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Study of the development of microbial biofilm within an experimental model, Galleria mellonella
Doubová, Dominika ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Dominika Doubová Supervisor: RNDr. Klára Konečná, Ph.D. Title: Study of the development of microbial biofilm within an experimental model, Galleria mellonella Aim of the thesis: The aim of this thesis is to introduce the invertebrate organism, Galleria mellonella (G. mellonella), that can be used within the scientific research as an alternative model organism to vertebrates. This model seems to be more financially favorable and more ethically acceptable, which is an essential fact in the current era of increasing pressure to abandon testing on vertebrates in particular. The experimental part of this thesis is focused on optimizing the utilization of the G. mellonella larvae as a model organism within the research of fungal burn wounds fungal infections induced by the yeast Candida albicans. Within these experiments, larvae were used to preparation of tissue explants on which the formation of a single- species yeast biofilm was investigated. Methods: Before obtaining of tissue explants, the larvae were decapitated. To reduce contamination of tissue samples primarily by the gut microbiota of the larvae, the approach of removing the digestive tube was optimized. Burn wounds were induced...
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Study of in vivo toxicity of selected compounds with the implementation of the invertebrate model Galleria mellonella
Turinová, Veronika ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
6 ABSTRACT Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Veronika Záveská Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Study of in vivo toxicity of selected compounds with the implementation of the invertebrate model Galleria mellonella Background: The aim of this diploma thesis was to monitor the influence of the weight of the larvae, or rather its developmental stage, on the results after the administration of selected doses of DMSO. In the experimental work, the toxic impact of DMSO on larvae was evaluated not only from the point of view of mortality. Much attention was also paid to the characteristic features that are commonly observed during tests on Galleria mellonella larvae, such as the degree of melanization, the impact on activity and cocoon formation. Methods: For individual experiments, three test groups of six individuals each were created based on weight, one group of larvae weighing 250 ± 10 mg, the second group weighing 350 ± 15 mg and the third group weighing 450 ± 15 mg. Using a Hamilton syringe, a volume of 10 µl of the test solution with the appropriate concentration of DMSO was administered to each larva directly into the haemocoel through the last left proleg. The administered doses...
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Comparative Analysis of Silk Proteins and Discovery of Novel Sericin Gene in Lepidopteran Moths
WU, Bulah Chia-Hsiang
This thesis focuses on the silk components of the Mediterranean moth, Ephestia kuehniella, and the discovery of a novel silk gene, P150/ser6, in the silkworm, Bombyx mori. We analyzed and described the cocoon silk components in both species. In the first publication, we combined transcriptomic, genomic, and proteomic approaches to identify silk proteins in E. kuehniella. In the second publication, we described the discovery of gene P150/sericin6 in B. mori based on microsynteny analysis.
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Study of the development of microbial biofilm within an experimental model, Galleria mellonella
Doubová, Dominika ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Dominika Doubová Supervisor: RNDr. Klára Konečná, Ph.D. Title: Study of the development of microbial biofilm within an experimental model, Galleria mellonella Aim of the thesis: The aim of this thesis is to introduce the invertebrate organism, Galleria mellonella (G. mellonella), that can be used within the scientific research as an alternative model organism to vertebrates. This model seems to be more financially favorable and more ethically acceptable, which is an essential fact in the current era of increasing pressure to abandon testing on vertebrates in particular. The experimental part of this thesis is focused on optimizing the utilization of the G. mellonella larvae as a model organism within the research of fungal burn wounds fungal infections induced by the yeast Candida albicans. Within these experiments, larvae were used to preparation of tissue explants on which the formation of a single- species yeast biofilm was investigated. Methods: Before obtaining of tissue explants, the larvae were decapitated. To reduce contamination of tissue samples primarily by the gut microbiota of the larvae, the approach of removing the digestive tube was optimized. Burn wounds were induced...
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Study of in vivo toxicity of selected compounds with the implementation of the invertebrate model galleria mellonella
Záveská, Veronika ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
6 ABSTRACT Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Veronika Záveská Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Study of in vivo toxicity of selected compounds with the implementation of the invertebrate model Galleria mellonella Background: The aim of this diploma thesis was to monitor the influence of the weight of the larvae, or rather its developmental stage, on the results after the administration of selected doses of DMSO. In the experimental work, the toxic impact of DMSO on larvae was evaluated not only from the point of view of mortality. Much attention was also paid to the characteristic features that are commonly observed during tests on Galleria mellonella larvae, such as the degree of melanization, the impact on activity and cocoon formation. Methods: For individual experiments, three test groups of six individuals each were created based on weight, one group of larvae weighing 250 ± 10 mg, the second group weighing 350 ± 15 mg and the third group weighing 450 ± 15 mg. Using a Hamilton syringe, a volume of 10 µl of the test solution with the appropriate concentration of DMSO was administered to each larva directly into the haemocoel through the last left proleg. The administered doses...
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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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Dissemination of Cordyceps fumosorosea by entomopathogenic Nematodes
HOPFGARTNER, Miriam
C. fumosorosea is an entomopathogenic fungus that has been used as a biocontrol agent against various insect hosts for some time. Its efficacy as an entomopathogen can be increased by joined application of the fungus with entomopathogenic nematodes. The aims of this thesis are to investigate the impact of the foraging strategy of different nematode species on the spore dispersal of C. fumosorosea and the significance the nematode's 2nd stage cuticle holds for spore dissemination. Additionally, novel approaches for the enhancement of spore transmission in soil environments via adhesives are explored.
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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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Transformation of potato with protease inhibitor gene SPI-2
Říhová, Barbora ; Fischer, Lukáš (advisor) ; Vítámvás, Pavel (referee)
The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....
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The effect of antimicrobial peptides on Saccharomyces cerevisiae and other yeast species
Makarova, Anna-Marie ; Hodek, Petr (advisor) ; Malcová, Ivana (referee)
The increased use of antibiotics, antifungal agents and disinfectants in the last decades has resulted in development of microbial resistance to these drugs. Candida species are the fourth most common cause of hospital-acquired bloodstream infection and kill 40% of those patients. Natural antimicrobial peptides are promising candidates for the development of new agents to treat yeast and bacterial infections, as their presumed mechanism of action differs significantly from the mechanism of action of current drugs. This work is focused on several peptides isolated from the venom of wild bees and their synthetic analogues and the identification of the most effective ones against non-pathogenic Saccharomyces cerevisiae and several pathogenic Candida species. Antifungal activity of eight cationic antimicrobial peptides was tested and compared under various conditions. The overall susceptibility of pathogenic yeast species to currently used antifungal drugs and the antimicrobial peptides was screened with the aim to identify potential synergistic and species-specific effects. The effect of antimicrobial peptides on membrane potential was measured by a fluorescent probe (diS-C3(3)), and the relative hyperpolarization of plasma membrane was shown for each peptide. The effect of antimicrobial peptides on...
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