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Operon structures inference in genome-wide analysis
Nejezchlebová, Julie ; Jurečková, Kateřina (referee) ; Schwarzerová, Jana (advisor)
The bachelor thesis is devoted to the problem of derivation of operon structures and creation of a software tool that allows prediction of operon structures. The tool both predicts operons based on gene expression information, but also refines already predicted operons with gene expression information. The tool is tested on the bacteria Escherichia coli BW25113 and Clostridium beijerinckii NRRL B-598. The theoretical part is devoted to description of operon structure and function, genome sequencing, transcriptome analysis, Clostridium beijerinckii NRRL B-598, Escherichia coli BW25113 and already available online tools for inferring operon structures. In the practical part of the thesis, the pre-processing of raw transcriptomic data to obtain a suitable format for the prediction of operon structures, testing of online tools and the actual implementation of the tool itself are discussed.
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The diversity of anaerobic ciliates from the subclass Scuticociliatia and their symbionts
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...
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Antibody-mediated rejection after kidney transplantation
Slatinská, Janka ; Viklický, Ondřej (advisor) ; Reischig, Tomáš (referee) ; Krejčí, Karel (referee)
Antibody-mediated rejection (AMR) is the main cause of the kidney graft dysfunction and its failure after transplantation. Antibodies lead to vascular damage that is either acute or chronic and manifests as sudden or progressive graft dysfunction. Risk factors for development of AMR are time spent on haemodialysis, retransplantation, previous sensitisation against HLA antigens, and persistence of panel-reactive antibodies. Diagnosis is based on detection of deposits of C4d component of complement in peritubular capilaries and presence of donor-specific antibodies (DSA). We can also observe injury caused by antibodies against non-HLA antigens without detection of anti-HLA DSA. Use of "molecular microscope" can be beneficial in diagnosis and stratification of the risk of graft failure. High expression of ENDAT (endothelial activation and injury transcript) improves prediction of kidney graft failure more than C4d staining. Based on gene expression, the AMR scoring system correlates with the diagnosis of AMR and predicts graft loss in the future. The main goal of our work was to recognize patients at risk of AMR. In our study, we confirmed the efficacy and safety of acute AMR therapy with plasmaphereses and administration of intravenous immunoglobulins for improving outcomes of kidney transplantation....
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De novo transcriptomics and its use in non-model organisms
Čalounová, Tereza ; Pluskal, Tomáš (advisor) ; Svatoňová, Petra (referee)
The rise of second generation sequencing enabled the study of non-model organisms. Without the requirement of having a reference genome, de novo transcriptomics allows the study of functional elements of their genomes. That way, the great complexity of non-model organisms can be explored. This thesis gives a comprehensive overview of the de novo transcriptomics experiment workflow from a bioinformatics perspective. The emphasis was placed on both theoretical background and practical approaches. This work also highlights new methods in de novo transcriptomics which may start to dominate in the near future. The practical part of the work presents transXpress - a de novo transcriptome assembly and annotation pipeline. Its use is demonstrated on a non-model plant long pepper (Piper longum) with medicinal potential. Keywords: transcriptomics, de novo transcriptomics, transcriptome, RNA-Seq, non-model organism, assembly
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Variability of the domestic chicken breeds in selected immunological traits of hen and egg
Bílková, Barbora ; Vinkler, Michal (advisor) ; Plachý, Jiří (referee) ; Trefil, Pavel (referee)
The avian immune system is a complex system of defence mechanisms that protect bird hosts against threats from ubiquitous pathogens. According to the co-evolutionary models, variability in immune traits of hosts is the key component providing ability to adapt and enhance their defence mechanisms in presence of constant selective pathogen pressure. Domestic chicken (Gallus gallus f. domestica) is used as a model organism in avian biology and also is one of the most important food-producing animals, not only for their meat but also for the egg production. Unfortunately, in research usually only inbred chicken lines are used and modern poultry husbandry is tight with unilateral breeding towards highly productive breeds. Those approaches decrease intra-population polymorphism in chickens. However, especially in case of farm animals, searching and extending the pool of immune variability and enhancing pathogen resistance is crucial for sustaining healthy and biologically secure populations and their products. Morphologically highly distinct traditional chicken breeds, which have evolved for hundreds years under different selective pressures, may represent this desirable immunological variability. In my thesis I described variability in chosen immunological traits, haematological parameters and proteomic...
