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Free living amoebae as causative agents of human infections.
Markovičová, Ivana ; Nohýnková, Eva (advisor) ; Doležal, Pavel (referee)
Free living amoebae as causative agents of human infections are world wide occuring organisms with the ability of dual lifestyle both in nature, which is their original habitat, so within host tissue. The aim of this theses is to summarize the basic available knowledge of the pathogenic species Naegleria fowleri and Balamuthia mandrillaris and of the pathogenic genus Acanthamoeba which invade CNS and are causative agents of rare but serious human infections, often leading to death of a patient. The exception of their deathly consequences is an Acanthamoeba keratitis infection in which non curing cases lead to a significant visual impairment and even to visual loss. In each of the chapters, the work is tend to summarize transmision ability, mechanisms of pathogenesis and pathophysiology of these three free living amoebae in order to describe their ability to cause infection.
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Testing of adhesive properties of plasticized polyesters using rotational rheometer
Ogadah, Chiazor Ugo ; Šnejdrová, Eva (advisor) ; Doležal, Pavel (referee)
Title of thesis: Testing of adhesive properties of plasticized polyesters using rotational rheometer Author: Ogadah Chiazor Ugo Department: Pharmaceutical Technology Supervisor: PharmDr. Eva Šnejdrová, Ph.D. In this thesis, the rheological and adhesive properties of three polyesters intended for use as drug carriers in polymeric drug delivery systems were studied. The theoretical section summarizes the polymers used as drug carriers. Plasticizers, mechanism of action, types and uses and finally bioadhesion mechanism, application of the bioadhesive preparations, and methods of testing bioadhesion are reviewed. In the experimental section, ethyl salicylate, ethyl pyruvate and triethyl citrate were selected as the plasticizers to decrease the viscosity of the polyesters for easy processability, and setting of the optimal adhesive properties. Flow properties of the plasticized systems was measured with a rotational rheometer at temperature 37o C and a shear rate range from 0.10 to 100 s-1 . The analysis of the viscosity curves revealed that the resulting plasticized systems are mostly Newtonian. All tested plasticizers decrease the viscosity of the systems. The most effective is ethyl pyruvate. The adhesive properties were determined by the "pull away test" on the rotational rheometer, and evaluated as...
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Traffic Shaping in High Speed Networks in DPDK
Doležal, Pavel ; Fukač, Tomáš (referee) ; Vrána, Roman (advisor)
This bachelor thesis is focused on traffic shaping in high speed networks. It presents framework DPDK, which can be used for fast packet processing. General traffic shaping mechanisms are described as well as traffic shaping in Linux using program tc. It also introduces a design and implementation of traffic shaper using DPDK framework for networks with 10 Gbps bandwidth. The traffic shaper uses a complex mechanism of hierarchical token bucket. The system was tested using high speed traffic generator Spirent.
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Technological aspects of solid solution formulations
Beneš, Michal ; Doležal, Pavel (advisor) ; Bělohlav, Zdeněk (referee) ; Dohnal, Jiří (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate PharmDr. Michal Beneš Supervisor Doc. RNDr. Pavel Doležal, CSc. Title of Doctoral Thesis Technological aspects of solid solution formulations Solid solutions are two- or multicomponent, single phase mixtures of solids, typically consisting of a polymeric carrier and an active substance (API) in amorphous state. The main advantage of solid solutions is the potential to increase the solubility of the API contained. The utilization of solid solutions is however complicated by many factors, which prevents its wider application. These include among other things the difficulty to determine solubility of the API in a polymeric carrier, poor predictability of physical stability as well as the complexity of appropriate selection of manufacturing process and its scalability. The experimental part is divided in three sections all associated with the technology of solid solutions. In the first of them, polymer films containing the API (dabigatran etexilate) were applied on glass plates and evaluated by spectral imaging techniques (infrared and Raman microspectrometry). Samples containing Kollidon VA64, Povidone K30 and Soluplus as polymer carriers, each loaded with 10, 25, 50 and 75 % (w/w) of the API...
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Introduction of APEX-based labeling for the research of mitochondrial organelles of Giardia intestinalis and Trichomonas vaginalis.
Drašnarová, Zuzana ; Doležal, Pavel (advisor) ; Rothová, Olga (referee)
Giardia intestinalis and Trichomonas vaginalis are parasitic protists living in the environment without oxygen. For this reason, their mitochondria were transformed into organelles lacking the oxidative phosphorylation. We do not know anything about the intermembrane space (IMS) of these mitochondrial organelles. Main reason is that it is hard to isolate IMS proteins using the classical methods of the molecular biology, as cell fractionation. Recently, the new ascorbate peroxidase (APEX) tag has been developed. This tagging is suitable for the fluorescence as well as the electron microscopy. APEX can also behave like biotin ligase when exposed to biotin-phenol and this allows the labeling of the proteins of the compartment as well as their isolation. We optimized the conditions for optimal APEX activity and using this technique we were able to visualize the IMS of hydrogenosomes of T. vaginalis and to isolate hydrogenosomal proteins.
