzheimer's disease and cancer. Alzheimer's disease is a neurodegenerative disorder, which is generally considered to be the most common form of dementia. Progression of this disease is accompanied with the deposition of amyloid plaques (AP) in the brain, which leads to neurodegeneration. The AP is a fragment released from amyloid precursor protein (APP) cleaved by secretases1 . High levels of CD were found in cerebrospinal fluid of the Alzheimer's patients2 . It was demonstrated that CD is able to cleave APP and produce the pathogenic AP. A genetic polymorphism in the CD gene was reported, which changes...
|
|
|
Expression and function of cellular prion protein in blood cells
Glier, Hana ; Holada, Karel (advisor) ; Živný, Jan (referee) ; Rusina, Robert (referee)
The cellular prion protein (PrPc) is essential for pathogenesis of fatal neurodegenerative prion diseases. Recently reported four cases of vCJD transmission by blood transfusion raise concerns about the safety of blood products. Proper understanding of PrPc in blood is necessary for development of currently unavailable blood screening tests for prion diseases. Flow cytometry is an attractive method for prion detection, however, the reports on the quantity of PrPc on human blood cells are contradictory. We showed that the majority of PrPc in resting platelets is present in the intracellular pool and is localized in α-granules. We demostrated that both, human platelets and red blood cells (RBC) express significant amount of PrPc and thus may play an important role in the transmission of prions by blood transfusion. Our results suggest a unique modification of PrPc on human RBC. Such modification of pathological prion protein could distort the results of blood screening tests for prions. Further we showed that the storage of blood prior to analysis and the choice of anti-prion antibody greatly affect the detection of PrPc by flow cytometry and we identified platelet satellitism as a factor contributing to the heterogeneity of PrPc detection in blood cells. Moreover, we demonstrated existence of...
|
|
|
The characterization of blood platelet cellular prion protein
Broučková, Adéla ; Holada, Karel (advisor) ; Matěj, Radoslav (referee) ; Suttnar, Jiří (referee)
The conformational conversion of the cellular prion protein (PrPc) to the misfolded isoform (PrPsc) is the central pathogenic event in the transmissible neurodegenerative prion diseases. The recently shown transmissibility of variant Creutzfeldt-Jakob disease by blood transfusion emphasizes the need for better understanding of the PrPc in blood. In the current thesis, we focused on blood platelet PrPc, which has not been very well described so far. In the first part of the thesis, platelet PrPc was characterized as glycosylphosphatidylinositol- anchored glycoprotein with dominant diglycosylated form. Platelet PrPc was shown to be sensitive to cleavage with proteinase K, which is a feature discriminating between cellular and pathological prion protein. We have confirmed that platelet PrPc binds copper ions by its N- terminal octapeptide repeat region. Regarding quantity of PrPc molecules expressed on blood elements we have proved that both platelets and red blood cells express considerable amount of PrPc and thus can not be neglected in the problematic of prions transmission by blood transfusion. The detailed study regarding PrPc localization in blood platelets is presented in the second part of the thesis. PrPc was shown to be expressed in -granules as well as on the cytoplasmic membrane of...
|
|
|
Molecular mechanisms in homocystinuria: spatial arrangement of human cystathionine β-synthase
Hnízda, Aleš ; Kožich, Viktor (advisor) ; Holada, Karel (referee) ; Jiráček, Jiří (referee)
Protein misfolding is considered to be the major pathogenic mechanism in homocystinuria due to cystathionine beta-synthase (CBS) deficiency. The aim of this work was to study molecular mechanisms underlying protein misfolding of CBS mutants. Firstly, we studied spatial arrangement of normal human CBS protein. Using data from differential covalent labeling of solvent-exposed aminoacid residues, we identified interdomain contact area between the catalytic core and the regulatory domain in human CBS, and we subsequently generated the structural model of the full-length CBS. In the next step, we studied evolutionary divergence of CBS protein structures. We performed phylogenetic analysis that revealed unique spatial arrangement of CBS enzyme in nematodes; the domain architecture of CBS in Caenorhabditis elegans was studied experimentally in more detail. Finally, we determined conformational properties of a representative set of human CBS mutants that exhibited in various extent affected formation of tetramers and decreased catalytic activity. Using thermolysin-based proteolytic techniques for analysis of nine mutants expressed in E.coli, we found that an unfolded structure is a common intermediate occurring in CBS misfolding. The importance of protein unfolding for pathogenesis of CBS deficiency was...
