|
|
Mucin hydrogels - artificial models of native mucus systems
Mikušová, Janka ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
The scope of this masters thesis is the preparation of a model mucin system and its utilization as an artificial model of the native mucus system. The creation of this model system, according to several designed methods was a part of experimental part of the thesis. The preparation of mucin system comprised of physical and chemical methods of hydrogel formation, screening and characterisation of the various physical conditions of the mucin properties on its molecular level, and the preparation of sorbent with sorption surface containing mucin. Methods of light scattering, namely dynamic light scattering (DLS), used for mucin particles size change monitoring, and electroforetic light scattering (ELS), used for Zeta potential change monitoring, were used for the screening of the impact of physical factors on the properties of mucin.For the characterisation of impact of the temperature on changes in mucin sctructure was, apart from monitoring of light scattering, used also a diferential scanning calorimetry (DSC), which registered temperature value, at which mucin thermal denaturation occurs. In the next part of the thesis we subdued the created sorption surfaces to various physical-chemical analyses, which task is the characterisation and projection of surface and confirmation of mucin presence.Substancial part in monitoring and characterisation of changes in surface sctructure of sorption surface was accomplished by Fourier transform infrared spectroscopy (FTIR). Scanning electron microscophy (SEM) was used for the final, more detailed, projection of the mucin enriched, sorbent surface structure. Suggested methods of mucin hydrogel, didnt prove sufficient results for the possibility of application of hydrogel as a artificial model of real mucus system, but the sorbent application was indicated as a suitable alternative and an instrument for the further mucin behaviour research and possibly subsequent bacterial adhesion, which represents the first step in the formation of the bacterial biofilm.
|
|
| |
|
|
Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
|
|
|
Features and functions of glycocalyx of trematode cercariae
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Kašný, Martin (referee)
Trematodes are parasites from phylum Platyhelminthes which have compex life cycles involving two to four hosts. This work focuses especially on trematodes of the family Schistosomatidae. Their cercariae which leave the snail intermediate host, actively penetrate the skin of definitive hosts and transform into schistosomula. This is accompanied by detachment of cercarial tail and emptying of penetration glands. During transformation, cercarial bodies undergo extensive ultrastructural and molecular changes. One of these changes is the loss of surface glycocalyx which represents a protective coat in the aquatic environment. In glycocalyx shedding, participation of proteolytic enzymes from cercarial penetration glands is expected during invasion of the host. Glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. This work describes the origin, ultrastructure, saccharide composition, function and shedding mechanism of cercarial glycocalyx.
|
|
|
Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
|
|
|
Synthesis of Neuraminidase binders suitable for theranostics
Berenguer Albiñana, Carlos ; Machara, Aleš (advisor) ; Cibulka, Radek (referee) ; Soural, Miroslav (referee)
Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...
|
|
|
Mucin hydrogels - artificial models of native mucus systems
Mikušová, Janka ; Obruča, Stanislav (referee) ; Sedláček, Petr (advisor)
The scope of this masters thesis is the preparation of a model mucin system and its utilization as an artificial model of the native mucus system. The creation of this model system, according to several designed methods was a part of experimental part of the thesis. The preparation of mucin system comprised of physical and chemical methods of hydrogel formation, screening and characterisation of the various physical conditions of the mucin properties on its molecular level, and the preparation of sorbent with sorption surface containing mucin. Methods of light scattering, namely dynamic light scattering (DLS), used for mucin particles size change monitoring, and electroforetic light scattering (ELS), used for Zeta potential change monitoring, were used for the screening of the impact of physical factors on the properties of mucin.For the characterisation of impact of the temperature on changes in mucin sctructure was, apart from monitoring of light scattering, used also a diferential scanning calorimetry (DSC), which registered temperature value, at which mucin thermal denaturation occurs. In the next part of the thesis we subdued the created sorption surfaces to various physical-chemical analyses, which task is the characterisation and projection of surface and confirmation of mucin presence.Substancial part in monitoring and characterisation of changes in surface sctructure of sorption surface was accomplished by Fourier transform infrared spectroscopy (FTIR). Scanning electron microscophy (SEM) was used for the final, more detailed, projection of the mucin enriched, sorbent surface structure. Suggested methods of mucin hydrogel, didnt prove sufficient results for the possibility of application of hydrogel as a artificial model of real mucus system, but the sorbent application was indicated as a suitable alternative and an instrument for the further mucin behaviour research and possibly subsequent bacterial adhesion, which represents the first step in the formation of the bacterial biofilm.
|
|
|
Sialic acid as a recognition motif for host-originated glycoproteins in Ixodes ricinus
SAMEK, Jakob
Glycans are found on the surface of every living cell and they are responsible for a countless number of functions. N-acetylneuraminic acid (often called sialic acid) is mostly found in vertebrate and only to a small degree in arthropods. The aim of this thesis was to find out if sialic acid is a recognition motif for the tick organism to take up and incorporate or recycle host originated proteins.
|
|
|
Synthesis of Neuraminidase binders suitable for theranostics
Berenguer Albiñana, Carlos ; Machara, Aleš (advisor) ; Cibulka, Radek (referee) ; Soural, Miroslav (referee)
Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...
|
|
|
Glycosylation and antigenic properties of Phlebotomus perniciosus and P. orientalis salivary proteins
Sumová, Petra ; Volf, Petr (advisor) ; Grubhoffer, Libor (referee)
The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...
|
guest ::
