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Interaction of Borrelia sp. with HL-60 cells and monocytes and cultivation of Anaplasma phagocytophilum in HL-60 cell culture
Marková, Lucie ; Hulínská, Dagmar (advisor) ; Beranová, Jana (referee)
Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are causative agents of Lyme disease and human granulocytic anaplasmosis. Their common vector in Europe are the ticks from the genus Ixodes. In our work, we focused on interaction of innate immune cells with the causative agent of Lyme diseases, that are insubstitutable in their function in the early phase of the disease. Anaplasma phagocytophilum is hard to cultivate, the only possibility is to cultivate it in cell cultures. Successful cultivation of Anaplasma phagocytophilum acquired from patients in our geographic area is crucial for following experiments and for diagnostics too. In our experiments, we used validated cell cultures of HL-60 cells, canine monocytes DH82 and murine monocytes P388D1. During our studies of interaction of the causative agent of Lyme diseases with cells, we used two strains of different species Borrelia. Borrelia garinii M192 and Borrelia burgdorferi sensu stricto B31. These strains vary in virulence. The strain M192 is virulent, but the strain B31 lost its virulence by passages. We specialised in study of morphological changes using light microscopy (observation of dyed and fixed preparates and observation in dark field), eventually by transmision electron microscopy. During our experiments, we concluded that HL-60...
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Dental pulp stem cells
Suchánek, Jakub ; Koberová Ivančaková, Romana (advisor) ; Kukletová, Martina (referee) ; Hampl, Aleš (referee)
The aim of this dissertation study was to optimize the isolation and long term cultivation protocols for human dental pulp stem cells. The protocols which showed best results were used for cultivation of dental pulp stem cell isolated from exfoliated teeth (SHED). Additional aims were to characterize DPSC and SHED and prove their ability to proliferate over Hayflick's limit and differentiate into mature cell lines (osteoblasts, chondroblasts and adipocytes). In order to find optimal protocols for isolation of dental pulp from tooth, we tried three different approaches. During optimization of cultivation protocol we focused on decreasing amount of fetal calf serum (FCS) from 10 % FCS in cultivation media (most often used in literature) into 2 % and thus get closer to cultivation conditions suitable for clinical usage. We compared DPSC cultivated in three different media (medium with 10 % FCS, 2 % FCS supplemented with growth factors and media with 2 % FCS supplemented with ITS and growth factors). For characterization of DPSC and SHED we used basic biological properties (proliferation activity, viability, morphology), their phenotype and karyotype. The study demonstrated that the best protocol for isolation of dental pulp from tooth was to break the roots and extract the pulp throw this aperture. We...
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Clostridium difficile - Importance and current options of laboratory diagnostics
Partlová, Simona ; Nyč, Otakar (advisor) ; Bártová, Markyta (referee)
This thesis mainly describes the question of Clostridium difficile diagnostic service. The first part briefly describes the characteristic of this bacterium. Further, the paper outlines common properties of the bacterium, the causes and manifestations of the diseases that it implicates and current opportunities of the laboratory service. The practical part of bachelors work deals with examination of toxin detection C.difficile in stool specimens, which was in case of positive result completed by cultivation. The cultivation and following Antimicrobial Susceptibility Testing (AST) is not routine task carried out in every clinical laboratory. PCR ribotyping is relatively new method that is provided rarely on the present. Generally, 195 patients have been tested in laboratory of Department of Medical Microbiology in Motol for Clostridium difficile toxin inherency in stool. Overall 30 samples have been tested as positive. The positive samples have been cultivated on special soil just for Clostridium difficile to acquire a clean culture. After that, the metronidazole susceptibility has been obtained from these gained isolates. Powered by TCPDF (www.tcpdf.org)
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Effect of enteral nutrition on the microbiota of children with Crohn's disease
Halbrštátová, Eliška ; Bunešová, Věra (advisor) ; Salmonová, Hana (referee)
Each year there are many facts being discovered, supporting the unexceptionable importance of intestinal microbiota on human health. One of these many fields of interest is the significant role of microbiota in inflammatory bowel diseases arising due to disruption to its balance. This bachelor thesis deals with one of the aforementioned inflammatory bowel diseases, namely Crohn's disease, whose occurrence increases not only in children, but in adults also. Because of the fact that there hasn't been discovered any effective drug yet, treatment is targeted on suppressing the inflammation, curtailment of the urgent phase and attaining long term symptomless period. One of treatment options is enteral nutrition, whose influence on microbiota will be investigated in this study. The research is focused on the influence of nutrition in children patients, which differs by its specifics from adult treatment.
Due to the difficulty of the study, from 50 originally classified patients, 15 children patients of Motol University Hospital were selected. Those were subjected to treatment by enteral nutrition for a period of 6 weeks, during which stool samples were taken. Fresh stool samples were used for cultivation and biochemical test.
