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Pathogenesis of nephrotic syndrome in children and predictors of corticosteroid treatment response
Bezdíčka, Martin ; Souček, Ondřej (advisor) ; Doležel, Zdeněk (referee) ; Reiterová, Jana (referee)
Nephrotic syndrome is a kidney disease caused by injury of the podocytes. It can be secondary due to infection, systemic disease or certain drugs, but it may also present as sudden primary nephrotic syndrome without obvious inducer. Current standard treatment has many severe adverse effects. In some patients that are resistant to the initial several-week-long glucocorticoid treatment it is possible to reveal the causative genetic aetiology of the disease, whereas in the rest of them aetiology remains unknown. Those who respond well to initial glucocorticoid treatment and achieve remission may later on develop repeated relapses requiring long-term glucocorticoid therapy. This work describes our original research studies focusing on the improvement of genetic diagnostics of nephrotic syndrome, on the exploration of molecular mechanisms of the second most common genetic cause of the steroid-resistant nephrotic syndrome (transcription factor WT1 mutants) and on the search of clinical and laboratory factors that could predict the resistence to glucocorticoid treatment. By combining Sanger and next-generation sequencing (NGS) we were the first to identify monogenic cause in 38 % of Czech and Slovak children with steroid-resistant nephrotic syndrome whose samples had been collected for 18 years. The most...
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Molekulární diagnostika fungálních patogenů z klinických vzorků
CIMICKÁ, Jana
This study is focused on optimization and use of molecular methods in mycological diagnostics. The patient's life often depends on early diagnosis, which current culture methods cannot provide. The solution is based on pan-fungal primers of ITS region and next generation sequencing (NGS). These methods were used for fungal detection from FFPE tissue samples from patients with ulcerative colitis (UC), Crohn disease (CD) and 10 controls without chronic inflammatory changes in the intestine. Results of this study have three levels. Firstly, the process of optimization highlights the importance of sterile DNA isolation in a laminar box which can prevent contamination by Malassezia spp. Secondly, raw data showed balanced rate of Ascomycota and Basidiomycota in CD samples similar to negative controls, while UC samples indicated higher representation of Ascomycota than other groups. Thirdly, detailed data showed fungal genera Malassezia, Cladosporium and Toninia in all groups, while genus Candida was found only in UC samples and genera Engyodontium and Ramularia were found in CD samples. In conclusion, mycobiome plays a role in inflammatory bowel diseases; however, its compartments are still questionable. The optimization of pan-fungal nested PCR and NGS helped with introduction of mycological diagnostics from FFPE samples to Bioptická laboratoř s.r.o. and is used for differential diagnostics.
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Virom of lower urinary tracts
Cirbusová, Adéla ; Saláková, Martina (advisor) ; Španielová, Hana (referee)
The human urinary tract was considered to be a sterile environment for many years. However, studies over the past decade have shown that urine harbours rich microbial community which includes also viruses. Nevertheless, there is only very little known about urinary virome so far. Optimised Next Generation Sequencing (NGS) protocol was used to describe the urinary virome of three individuals. However, characterization of the virome from urine samples using NGS proved to be quite challenging, mainly due to observed viral genomes fragmentation. Despite this problem, it was possible to identify human endogenous retroviruses in all individuals and also JC polyomavirus in two of them. Quantitative PCR was further used to characterize part of the urinary virome represented by human DNA viruses. Possible differences in prevalence and viral load of human DNA viruses were observed in individuals with and without bladder carcinoma (bc). Urine of these patients was obtained from different sites of the urinary tract to further establish, if there is a difference in these samples. Torque Teno virus and JC polyomavirus were found as the most common viruses. Torque Teno virus was detected in 75 % patients with and 60 % patients without bc, JC polyomavirus in 43,8 % patients with and 50 % patients without bc. BK...
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Molecular identification, typing and adaptation of Achromobacter spp. during the course of chronic infection
Učíková, Barbora ; Dřevínek, Pavel (advisor) ; Nemec, Alexandr (referee)
Achromobacter spp. is an emerging pathogen, especially in chronic respiratory infections in patients with cystic fibrosis. MALDI-TOF mass spectrometry provides reliable identification only at the genus level. The nrdA gene sequence is used for species identification of representatives. Clonality studies using multilocus sequence typing can determine whether a patient is still infected with the same clone or whether reinfection with a new strain occurs over time. Time-collected isolates of Achromobacter spp. from patients with cystic fibrosis were included in our study. Patients were divided into three groups according to the time interval between collections. In the first group, the external interval between collections was approximately 10 years, in the second group 7 to 12 months, and the remaining group consisted of single isolates. In the course of chronic infection, Achromobacter spp. adapt to the exposed antibiotics and to the host. Isolates sampled at an interval of 10 years showed a higher number of mutations than isolates with a sampling interval of up to one year. During chronic infection, loss of motility occurs, which we demonstrated phenotypically at the level of motility, reduction in flagella number and changes in flagellar genes. Increased resistance was observed in some isolates by...
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The differences in the virome of different populations of honey bee (Apis mellifera)
Kadlečková, Dominika ; Tachezy, Ruth (advisor) ; Votýpka, Jan (referee)
European honey bee (Apis mellifera) is major pollinator for agriculture and vital for food production. Large number of viruses infecting A. mellifera have been discovered over the years, but it isn't yet known if they are pathogenic for their host. However, presence of non-viral pathogens like Varroa destructor can greatly increase their virulence and have fatal consequences for the colony. The aim of this study was to test and verify robustness of the method for virome detection on healthy honey bees from the Czech Republic. Last but not least we aimed to detect non-viral parasites and correlate their presence with detected viruses. We have successfully identified large number of viral sequences from different viral families. Viral composition was found to be influenced mainly by colony from where the honey bees were collected. That was mainly given by a large amount of bacteriophages in the samples. However, analysis of individual viruses, known to infect honey bee, indicated that viral prevalence and viral loads of specific viruses is quite different among individual honey bees from the same colony. Interestingly we were able to find highly diverse Lake Sinai viruses. We were able to observe correlations either between individual viruses or viral other non-viral pathogens. Further analysis is...
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Contribution of Next Generation Sequencing for Laboratory Diagnostics
Votýpka, Pavel ; Beránek, Martin (advisor) ; Macháček, Miloslav (referee)
5 ABSTRACT Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Pavel Votýpka Supervisor: Doc. PharmDr. Martin Beránek, Ph.D. Consultant: Mgr. Nikola Ptáková Title of diploma thesis: Contribution of Next Generation Sequencing for Laboratory Diagnostics The endeavor to sequence the whole human genome lead not only to the knowledge acquisition regarding the human genetic information but as well to the development of new sequencing methods and technologies. In order to keep up with progress in genetic field in many clinical and research laboratories the new massive parallel sequencing equipment is being utilized. On the market are currently established four leading platforms - Illumina, Solid, Ion Torrent and 454 Life Technologies. The process of sequencing analysis can be summarized into three main steps - the sequencing library preparation, sequencing itself, variant calling and data analysis. Each part of the sequencing analysis exhibits certain specifics, we need to count with and as well its pitfalls, we need to avoid or to minimalize their impact on the analysis final result. Recently new methods termed sequencing of the 3rd generation are being developed, enabling sequence of a single DNA molecule to be determined without previous...
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