|
|
|
Utilization of Cannabis Sativa in Teaching Chemistry
Mušková, Adéla ; Holada, Karel (advisor) ; Beneš, Pavel (referee)
The diploma thesis deals with Cannabis sativa. The aim is to focus on possible utilization of Cannabis sativa in teaching chemistry. The theoretical part describes the botany of cannabis, overviews phytocannabinoids, substances contained in cannabis, and it also includes wide use of cannabis in agriculture and industry. The practical part deals with the selection and arrangement of a possible curriculum about chromatography as the most affordable technique for analysis of cannabis at school, including the formulation of school experiment methodology using the thin-layer chromatography of Cannabis sativa extract to demonstrate the presence of phytocannabinoids. The practical part also includes the scheme of an educational project on cannabis and describes the latest techniques, as well as the methodology of work with them, which are suitable for analysis of phytocannabinoids.
|
|
|
Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
|
|
|
The Role of Cellular Prion Protein in Erythroid Differentiation
Panigaj, Martin ; Holada, Karel (advisor) ; Živný, Jan (referee) ; Rusina, Robert (referee)
4 Abstract The cellular prion protein (PrPC ) is evolutionary conserved protein expressed in cells of various origins. Although PrPC plays a basic role in the pathogenesis of the fatal neurodegenerative prion disorders, its physiological role remains enigmatic. Prion diseases are characteristic by long latency period during which they are not identifiable by any conventional methods. Although the blood is an ideal material for developing of screening tests, little is known about traits of PrPC and its role in blood cells. We showed that human erythrocytes express low amounts of PrPC per cell, but due to the high numbers of erythrocytes, they are major contributors to the pool of blood cell-associated PrPC . Based on our biochemical characterization we propose that PrPC on human erythrocytes is uniquely modified. Such a modification in abnormal prion protein may complicate screening tests for prion diseases in blood. It was reported that prion diseases deregulate the transcription of erythroid genes, and PrP-/- mice demonstrate a defective response to experimental anemia. To investigate the role of the PrPC in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and in vitro. We showed that surface expression of PrPC on erythroid precursors in bone marrow and spleen...
|
|
|
Study of the photodynamic inactivation of prions by phthalocyanines.
Kostelanská, Marie ; Holada, Karel (advisor) ; Zimčík, Petr (referee) ; Kolářová, Hana (referee)
Transmissive spongiform encephalopathies, also called prion disorders, are fatal neurodegenerative diseases affecting mammals. In patients, the pathological prion protein (PrPTSE ) accumulates in CNS and causes death. Prions possess high binding affinity to surfaces. Moreover, they are highly resistant to conventional sterilization procedures which rise the risk of nosocomial transmission from patients in subclinical stage of prion disease through medical tools. In the thesis, we evaluate the efficiency of photodynamic inactivation (PDI) for prion decontamination. The PDI is induced by photoactivation of phthalocyanine (Pc) derivates AlPcOH(SO3)2, SiPc(OH)2(SO3)1-3 or ZnPc(SO3)1-3. Pc exposed to light generate reactive oxygen species, mainly singlet oxygen (O2(1 ∆g)). Production of O2(1 ∆g) in aqueous solution was confirmed by iodide method, quenching by NaN3 and oxidative degradation of uric acid. The photoactivation of Pc in infectious brain homogenate led to elimination of PrPres signal (= proteinase K-resistant PrPTSE fragment) below the detection limit of western blot by using nanomolar AlPcOH(SO3)2 concentration. The complete elimination of PrPres signal was accompanied with total protein concentration decrease by a maximum of 20% in brain homogenate No signs of protein fragmentation or...
|
guest ::