Cultivated and quantified were the groups of gramnegative bacteria, coliform bacteria and Escherichia coli, total amount of anaerobic bacteria and bacteria of the genus Bifidobacterium, Lactobacillus and Enterococcus. Biochemical tests were further conducted, those were targeted on the detection and identification of the genus Bifidobacterium in the patient's stool. For identification, the commercial diagnostic system API ZYM was used. With the aim of promoting the conclusiveness of study, samples were preserved for the isolation of bacterial DNA and further research.
A review of the literature proved the fact that in Crohn's disease some of the beneficial bacterial groups are reduced, while, on the other side, quantity of some bacterial groups increases, which converts otherwise beneficial and ordinarily inhabiting groups of the intestinal microbiota to pathogenic ones. So enteral nutrition treatment should support increasing the number of reduced groups and the pathogenic ones should be reduced to benefit of reduced groups.
Summing up the results of our study comes to the conclusion, that expected increase bacteria of the genus Enterococcus a Lactobacillus was achieved, thus confirming efficiency of enteral nutrition treatment. On the contrary, the growth of the bacteria of the genus E. coli and the group of coliform bacteria, together with the downturn of bacteria of the genus Bifidobacterium, refutes the efficiency of enteral nutrition treatment.
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Factors influencing the quality of red wine
Zechmeisterová, Lucie ; Vránová, Dana (referee) ; Omelková, Jiřina (advisor)
In my thesis, I focused on monitoring of microorganisms in the sample of red grape juice and on the interactions between yeasts, bacteria and filamentous fungi. Three different media were applied for the cultivation of microorganisms; firstly for monitoring of total volume of microorganisms, secondly for yeasts and third time for lactic acid bacteria. The indirect method was used for the determination of the amount of viable cells. This method consists in enumerating of visible macroscopic colonies grown up on agar plates. When the cells grew up, the forms of colonies were analyzed visually and the morphology of microorganisms was detected microscopically. The operating time of enzymes in grape juice in the production of red wine was monitored after application of commercial enzymatic preparation. The enzym action in grape juice was observed on the basis of the process of degradation of high – molecular substrate by enzymes through the use of Ubbelohd´s viscometer. The research findings provided a lot of knowledge about the occurance of microflora in the process of production of red wine. The commercial preparations added to grape juice played a significant role.
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Infection of salmonelosis, their source and diagnosis.
Valkus, Martin ; Veselá, Mária (referee) ; Vránová, Dana (advisor)
This bachelor‘s thesis deals with infection of salmonelosis, it’s source, prevention and diagnosis. Theoretical part includes general information about Salmonella species and its morphology. It describes pathogenesis, source of infection, clinical symptoms, treatment and diet recommendations. It analyzes statistics of number of infection occurrences in Czech Republic. It also deals with prevention of infection origin on schools, population foreknowledge and climatic effects. Experimental part of this thesis contains description of optimization procedure of identification Enterobacteriaceae by adjusting of pH of prepared medium MIU.
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Nožnosti kultivace komerčních zón v okrajových částech sídel
Šabata, Ondřej
Cultivation of Shopping Center spaces and it's alternatives in peripheral urbs is a complex theme, which is devided into theoretical part and a study of a particular situation in practice. The origin, development and context of the peripheral urbs related on development of regional planning is described at the first part of this paper. Then current situation is described, standardized and demonstrated on several examples of Shopping Centers in Europe, focused on landscape continuality. Second part of this paper is focused on landscape study in practice on example of Cerna Pole Shopping Center in Brno, CZE. Process of analyzing and creating of a solution is described, including pictures of the area and examples of particular architectural design. In conclusion, the sumary is completed by several options of cultivation of commercial zones and public spaces. Plans and designs of an architectural study are included separately at the end of this script.
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In vitro expansion and activation of natural killer cells for cell therapy.
BENEŠOVÁ, Monika
NK cells are part of the non-specific immune response and are one of the main components of antitumor immunity. They do not need antigen stimul for thein activation but recognize damaged ( transformed ) cells by characteristic decreased expression of MHC I molecules. These natural killers become subjekt of many clinical studies based on the use of anti-tumor activity of NK cells for both solid tumors and in hemato - oncological diseases. The aim of this study was to find optimal conditions for in vitro expansion and activation of NK cells. NK cells were isolated from mononuclear cell fraction by magnetic separation and cultured in two types of media SCGM and X - VIVO 10 with the addition of interleukin-2 respectively OKT3 antibody. The influence of the mononuclear cells on proliferation of NK cells was tested. After a 6- day culture , the cells were passaged and growth of NK cells was determined using hematological analyzer and flow cytometry. Expression of the activation markers CD25 and CD336 (NKp44) was observed. All experiments were conducted under conditions of good manufacturing practice. Slightly higher gain of NK cells was observed in SCGM media without a significant differences in the other additives. Much higher number of NK cells was observed in culture with supporting irradiated mononuclear cells. There were no differences in cultures with autologous or allogeneic cells. We found that NK cells with higher proliferative potential express increasingly CD25, unlike the cells with decreased proliferation which had increased expression of CD336 marker. The work led to the definition of the optimal culture conditions for NK cells and became the basis for the further development of the medicinal product from in vitro activated NK cells.
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