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Structure and function of mitochondrial secretin.
Klápšťová, Veronika ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
Type II secretion system (T2SS) is one of the secretion systems found in gram-negative bacteria that provides transport of some bacterial proteins across the outer membrane. The passage through the membrane is mediated by a pore assembled from subunits called GspD or secretin. Together with three other components of T2SS, GspD was discovered in the genome of several protists including Naegleria gruberi, Andalucia godoyi, Reclinomonas americana, Neovahlkampfia damariscottae or in s species of genus Malawimonas. Previously it was found out that these proteins localize into the mitochondria. If found functional and with analogous topology to the bacterial system, the eukaryotic T2SS would represent unique mitochondrial protein export system. Secretin is essential subunit of T2SS which is not only the passive membrane channel, but also participates in the recognition of the substrate. Therefore, the research of the eukaryotic secretin could bring a valuable knowledge about the function of the mitochondrial T2SS. The experimental part of this thesis tries to characterize the eukaryotic secretin and it focuses on (i) the assembly of the secretin channel, in both, the bacteria and in the artificial membranes, (ii) the interactions of GspD with the other subunits of T2SS and (iii) the mechanism of import...
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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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Nanodispersion application system.
Pekárková, Veronika ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Diploma thesis summarizes the current information about the biopharmaceutical pharmaceutical classification system, disperse systems and solid dispersions in the field of drug delivery dosage forms. The experiment is directed to an in vitro dissolution evaluation of nanofibre membranes produced by electrospinning. The membranes contained a mixture of 10% bovine albumin and FICT albumin in a ratio of 1: 1 and further polyethylene oxide and polyvinyl alcohol. The main task was to verify the transmucosal in vitro permeation of albumin using sublingual membrane in vitro. The acceptor phase was phosphate buffer pH 7.4. The dissolution experiment confirmed a very rapid dissolution of the carrier nanofibrous membrane containing albumin. The most of the albumin was available for analysis already in 3, resp. 5 minutes. The time course of in vitro permeation of albumin through the sublingual porcine membrane showed 24 hr continuous linear period, either during permeation of nanofibrous membrane or a comparative solution. Average values of albumin fluxes under these conditions were ca J = 0.81 [ng / cm2 / h] SEM = 0,3 when 64layered nanomembrane and donor adjusted at pH 6.8, J = 0.40 [ng / cm2 / h], SEM = 0.34 or when nanomebrane was adjusted in donor at pH 7.4, and J = 0.77 [ng / cm 2 / h] for solution at pH...
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The effect of addition of thickening agents on nanoemulsion properties II
Dostálová, Alžběta Dorota ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Non-transparent nanoemulsions are determined by particle size of discontinuous phases in range of 200 - 300nm. Contrary to microemulsions that are transparent and thermodynamically stable, nanoemulsions are affected by number of common destabilizing processes as macroemulsions including Ostwald ripening. Adding of thickening agents to the nanoemulsions shall affect stability of the system. Generally, as the viscosity of continuous phase increases, so does the physical stability of the emulsion system. However, the system may be destabilized if the viscosity of nanoemulsions is increased due to the effect of addition of polymer thickening agent and if certain concentration of polymer thickening agent is exceeded. This phenomenon is utilized in the thesis in order to select appropriate oil phase of nanoemulsions. Two cellulose derivatives hydroxypropylmethylcellulose (HPMC) and hydroxyethylcellulose (HEC) were selected as thickening agents which were added to nanoemulsions at the different concentrations assessed as follows: 0.083; 0.167; 0.333 and 0.667 ‰. Samples were then examined using the DLS (Dynamic Light Scattering) method in order to measure size of dispersed particles of oil phase and by the capillary viscosimetry. In order to test stability of the samples the thermal stress test was used...
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Type II secretion system in the mitochondria of Naegleria gruberi.
Krupičková, Alžběta ; Doležal, Pavel (advisor) ; Hampl, Vladimír (referee)
Several types of the transport (secretion) systems can be found in the membranes of gram-negative bacteria. These systems export proteins into the extracellular milieu of bacteria. One of them, type II secretion system (T2SS), exports the folded proteins through the outer bacterial membrane. The core subunits of T2SS were discovered in the genomes of several protists belonging to the Discoba and Malawimonada groups, including Naegleria gruberi and Malawimonas sp. str. 249. Our experiments suggest that these subunits are localized in the mitochondria of these protists. The discovery of these genes in eukaryotes is surprising. If the T2SS was active in these mitochondria, it would represent an evolutionary intermediate stage of the organelle, which can export and import the proteins. In experimental part of the thesis, we studied how the proteins of N. gruberi and M. sp. str. 249 interact. We demonstrated several interactions, which correspond to the function of bacterial T2SS. To this aim, we employed bacterial and yeast two-hybrid systems. Further, we explored the import of the core subunit of T2SS, pseudopilin, of N. gruberi into the inner mitochondrial membrane. And we also investigated the import of M. sp. str. 249 secretin into the outer mitochondrial membrane.
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