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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 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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Preparation and characterization of chicken antibodies against pathogenic yeast Candida albicans
Vodecký, Matúš ; Hodek, Petr (advisor) ; Dračínská, Helena (referee)
The increase of infections caused by yeast pathogens is an alarming global public health problem. People with compromised immune systems, such as after infection with human immunodeficiency virus or diabetes mellitus, are most at risk. Diagnosis is often inadequate, and treatment relies on a limited selection of antifungal drugs, to which resistance is also increasing. These aspects represent a major problem, as second-choice drugs, which may have toxic side effects (e.g., nephrotoxicity in the case of amphotericin B), must then be applied to resistant pathogens. Given the severity of yeast infections, scientists are trying to find new ways of protection against these pathogens. One option is passive immunotherapy. In this bachelor thesis, antibodies from hen's eggs were chosen for this purpose, as they have the potential for wide therapeutic use. The preparation of hen's egg yolk IgY antibodies is both ethically and economically acceptable. Moreover, egg yolk antibodies show relatively high stability. Polyclonal antibodies were isolated from eggs of hens immunized with either cell wall or whole C. albicans cells in a medium simulating the vaginal environment (VSF). Antibody reactivity was tested by ELISA. The cell walls of C. albicans in VSF medium or YPD medium were used as antigen. The results...
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Chicken antibodies against the pathogenic yeast Candida albicans
Konečná, Lucia ; Heidingsfeld, Olga (advisor) ; Kubíčková, Božena (referee)
5 Abstract Candida albicans belongs to the most widespread pathogenic yeasts. As an opportunistic pathogen, it mainly colonises the mucous membranes of the host and causes infection, especially in individuals with a compromised immune system. The most common infection caused by C. albicans is vaginal mycosis, which at least 3 out of 4 women will experience during their lifetime. Antifungal drugs are used to treat this condition, but the yeasts are becoming less susceptible. For this reason, there is an effort to develop new treatment options for (not only) infections caused by C. albicans. Although vaginal mycosis is not a severe condition in most cases, it is a widespread infection that occurs repeatedly in many women, sometimes several times a year. Since vaginal mycosis reduces the quality of life in many women, we decided to focus on this disease in this paper. This project used hen antibodies, which have a number of advantages over mammalian antibodies. In oviparous animals, antibodies are not only found in the blood serum, as seen in mammals but are also secreted into their eggs. This makes the isolation of hen antibodies non-invasive and ethical. Moreover, the methods of isolating antibodies from eggs are also simpler than isolation from blood serum. Ultimately, hen antibodies are also more...
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Roles of antigen presenting cells in regulation of Th17 response against Candida albicans
Böhmová, Helena ; Dobeš, Jan (advisor) ; Kostovčíková, Klára (referee)
Candida albicans is a common human pathobiont that inhabits mucosal surfaces throughout the body. In healthy individuals, it behaves as a benign member of the microfora. However, in immunocompromised individuals Candida becomes pathogenic and causes extensive mucosal infections. In the most severe cases, Candida translocates into the bloodstream and causes life-threatening deep tissue infections. Although the innate immune components involved in early anti-Candida immune response are relatively well defned, our knowledge regarding adaptive T cell responses to Candida is limited. Several populations of antigen-presenting cells (APCs) have been implicated in the induction of protective Th17 response against Candida - including innate lymphoid cells type 3 (ILC3s), conventional dendritic cells (cDCs) and CX3CR1+ mononuclear phagocytes (MNPs). The cellular and molecular mechanisms by which Candida-specifc T cells are induced have not yet completely been identifed. Presented thesis focuses on the involvement of direct antigen presentation by these APC populations in mounting the anti-Candida adaptive immune response. Furthermore, this is investigated in the context of both gastrointestinal colonization and bloodstream infection by C. albicans. In the frst part, published data concerning the immune...
