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Nanofibre membranes as drug carriers 12
Fojtíková, Veronika ; Doležal, Pavel (advisor) ; Dittrich, Milan (referee)
The main advantages of sublingual systemic administration of drugs can be linked with a rich blood supply and very small thickness of the non-cornified biological sublingual epithelium. The theoretical part partially corrects the fact that the hitherto little addressed issue of sublingual administration route is connected with the influence of saliva and mucosal coating. The goal of the experiment was to verify the possibility of using albumin (BSA) as a part of potential drug carriers in the nanofiber membranes for sublingual administration. Sublingual membranes were obtained by dissection of porcine tongue (Sus scrofa) followed by rapid freezing in liquid nitrogen and further storage at -20 ř C. In vitro experiments involved the use of saliva as a medium of intermediate contact of drugs with the sublingual mucosa epithelium. Permeation of fluorescently labelled bovine serum albumin (FITC-BSA molecular weight of about 68. 000) was evaluated. Its application in the form 32-layered and 16-layered nanofiber carriers with the content of 30 percent FITC- BSA by weight was compared to permeation of the solutions of equivalent concentrations of lyophilized FITC-BSA and yielded the following findings: 1. FITC-BSA penetrated sublingual porcine membrane always by the flux J in the order of 101 µg/cm2 . h-1...
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Nanofibrous Membranes as Drug Delivery Systems
Vrbata, Petr ; Doležal, Pavel (advisor) ; Vetchý, David (referee) ; Podzimek, Štěpán (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Pharmaceutical technology Candidate: Mgr. Petr Vrbata Supervisor: Doc. RNDr. Pavel Doležal, CSc. Title of Doctoral Thesis: Nanofibrous membranes as drug delivery systems Nowadays, nanofibrous membranes are very quickly developing systems with proportions in nanometre scale. They are intensively studied and used in many technical disciplines, mostly thanks to their unique properties, among them great surface area to volume as a highlight. Their use in the field of biomedicine and pharmacy as drug delivery carriers, wound dressings and tissue engineering scaffolds is currently a topic of great interest. The doctoral thesis deals with newly formulated drug loaded nanofibrous membranes made of several selected polymers with diverse properties produced by electrospinning technology for large-scale production. The purpose was an experimental investigation and demonstration of potential benefits from application of nanofibrous membranes to selected application sites and dosage forms. The prepared membranes were evaluated by scanning electron microscopy and differential scanning calorimetry that proved successful incorporation of the drugs into the structure of the polymeric nanofibers. Moreover the measurements proved that the...
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Export of biomolecules from mitochondria
Pelc, Josef ; Doležal, Pavel (advisor) ; Kovalčíková, Jana (referee)
Mitochondrion is cellular organelle evolved from the endosymbiotic bacteria. Because of its bacterial origin mitochondrion has a number of unique properties and metabolic pathways. Mitochondria or its reduced forms are present in all eukaryotic organisms except a single exception. During the course of evolution most of mitochondrial DNA was transferred to the nucleus. Despite this, mitochondria still encode several proteins that are part of the electron transport chain and the ATP synthase. These proteins are transported from the mitochondrial matrix into the inner mitochondrial membrane. Misfolded proteins of the inner membrane are degraded by mitochondrial proteases, which leads to the release of generated peptides. Fe-S clusters are assembled in the mitochondrial matrix. Still unknown molecule, which is product of ISC pathway is transported into cytosol and participates in the assembly of cytosolic and nuclear iron-sulfur proteins. Other transport pathway ensures the exchange of lipids between the endoplasmic reticulum and mitochondria. Both mitochondrial membranes have different lipid composition. To maintain their properties specific lipid transport is required between the two mitochondrial membranes. This transport pathway is also used for the lipid synthesis in the inner mitochondrial membrane.
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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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