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Nanofibrous membranes as drug delivery systems II
Stránská, Denisa ; Doležal, Pavel (advisor) ; Dittrich, Milan (referee) ; Maťha, Vladimír (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Pharmaceutical technology Candidate: Ing. Denisa Stránská Supervisor: Doc. RNDr. Pavel Doležal, CSc. Title of Doctoral Thesis: Nanofibrous membranes as drug delivery systems Nanofibres are material structures of unique properties that are well-useable in many exciting areas, including medical products. In the field of air filtration, different types of nanofibre filters are produced industrially. On the other hand, in the area of medicated nanofibres, there is still a major barrier for production, and hence the commercial use of nanofibres, especially due to the low weight and required content uniformity of the final products. Electrospinning technology is the most promising in this context, however, no results on the evaluation of nanofibrous products intended for the systemic administration of drugs obtained by certified manufacturing process have been published. In this doctoral thesis the individual steps of the nanofibrous products production are described, experimentally evaluated in compliance with good manufacturing practice and documented with an emphasis on the validation procedures and thus on the desired repeatability of all steps leading to the production of the final product. Validation processes included...
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Dynamics of Giardia intestinalis encystation.
Vinopalová, Martina ; Doležal, Pavel (advisor) ; Verner, Zdeněk (referee)
Giardia intestinalis is an anaerobic parasite, that colonizes the small intestine of humans and other vertebrate hosts. This cosmopolitan parasite, which causes diarrhoea, is transmitted by contaminated water or food via a resistant stage, the cyst. The encystation process involves a number of events that lead to a complete reconstruction of the cell into the form of infectious cyst. The aim of this work was to visualize these modifications in vivo by means of enzymatic labelling of proteins. For the purposes of this work, enzymatic tags Y-FAST and HaloTag were chosen, as they enable visualizing live cells under anaerobic conditions. Chimeric protein constructs were created to visualize the dynamics of the encystation vesicles, the structures of endoplasmic reticulum, the adhesive disc and mitosis. Using the developed constructs, we successfully followed the dynamics of the encystation vesicles and the adhesive disc in vivo. Finally, this work has provided novel molecular tools, which will be used to follow the overall redesign of the parasite cell during encystation.
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Investigation of newly discovered protein GL50803_16424 in Giardia intestinalis.
Pelc, Josef ; Doležal, Pavel (advisor) ; Pyrih, Jan (referee)
The anaerobic unicellular eukaryotic organism Giardia intestinalis is a worldwide parasite. Giardiasis, the intestinal disease caused by Giardia, is one of the most common parasitic disease in the developed part of the world, that causes health problems not only to humans but also to animals. This organism is also interesting for its many unique cellular features. One of them is the presence of mitosomes - the organelles derived from mitochondria. Analogously to mitochondria, mitosome is limited by two membranes and shares the mode of the protein transport. However, mitosome does not have its own genome and as far as we know, there is only one pathway of the iron-sulfur cluster biosynthesis in this organelle. Using the in vivo enzymatic tagging technique, several novel mitosomal proteins were identified, including GL50803_16424. The protein GL50803_16424 attracted our attention by interacting with components of all mitosomal subcompartments: the outer membrane, the membrane and the matrix. In addition, the expression of HA-tagged GL50803_16424 resulted in the formation of peculiar structures near the mitosomes never seen before in G. intestinalis. Bioinformatic approaches revealed that the GL50803_16424 has domain similar to the myelodysplasia- myeloid leukemia factor 1-interacting protein. Our...
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Structure and function of the invasion apparatus of microsporidia
Dohnálek, Vít ; Doležal, Pavel (advisor) ; Vávra, Jiří (referee)
Microsporidia are obligate intracellular parasites that cause significant economic damage and can infect humans. They had evolved highly specialized invasion apparatus that is unique to them. During the invasion the sporoplasm is released from the spore wall and it is transferred to the host cytoplasm through the polar tube. Current research on the invasion apparatus has been focused mainly on the polar tube that is its most prominent structure. Polaroplast and posterior vacuole remain out of the main interest, although they are necessary for the activation and execution of the invasion. If the right combination of environmental factors occurs, the organization of the polaroplast begins to change, the posterior vacuole starts to swell and the polar tube is discharged. Sporoplasm is eventually pushed through the tube into the host cell by growing posterior vacuole. The mechanism has not been explained yet however plenty of theories are trying to explain the germination. This work summarizes current theories and knowledge of structure and function of particular parts of the invasion apparatus. Key words: Microsporidia, invasion, polar tube, intracellular parasitism, germination
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HPMC-based liposomal mucoadhesive films with model peptide as target API
Belcáková, Hedviga ; Nobilis, Milan (advisor) ; Doležal, Pavel (referee)
Charles University, Faculty of Pharmacy in Hradec Kralove Department of Pharmaceutical Chemistry and Pharmaceutical Analysis Candidate: Hedviga Belcáková Supervisor: Prof. Milan Nobilis, CSc., Veronika Fidelj (Heidelberg University, Insitute of Pharmacy and Molecular Biotechnology) Diploma thesis title: HPMC-based liposomal mucoadhesive films with model peptide as target API This thesis describes the preformulation stage of mucoadhesive films intended for liposomal peptide delivery via buccal membrane. The evaluation consisted of thickness, maximum tensile strength, strain, moisture content, in vitro swelling and liposome integrity measurements. The chosen polymer (hypromellose, HPMC) was found to perform optimally in concentrations of 10 % with PEG 400 (5 %) acting as plasticizer and liposome concentration of 2 %. The developed preparation method showed good reproducibilty with room for improvement in the homogenization area. The choice of medium (H2O vs. PBS) showed strong influence on formulation's mechanical properties resulting in significant loss of elasticity and mucoadhesive strength. The addition of liposomes in the third stage had been carried out successfully with only occassional effect on their integrity after dissolution.
