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The role of mitochondria in the pathogenesis
Marková, Lenka ; Doležal, Pavel (advisor) ; Verner, Zdeněk (referee)
This thesis is focused on the existing data referring to role of mitochondria in the pathogenesis of several important diseases. It introduces mitochondrial proteins, their role and metabolism, dysfunction of which is behind pathogenetic processes. The thesis also summarizes possible mitochondrial damage, its progress and consequences, which can lead to diseases or aggravate their process. It also pays attention to the role of mitochondria during oncogenesis as well as important neurodegenerative disorders such as Alzheimer's or Parkinson's disease, Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis or autosomal dominant optic atrophy. The thesis mentions the role of disrupted mitochondrial dynamics in type 2 Diabetes. In conclusion the thesis mentions the role of mitochondria and their damage in relation to infection by the parasites Toxoplasma gondii and Trypanosoma cruzi.
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Mitochondrial unfolded protein response
Zemanová, Tereza ; Doležal, Pavel (advisor) ; Mach, Jan (referee)
Mitochondrion is an organelle of the eukaryotic cell responsible for the crucial metabolic processes and the apoptosis. Therefore, it is necessary for the survival of the cell to maintain mitochondrial functionality. Hence, the mitochondria have specific signalling stress-activated pathways for re-establishing its homeostasis. Unfolded protein response is a stress response to the presence of unfolded proteins that includes signalling from the organelle to the nucleus and activating specific genes for chaperones and proteases. This thesis summarizes current knowledge of the mitochondrial UPR, in the matrix as well as in the intermembrane space. It describes different types of the mitochondial chaperones and proteases, the reasons and the impacts of the mitochondrial stress causing the UPR and also briefly covers the problematics od the UPR in the cytosol and the ER.
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Nanofibre membranes as drug carriers 11
Swierkoszová, Martina ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Sublingual and buccal routes of represent alternative approaches of system drug administration. After a brief summary of essential information about the ways of drug penetration into the body the theoretical part of the work possess information on oromucosal barriers, particularly in relation to possible problems related to the physical properties of saliva and mucus. The experiment was to verify the release and subsequent permeation albumin (BSA) through sublingual membrane in vitro was performed. Sublingual porcine (Sus scrofa, var. domestica) mucosa stored at - 20 ř C was used as biological membrane. The content of the in vitro experiments was release and permeation using 16-fold and 32-fold layered nanomembrane carriers containing polyvinyl alcohol, polyethylene oxide and 30 percent of a mixture (1: 1) albumin BSA and fluorescently labelled bovine serum albumin (FITC-BSA). The liquid medium for the release was citrate-phosphate buffer pH 6.8, respectively its mixture with human saliva (1: 1) and the acceptor medium was HEPES buffer pH 7.4. Multiple layered nanofibrous membrane was perfectly wetted immediately after the first contact with a small amount of buffer pH 6.8, resp., with saliva diluted by buffer. Release FICT-BSA was macroscopically observable due to yellow color arisen. Sublingual...
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Interactions of Mitochondria with Other Cellular Structures
Vinopalová, Martina ; Doležal, Pavel (advisor) ; Voleman, Luboš (referee)
In cells, mitochondria fulfil diverse set of roles, including a production of energy-rich molecules, which are necessary for proper functioning of the cell, calcium homeostasis, apoptosis and even biosynthesis of Fe-S centres, heme and steroids. To coordinate some of these processes with events occurring in the rest of the cell, mitochondria need to communicate with the other cellular structures through their physical contacts. Resulting intracellular platforms give rise to additional mitochondrial functions. This thesis summarizes current findings from the cells of mammalian model organisms and the budding yeast Saccharomyces cerevisiae about the interactions of this semiautonomous organelle with other cellular components and about the functions, which these interactions mediate.
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Mitochondrial protein import.
Drašnarová, Zuzana ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
Most of mitochondrial proteins are synthesized in the cytoplasm and after that transported to the outer or the inner membrane or to the intermembrane space and the mitochondrial matrix. All mitochondrial proteins cross the outer membrane via the TOM complex. From here different populations of proteins follow distinct transport routes: (i) β-barel proteins are assembled in the outer membrane with the help the SAM complex, (ii) after the passage through the TOM complex the intermembrane space proteins are bound by the MIA pathway, (iii) the mitochondrial carrier proteins of the inner mitochondrial membrane require the activity of the TIM22 complex and finally (iv) the matrix proteins as well as the small sub-population of the inner membrane proteins are transported via the TIM23 complex. Whereas the transport across the outer mitochondrial membrane does not require the additional energy, the transport across the inner membrane depends on ATP and/or the membrane potential. The transported proteins carry targeting sequences which are recognized by the outer membrane receptors. Key words: protein import, mitochondria, translocase, membrane, matrix, intermembrane space
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Type II secretion system in gram negative bacteria.
