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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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Nanofibre membranes as carriers of drug 10.
Nguyenová, Jana ; Doležal, Pavel (advisor) ; Dittrich, Milan (referee)
The theoretical part of thesis provides information on the biopharmaceutical classification system of drugs and its context in the research and development of pharmaceuticals. The methods used to increase the solubility and acceleration including electrospinning are presented. The experimental part is the pilot study on the evaluation of conditions suitable for dissolution testing of newly prepared nanofibers made from polyvinylpyrrolidone membranes with a high content (up to 35 per cent) gatrin as a substance poorly soluble in aqueous vehicles. The parameters of determination of gatrin by HPLC using C18 sorbent and a mobile phase of acetonitrile: phosphate buffer pH 8 were preliminarily evaluated as perfectly applicable to vehicle type-phosphate buffer pH 6.0. The same conditions were found to be in a severe collision with a polymer material of nanofibrous membrane during the dissolution evaluation or with acetonitrile in the mobile phase, an accurate determination of gatrin was not obtained in this case. These findings lead to the proposal to change the formulation of the nanofiber membranes using polymer different from polyvinylpyrrolidone (eg. hydroxypropylcellulose) or the replacement of acetonitrile for methanol at the mobile phase for HPLC. However, in all cases, all the analytically...
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AFM in Pharmaceutical Technology 3.
Ščuryová, Veronika ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Student: Veronika Ščuryová Supervisor: doc. RNDr. Pavel Doležal, CSc. Title of thesis: AFM in Pharmaceutical Technology 3 The theoretical part deals first with the construction of AFM microscope, the principle of the method, determining the surface topography and regimes which can be used. Described therein are distinct advantages over previous traditional methods but also its pitfalls. Next, I compare the results of measurements using AFM and declared size and devote also determine the shape of the particles. Experimental part is focused first on the detailed description of sample preparation for AFM measurement of nanoparticles. This procedure was followed by practical use to characterize the magnitude of the four types of commercially available nanoparticles Chromeonov (Sigma-Aldrich) using atomic force microscopy. The laboratory prepared Ag- nanoparticles could not be evaluated due to of technical and methodological reasons. The magnitude of the measured results nanoparticles were processed in histograms, which provide a description of the distribution of the measured values of the nanoparticle size. I found that compared to the size of the nanoparticles declared by the manufacturer are...
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Parasite's life within the host cell.
Krupičková, Alžběta ; Doležal, Pavel (advisor) ; Štáfková, Jitka (referee)
Plasmodium falciparum is one of the most dangerous human intracellular single-celled pathogen. This thesis describes the invasion, the survival and the egress of the pathogen from the host cell (erythrocyte). The main topic of the thesis is the parasite's life within the host cell in a newly created compartment, parasitophorous vacuole. By exploding hundreds of proteins Plasmodium falciparum establishes number of unique modifications inside as well as on the surface of the host cell. The thesis finishes with the chapter on the therapy and possible development of new drugs and the chapter on the parasite evolution. Powered by TCPDF (www.tcpdf.org)
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Suppositories in current therapy II
Krimová, Barbara ; Musilová, Marie (advisor) ; Doležal, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Author: Barbara Krimová Supervisor: RNDr. Marie Musilová, CSc. Title of diploma thesis: Suppositories in current therapy II Suppositories as dosage forms are still in use at present. For certain medicinal substances they are even one of the few possible applications. Therefore it is still necessary to deal with their proper formulation and storage. In this thesis, we followed up the influence of storage temperature of suppository bases on its calorimetric characteristics. As a basic method of thermal analysis we used differential scanning calorimetry. We determined the content of the solid phase in the suppository bases stored at temperatures of 26 řC and 5 řC. We evaluated the changes that occurred within one year after the preparation of suppositories. The obtained data shows increase in share of solid phases during the storage and the major differences are just in storage temperature. Thus we consider appropriate to set maximum temperature limits for storage of suppositories.
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Comprehensive investigation of penetration enhancers with complementary analytical techniques.
Vidlářová, Lucie ; Doležal, Pavel (advisor) ; Musilová, Marie (referee)
Transdermal drug delivery system is in the centre of attention in recent years. For efficient dermal drug delivery the drug has to overcome the barrier of the outermost layer of the skin, the stratum corneum. For facilitating dermal drug transport, the barrier properties of the stratum corneum can be varied by applying chemical penetration enhancers. The aim of this work was to characterize various penetration enhancers and investigate their mechanism of action. We combined well established techniques like differential scanning calorimetry (DSC) and infrared spectroscopy (IR) with confocal Raman microscopy (CRM) as an upcoming technique in skin research. CRM offers the possibility of label-free and non- destructive, chemically selective analysis of stratum corneum lipids and proteins. We used isolated human stratum corneum for incubation with the penetration enhancers. As a novel approach, the samples of treated stratum corneum were freeze dried to avoid any discrepancies which might come up with differences in the hydratation state of stratum corneum (SC). Furthermore, the structure of lipids and proteins in the stratum corneum was analyzed. In our study, stratum corneum was treated with dimethyle sulphoxide, propylene glycol, ethylene glycol, ethylene glycol-d4 and oleic acid. We observed that...
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Use of DSC to Determinate Volume of Active Substances II
Víchová, Eva ; Musilová, Marie (advisor) ; Doležal, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Department of Pharmaceutical Technology Candidate Mgr. Eva Víchová Consultant RNDr. Marie Musilová, CSc. Title of Thesis Use of DSC to determinate volume of active substances ll This thesis deals with the determination of paracetamol in commercially produced Panadol Baby suppositories. Differential scanning calorimetry was used for this evaluation. This method detects the amount of heat needed to melt paracetamol. The heat quantity is directly proportional to the amount of the drug in the sample. The content is then calculated from the equation of the calibration curve. The thesis includes the measurement record of total twenty samples and the samples deals with the calibration curve. There was found higher content of paracetamol in the tips than in the circular bases. For all samples there was found lower content of drug than was declared. We recommend to compare the results obtained by DSC with another analytical method.
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Use of DSC to Determinate Volume of Active Substances I.
Chlubnová, Hana ; Musilová, Marie (advisor) ; Doležal, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Use of DSC to determinate volume of active substances I. Mgr. Hana Chlubnová This paper studies the determination of active substances volume in suppositories with differential scanning calorimetry. This method is more expensive than other used methods but its advantage is in a speed and no separation of active substance from suppository base needed. We used suppositories Ben-U-Ron containing 125mg and 1000mg of paracetamol for our measurements. The samples were taken for the base and the tip of suppositories. On the ground of the results we can state it is necessary to repeat DSC measurements several times to get values of substance volume. These values are in certain interval and the right result cannot be achieved from one single measuring. It was also found that suppository with 125mg of paracetamol contained less amount of tested substance than is proclaimed by producer. This was very small reduction in a border of analytical mistake but it was in every tested samples. It is probably caused by melting of paracetamol in suppository base due to higher temperature (180řC). This was only in case of 125mg suppository, 1000mg suppository samples were all in settled intervals.
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