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Bioactive molecules involved in blood processing by haematophagous monogeneans of the family Diplozoidae
Jedličková, Lucie
Monogeneans from the family Diplozoidae (subclass Heteronchoinea) are bloodfeeding ectoparasites inhabiting gills of common carp. Digestion of blood in diplozoids is an intracellular process taking place in gut cells within lysosomal cycle in the presence of parasite's peptidases. However, information about the blood digestion comes only from ultrastructural and histochemical analyses. Therefore, I have focused in this work on biochemical and molecular characteristics of bioactive molecules which may participate in blood processing by E. nipponicum adults, especially cysteine peptidases of cathepsin L- and B- types, aspartic peptidases of cathepsin D-type, and Kunitz-type inhibitors of serine peptidases. In homogenates and excretory/secretory (E/S) products of E. nipponicum adults, an activity of cysteine peptidases of cathepsins L-type dominated, followed by an activity of cathepsin D-like aspartic peptidases and a minor cathepsin B-like activity. Inhibitors of the abovementioned peptidase types completely blocked hemoglobinolytic activity in the samples. In the transcriptome of E. nipponicum adults, ten cathepsin L-coding transcripts were found and only one cathepsin B-coding transcript. Primary structures of the encoded enzymes were bioinformatically and phylogenetically compared. Two abundant...
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Prediction of graft function development and rejection of transplanted kidney
Wohlfahrtová, Mariana ; Viklický, Ondřej (advisor) ; Zadražil, Josef (referee) ; Reischig, Tomáš (referee)
Improving the short-term results of kidney transplantation did not result in improving the long-term function and survival of kidney allograft. Organ shortage and increasing number of marginal donors remains the key problem in transplant today. The quality of donor organ is critical for graft function development and survival. The aim is to improve understanding to ischemia/reperfusion injury and its consequences, predict delayed graft function and rejection, improve organ allocation strategy and identify patients suitable for safe drug minimization or complete withdrawal of immunosuppressive therapy. Analysis of donor kidneys identified poor tubular cell quality and low survival factor, Netrin-1 expression levels, to be associated with delayed graft function. We confirmed that reperfusion phase of ischemia/reperfusion injury leads to minimal morphological but significant molecular abnormalities. Dissociation observed in histology and molecular pathology finding calls for an integrated approach in donor quality organ evaluation and allocation for transplantation. Significant heterogeneity within donors with expanded criteria was shown and subgroup of organs at low risk of delayed graft function was identified. We suggested donor biopsies to be performed as a routine praxis in all kidneys...
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The diversity of anaerobic ciliates from the subclass Scuticociliatia and their symbionts
Poláková, Kateřina ; Čepička, Ivan (advisor) ; Fiala, Ivan (referee)
Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...
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Comparison and characterization of salivary proteins from Sergentomyia and Phlebotomus sand flies
Polanská, Nikola ; Volf, Petr (advisor) ; Martin-Martin, Ines (referee) ; Chmelař, Jindřich (referee)
Sand flies (Diptera, Phlebotominae) are small biting insects and vectors of Leishmania spp. which cause medically and veterinary important disease - leishmaniasis. During the piercing of the host skin, sand fly females inject saliva to facilitate the blood feeding. The sand fly saliva is composed of many bioactive molecules which were shown to possess anti-inflammatory and anti-haemostatic functions. The saliva affects host's immunity in the bite site and consequently enhances the survival and development of transmitted pathogens. Most of the studies focus on salivary proteins and enzymes of sand flies belonging to Phlebotomus and Lutzomyia genera, while salivary proteins from sand flies of the third genus Sergentomyia were neglected so far. In this thesis we focused on comparison of salivary proteins from two Phlebotomus species, namely Phlebotomus perniciosus and Phlebotomus orientalis, and Sergentomyia schwetzi. These sand fly species differ not only by the ecology and geographical distribution but also by host preferences. Both Phlebotomus species prefer large or medium-size mammals as the bloodmeal source, particularly rabbits, hares and dogs for P. perniciosus and cattle, goats, sheep and humans for P. orientalis. Contrarily, Sergentomyia sand flies are known for preferred feeding on reptiles...
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Sekvenování transkriptomu pro studium exprese genů u živočichů
Toufar, Jiří
In the introductory chapters, the thesis deals with the best description of the gene expression regulation process, which is an essential prerequisite for understanding the following chapters. Gene expression regulation covers regulation of transcription initiation, transcription elongation control, post-transcriptional modification and RNA editing. In the following chapters, the thesis deals with nucleic acid sequencing. Furthermore, classical sequencing methods, sequencing of the new generation, such as Illumina sequencing and the most modern third-generation sequencing methods such as SMRT-seq or Oxford Nanopore are also analyzed. Moreover, the thesis contains information on the comparison of selected methods. In the following sections, the thesis explains the term transcriptome and general procedure of the RNA sequencing experiment including single cell isolation, single-cell sequencing methods, and animal tissue sequencing. In the final chapters, the thesis explores the use of RNA sequencing and RNA-seq methods for specific purposes. In the conclusion, a comparison of RNA-seq with DNA/RNA microarrays was performed.
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