Klápšťová, Veronika ; Doležal, Pavel (advisor) ; Mach, Jan (referee)
This thesis describes the structure and the assembly of the type II secretion system which is found in some gram negative bacteria such as Vibrio cholerae, Pseudomonas aeruginosa, Legionella pneumophila or Klebsiella oxytoca and which transports their exoproteins across the outer membrane. The thesis also presents some important substrates translocated by this system - toxins, enzymes and proteins involved in bacterial metabolism or motility - and hypothetical mechanism of the secretion. Conclusion is focused on the discovery of the secretion system in mitochondria of some protists and its importance for the research of the evolution of eukaryotic organisms.
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Permeation of nanoparticles across sublingual membrane 3
Šašurová, Michaela ; Doležal, Pavel (advisor) ; Vachek, Josef (referee)
4 ABSTRACT The theoretical part describes anatomical and physiological aspects of the oral cavity, tongue and salivary glands. It captures the issue of sublingual administration of drugs. Through a brief history of nanotechnology it comes to characterize nanoparticles as basic building blocks of nanostructures. It describes the production and properties of nanoparticles, shows the advantages of using nanoparticles as a drug delivery system, but also point out their potential toxicity. At the end of the theoretical part principles and methods of measurement used in the experiments are summarized and characterized. In the experimental part in vitro permeation experiments are described and evaluated. Permeation of nanoparticles marked Chromeon 470 through porcine sublingual membrane in area of 1 cm2 from the donor phase of pH 6.6 to acceptor phase pH 7.4 was observed in these experiments. For the permeation experiments were used both fresh and long-term frozen sublingual membranes fixed in Franz diffusion cells with glue or silicone urease. Before starting the permeation experiments the size of nanoparticles or agglomerates were characterized using dynamic light scattering and by fluorescence microscopy. It was verified that the two evaluated methods of fixing of membranes in diffusion cells have no...
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Reverse genetics in anaerobic protists
Stojanovová, Darja ; Tachezy, Jan (advisor) ; Doležal, Pavel (referee)
This work is focused on reverese genetics of anaerobic protists, mainly T. vaginalis, G. intestinalis and E. histolytica and deals with techniques and experimental procedures of genome manipulation in these parasites. Both DNA and RNA can be manipulated and the gene function can be disclosed using methods of reverse genetics. The knowledge gained is useful in many ways. For example, using these techniques crucial aspects of biology of parasitic prostist are studied, providing basis for potential development of new drugs. Utilization of such methods also helps to understand the cellular and metabolic pathways and mechanisms, that could be very diverse or reduced in protists. The methods of reverse genetics that result in permanent and inheritable changes in DNA are, for instance, homologous recombination or DNA integration. There is also a transcriptional gene silencing (TGS) technique to stop gene expression even though the coding DNA remains unchanged. TGS could be realized by several mechanisms, for example by RNA interference. RNA interference pathway, commonly known as posttranscriptional gene silencing mechanism, causes the breakage of mRNA or stops its translation. Other techniques of gene silencing involve, e.g., the expression of antisense RNA, oligonucleotudes and ribozymes.
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AFM in pharmaceutical technology 2.
Princová, Tatiana ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
The theoretical part deals with topics related to the formation of nanofibers and nanomembranes by different ways of electrospinning. The literary search focused on "medicated nanofibrous membrane" gives recent information on nanomembranes containing drugs and also shows the perspective of the use of nanofibers in this area. The experimental part deals with AFM parameters needed for characterisation of the samples of six selected polymer nanomembranes with the content of naproxen, folic acid and diosmin. The appearance and thickness of the nanofibers was examined. The set up parameters of the AFM measurements allowed to observe the distribution of the drug in non- crystalline state within the nanofibers, regular fibrous shapes of crystal-like nanofibers as well as distinguished nanoingots of the polymers. The captured scans are stored and available for further analysis. Keywords: electrospinning, nanomembrane, naproxen, AFM, drug-loaded nanofibers
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Permeation of nanoparticles across sublingual membrane 4
Horejšová, Lenka ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Charles University in Prague, Faculty of Pharmacy in Hradci Králové Department of: Pharmaceutical technology Consultant: doc. RNDr. Pavel Doležal, CSc. Student: Lenka Horejšová Title of Thesis: Permeation of Nanoparticles Across Sublingual Membrane 4 The theoretical part describes the anatomy and physiology of oral cavity and sublingual drug administration. It also describes theory of nanotechnology and the characterization of nanoparticles with a focus on nanoparticle drug forms and there are also mentioned methods and mesuring device. . The experimental part describes in vitro permeation experiment of nanoparticles (Nano beads based on PD, Chromeon 470 marked, carboxylated). Permeation of this nanoparticles through porcine sublingual membrane (1 cm2) . Thesis compares nanoparticles transfer of phosphate buffer pH 6.6 and citrate buffer pH 6.6 to the acceptor phase consisting of phosphate buffer pH 7,4. First permeation experiment works with frozen sublingual membrane, a second experiment with the native membrane. The dimension of nanoparticles and aggregates of nanoparticles in dispersions were measured before the start of permeation by the dynamic light scattering. This work demonstrates that the choice of donor buffer has not an important influence. Key words: nanoparticles, fluorescent...
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