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Development od methods for analysis of interactions of the antiviral drug ritonavir with lanosterol-14- α-demethylase of pathogenic yeasts
Burdová, Tereza ; Heidingsfeld, Olga (advisor) ; Dostál, Jiří (referee)
Ritonavir is an HIV protease inhibitor, and has been used as a part of drug cocktails for highly active antiretroviral therapy (HAART). Oropharyngeal candidiasis is one of the most common opportunistic infections among AIDS patients. The research showed that those who were treated with cocktails containing HIV protease inhibitors including ritonavir suffered from oropharyngeal candidiasis to a much lesser extent than patients who received drugs without protease inhibitors. The reason may be the fact that HIV protease inhibitors also inhibit the proteases of pathogenic yeasts, despite high Ki. Another explanation may be that some of these inhibitors block lanosterol-14-α- demethylase, a key enzyme in the biosynthesis of ergosterol, which is an important component of yeast membranes. Aim of the thesis is the isolation of the microsomal fraction of the pathogenic yeasts Candida albicans and Candida parapsilosis and optimization of methods for analysis of the inhibition of yeast lanosterol-14-α- demethylase by ritonavir. Key words: ritonavir, Candida albicans, Candida parapsilosis, lanosterol-14-α- demethylase, microsomal fraction
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Evolution of mixed cultures of the pathogenic yeasts Candida albicans and Candida guilliermondii
Hirko, Dominik ; Heidingsfeld, Olga (advisor) ; Schierová, Michaela (referee)
The objective of this thesis was to investigate how cocultures of Candida albicans and Candida guilliermondii change overtime under control conditions and under the influence of fluconazole. These species are opportunistic fungal pathogens but widely differ in their susceptibility to antimycotic of interest - fluconazole. After a brief introduction to special commonalities, the mechanism of pathogenesis, and the treatment of infection, this work explores each organism's growth curves under selected conditions and the process of artificial evolution using the model of passaging of cocultures. Afterwards, these populations of C. albicans and C. guillieromondii were investigated using qPCR and chromogenic media. qPCR analysis revealed that under control conditions, C. albicans (CA) prevails; the possible reason behind this is a 20% shorter generation time, as revealed by the growth curve. In the presence of fluconazole, two trends occurred. One is related to the innate resistance of C. guilliermondii (CG), where CG was dominant by the end of passaging. The second trend led to CA being the dominant one, despite its susceptibility. This is a result of a heightened resistance, where minimal inhibitory concentration 50 (MIC50) increased almost 10-fold, possibly due to mutations. The change in populations...
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Ammmonium transport in yeast
Faltýnková, Kateřina ; Palková, Zdena (advisor) ; Princová, Jarmila (referee)
Bachelor thesis - Kateřina Faltýnková - Ammmonium transport in yeast Ammonium and ammonia are an essential nutrient for every yeast cells, not only in metabolism, for example in amino acid synthesis, but also as signalling molecules that serve for communication between colonies or for the regulation of pseudohyphal growth. Transport of ammonia and ammonium ions requires active transport, which is provided by MEP permeases inside the cell likely by exporters ATO proteins out of the cell. In this work there are described families of genes MEP and ATO with main focus on their importance for uptake and export of ammonium ions by yeast and also the regulation of these two gene families in the yeast Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans a Yarrowia lipolytica.
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Detection of resistance to echinocandins antifungal agents in Candida sp. using molecular biological methods
Vitáčková, Petra ; Chrenková, Vanda (advisor) ; Nyč, Otakar (referee)
Invasive diseases due to Candida sp., especially by C. albicans, represent very severe complication in immunocompromised patients. More over the presence of antifungal resistance leads to delay of targeted antifungal therapy and increases morbidity and mortality in this group of patients. Therefore the aim was to introduce a rapid method of caspofungin resistance detection by the mass spectrometry MALDITOF. The tests were performed by the use of reference strain C. albicans CCM8261 and caspofungin resistant strain C. albicans M30. Different settings of mass spectrometer were used for the measuring. The obtained spectra were evaluated by correlation and cluster analysis (dendrogram). By cluster analysis it was possible to differenciate the susceptible and the resistant strain. During the analysis we have found, that mass spectrometer settings are unique for each machine and we cannot use the published data. We did not succeed to determine the similarity by correlation analysis. The quality of obtained spectra was quite poor, probably because of non-suitable culture medium used in the test The cluster analysis confirmed the possibility of resistance detection by mass spectrometry; nevertheless more testing profiting from current experience is needed for introduction of this test in routine. Powered by TCPDF...
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CUG Codon in Pathogenic Yeasts of the Genus Candida
Marečková, Lucie ; Heidingsfeld, Olga (advisor) ; Půta, František (referee)
2. Abstract In many Candida species the standard leucine CUG codon is translated as a serine, although not in 100% cases. This dual specifity of the CUG codon has evolved through a mechanism that required codon ambiguity mediated by a unique tRNACAG, which is in vitro aminoacylated more often by serine than by leucine. This codon ambiguity has been tolerated for more than 170 million years. The explanation at least for now is that the CUG codon reassignment could have generated genetic diversity that facilitated occurrence of new phenotypes resistant to stress. Beside this, an important step was to reduce negative impact of the codon ambiguity by crucial mutations in the structure of the ser-tRNACAG. Candida species became a valuable experimental model for elucidation of the genetic code changes. While consequences of the CUG codon reassignment have been extensively studied in Candida albicans, this topic has not yet been addressed in Candida parapsilosis. Solving the structure of C. parapsilosis secreted proteinase Sapp1p provided a tool to carry out a "case study" of possible effects of the CUG codon ambiguity. The SAPP1 gene contains one CUG codon, and the respective serine is located on the loop in the close proximity of the active site of the proteinase.
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