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The role of CRL4 ubiquitin ligase complex in homeostasis and regeneration of gastro intestinal tract
Daněk, Veronika ; Procházka, Jan (advisor) ; Janečková, Lucie (referee) ; Čermák, Lukáš (referee)
Ubiquitination represents a central regulatory mechanism for protein function utilization. Dysfunction of ubiquitin ligases is associated with the development of various pathological conditions. However, the full complexity of the ubiquitinating network in tissue homeostasis and carcinogenesis remains to be elucidated, limiting its potential as a therapeutic target. In my PhD project, I aimed to reveal the role of E3 ubiquitin ligases, particularly CUL4A, in the alteration of regulatory pathways that result in gastrointestinal (GIT) homeostasis disorders and tumor expansion. By using a novel semantic biclustering technique, we performed expression profiling of the E3 ubiquitin ligases and uncovered the potentially redundant features of GIT-specific ubiquitin ligases based on their tissue-specific expression and ontological relationships. Testing the identified compensatory network on the mouse model showed that genes from the same ontology group simultaneously altered their expression pattern after induced epithelial damage, exposing their complementary role during tissue regeneration. Next, I focused on CUL4A, a scaffold of the Cullin4-RING E3 ubiquitin ligase complex (CRL4). We described that CUL4A is expressed by populations of secretory cells in the small intestine and colon. CUL4A deficiency...
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Classification based on mixture models
Janečková, Lucie ; Komárek, Arnošt (advisor) ; Maciak, Matúš (referee)
This thesis deals with classification based on mixture models, mainly on models finite normal. At first, there are introduced basic definitions and characteristics of finite mix- tures. Afterwards there is described the maximum likelihood method and her obstacles in context of finite mixtures, which we are using for unknown parameters estimation. Then there is described EM algorithm, that is used to obtain the maximum likelihood estimator and there are calculated the formulae for one iteration of EM algorithm. In the last part there is shown, how can finite normal mixtures be used for classification. 1
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Tcf4 transcription factor in the intestinal epithelium renewal and pathology
Müllerová, Tereza ; Janečková, Lucie (advisor) ; Vávra, Jiří (referee)
TCF4 (T-Cell Factor 4, sometimes also TCF7L2) is an important effector of the canonical Wnt signalling pathway. The transcription factor is produced in many different isoforms with tissue-specific expressionand diametricallyopposing functions.In the intestine,TCF4 is a key factor in epithelial regenerationandmaintenance of stem cell homeostasis. Its depletion causes loss of the proliferatingcrypt compartment and complete breakdown of the intestinal mucosal architecture. The processes in which it acts are therefore vital and must be strongly regulated by the Wnt signalling pathway. Disturbances in TCF4 expression or alternative splicing often lead to a wide range of complex pathologies such as colon cancer, ileal Crohn's disease and type 2 diabetes mellitus.
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In vitro 3D organization of the mammalian testes
Zahradníková, Hana ; Tlapáková, Tereza (advisor) ; Janečková, Lucie (referee)
A testicular organoid that would sufficiently recapitulate the architecture of the testicular tissue and at the same time be able to provide the complete process of spermatogenesis in vitro has not yet been created. Achieving this goal would mean the development of a 3D model of the testis, which would mimic the in vivo situation in terms of cell arrangement and could thus contribute to a deeper understanding of the physiological functioning of the testicular microenvironment. Among other things, such a model has a great potential for clarifying the causes of male infertility and finding treatment options. This thesis dealt with the generation of organoids from mouse or human testicular cell suspensions, which can also be used, for example, to study de novo organogenesis. A total of four 3D culture systems were tested, of which the soft agar culture system (SACS) achieved the best results. Furthermore, as part of this thesis, the procedure for preparing testicular organoids for light sheet microscopy was successfully introduced and optimized, enabling the evaluation of their internal structure. From the testicular cell suspension of a 5-day-old mouse, it was possible to prepare testicular organoids, the structure of which in some respects resembled the organization of the testis in vivo. The...