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Proteome of the encystation-specific vesicles in Giardia intestinalis.
Marková, Lenka ; Doležal, Pavel (advisor) ; Tůmová, Pavla (referee)
The process of encystation is a vital and important part of life cycles of many organisms. One of them is Giardia intestinalis, a significant single cell parasite of vertebrates, including man. During the process of encystation there are unusual organelles, called encystation vesicles. These serve to accumulate material for the future cyst wall. The main proteins found in encystation vesicles that compose the cyst wall, are CWP1, CWP2 and CWP3. This work is focused on these proteins. The structure and properties of these proteins were used to study the main aim of this work, that is the identification of other proteins associated with the encystation vesicles. In the experimental part, a system was created, that allows targeted characterization of proteins and cell compartments, by utilization of biotin ligase of Escherichia coli. For this systém, we created specific constructs, that were successfully inserted into plasmids and into G. intestinalis cells. For the intention of cloning, a method of direct site mutagenesis was optimalized. The expression of chosen proteins was detected successfully during the in vitro encystation. Aside, it was proven that CWP1 forms complexes with an unknown protein. This work relates the composition of cyst wall of G. intestinalis with other components identified in...
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Structure and mechanical properties biodegradable Mg-3Zn-2Ca alloy processed by ECAP
Havlíček, Štěpán-Adam ; Podrábský, Tomáš (referee) ; Doležal, Pavel (advisor)
Zaměření této bakalářské práce je na mechanické vlastnosti a mikrostrukturu biodegradabilní hořčíkové slitiny Mg-3Zn-2Ca zpracované pomocí uhlového protlačování skrze shodné kanály (ECAP). Každý vzorek se liší počtem protlaku ve směru Bc. Zpracovaná hořčíková slitina ukazuje zlepšení napěťových a deformačních charakteristik, avšak s rostoucím počtem protlaků začínají klesat deformační charakteristiky. Aplikace ECAP metody způsobila v mikrostruktuře zjemnění zrn a ve směru protlačování prodloužení zrn. Ke studiu mikrostruktury bylo využito světelné mikroskopie a studium tahových vlastností bylo provedeno tahovými zkouškami.
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Zn and Mg based bulk materials for biomedical applications
Ryšťák, Jaroslav ; Fintová, Stanislava (referee) ; Doležal, Pavel (advisor)
Topic of the diploma thesis is Zn-Mg bulk material preparation by powder mixtures sintering at hot pressing. Structure, porosity and physically mechanical properties of prepared bulk materials were evaluated. Obtained results and their interpretation were served as feedback for following optimization of individual processing parameters of bulk materials preparation. Solution of diploma thesis is focused on study and control of processes during bulk material preparation and processes description from physical-chemical point of view with respect to structure creation and final material properties.
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Biogenesis of Giardia intestinalis mitosomes
Voleman, Luboš ; Doležal, Pavel (advisor) ; Faso, Carmen (referee) ; Dawson, Scott C. (referee)
7 ABSTRACT Mitochondria of opisthokonts undergo permanent fusion and fission throughout the cell cycle. Keeping these two processes in balance is vital for various aspects of mitochondrial and cellular homeostasis. Both mitochondrial fusion and division mechanisms are controlled by highly conserved dynamin-related GTPases that are present in all kingdoms of life. The aspects of mitochondrial dynamics outside the opisthokonts is, however, almost completely unexplored phenomenon. In our work, we introduced a tool for live imaging of the reduced forms of mitochondria into model organisms Giardia intestinalis and Trichomonas vaginalis, anaerobic protist parasites from the Excavata supergroup of Eukaryotes. Using this technique, we investigated the dynamics of the mitosomes, the simplest forms of mitochondria, of G. intestinalis. The division of mitosomes is restricted to Giardia mitosis and is absolutely synchronized with the process. The synchrony of the nuclear and the mitosomal division persists also during the encystation of the parasite. Surprisingly, the sole dynamin-related protein of the parasite seems not to be involved in mitosomal division. However, throughout the cell cycle mitosomes associate with the...
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Characterization of the protein import into Giardia Intestinalis mitosomes
Pyrihová, Eva ; Doležal, Pavel (advisor) ; Tsaousis, Anastasios (referee) ; Stairs, Courtney (referee)
Mitochondrial endosymbiosis was a key event in the evolution of eukaryotes. Its proteome evolved into a unique combination of inherited bacterial components as well as novel eukaryotic inventions. Today, mitochondria show a huge variety across eukaryotic species - from aerobic mitochondria with cristae and complex protein apparatus for maintaining its own genome to hydrogen-producing hydrogenosomes and tiny anaerobic mitosomes without their own genome and with only a single metabolic pathway. Comparing the existing spectra of mitochondria is beneficial for studying their evolution. The only ubiquitous and unifying features are double membrane, ISC pathway for iron-sulfur cluster synthesis and the core of protein import pathway. Therefore, these features could be considered as truly ancestral and essential to mitochondria. Mitosomes of various parasitic protists have evolved independently from complex mitochondria, since they are present in completely unrelated species and yet their evolution led in a surprisingly similar composition of protein import pathway. These retained components are thus believed to be functionally essential and for some reason hard to be replaced by alternate proteins. Mitosomal import pathways were considered very minimalistic for a long time. Nevertheless, the latest...
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