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Study of the substrate specificity of the LACTB tumour suppressor enzyme
Baudyšová, Alžběta ; Kečkéšová, Zuzana (advisor) ; Janečková, Lucie (referee)
Serine beta-lactamase-like protein (LACTB) is a tumour suppressor that modulates mitochondrial lipid metabolism and induces differentiation of breast cancer cells. This is achieved by the LACTB-dependent downregulation of phosphatidylserine- decarboxylase (PISD) which subsequently leads to decreases in the amounts of phosphatidylethanolamines and lysophosphatidylethanolamines in mitochondrial membranes. However, PISD was shown to not be a direct substrate of the LACTB enzyme what leaves the identity of the LACTB substrate an open question. To fill this important gap in the mechanism of the LACTB tumour suppressive pathway, this diploma thesis was focused on finding a physiological substrate of LACTB via Proteomic Identification of protease Cleavage Sites (PICS) assay. For this purpose, the other sub-aims of this project were to isolate recombinant wild-type LACTB and its catalytic mutant, to reveal ideal in vitro conditions for LACTB activity and to find out the requirements needed for LACTB multimerization. My results show that in vitro activity of LACTB is increased in the presence of higher pH and calcium ions. I also show that higher LACTB multimeric forms are bound together via disulfide bonds as they disintegrate after treatment with dithiothreitol. Furthermore, and most importantly, I show...
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Classification based on mixture models
Janečková, Lucie ; Komárek, Arnošt (advisor) ; Maciak, Matúš (referee)
This thesis deals with classification based on mixture models, mainly on models finite normal. At first, there are introduced basic definitions and characteristics of finite mix- tures. Afterwards there is described the maximum likelihood method and her obstacles in context of finite mixtures, which we are using for unknown parameters estimation. Then there is described EM algorithm, that is used to obtain the maximum likelihood estimator and there are calculated the formulae for one iteration of EM algorithm. In the last part there is shown, how can finite normal mixtures be used for classification. 1
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Novel mechanisms of T cell-mediated intestinal autoimmunity to Paneth cells
Brabec, Tomáš ; Filipp, Dominik (advisor) ; Janečková, Lucie (referee)
(En) Paneth cells are one of the major player in the maintenance of the homeostatic relationship between intestinal microbiota and the immune system. This function is largely achieved by their production of bactericidal enteric α-defensins (ED) and other antimicrobials. Disruption of Paneth cell functions is associated with severe human disorders such as Crohn's disease (CD) and Autoimmune Polyendocrinopathy- Candidiasis-Ectodermal Dystrophy (APECED). However, there is only a very limited information regarding the interactions and regulatory circuits operating between Paneth cells and intestinal immune system in either health or under pathological conditions. The previous study conducted in our laboratory described a new mechanism for the initiation and maintenance of Paneth cells targeted autoimmunity. The suggested model was that ED-specific T cells escape the selection in the thymus, infiltrate the intestine and diminish Paneth cell numbers through autoimmune destruction. This process also lead to the accumulation of inflammation- inducing bacteria, which were implied to exacerbate the inflammatory autoimmunity. Since this model of intestinal autoimmunity is of correlative nature, its intrinsic mechanism and functional relationships between immune system, Paneth cells and microbiota are largely...
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Aluminium fractionation in water treatment.
Houšková, Lucie ; Benešová, Libuše (advisor) ; Janečková, Lucie (referee)
Aluminium and natural organic compounds are common components of surface water in our country. Removal of these substance represents one of basic problems of water treatment. Chemical treatment process is based on particles destabilisation and aggregation. The efficiency of treatment is influenced by many chemical and physical factors (the reaction conditions, type and dosage of destabilisation reagens, reactive pH, the mean velocity gradient and the period of its duration). Common water treatment technologies are based on the reagent doeses optimisation. Properties of formed aggregates (namely their size, shape and density) and their separation capabilities are influenced by the magnitude of the velocity gradient and the period of its application. This thesis is focused on the optimisation of doses of alum - Al2(SO4)3.18 H2O and the properties of aggregation. For increasing of alkalinity the lime water was used as an alkalisation reagent. Laboratory tests were conducted by jar test using the variable speed paddle gang stirrer. The result showes that Al2(SO4)3.18 H2O is suitable destabilisation reagens, but salts of the Fe3+ ionts are better.
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Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii
Žídek, Radim ; Kozmik, Zbyněk (advisor) ; Janečková, Lucie (referee) ; Macůrková, Marie (referee)
Radim Žídek "Wnt/β-catenin signalling in the development of the marine annelid Platynereis dumerilii" (dissertation) Abstract: Wnt/β-catenin signalling is absolutely crucial for the early embryonic development of metazoan animals from the establishment of body axes, through the specification of germ layers and tissues to the development of organ systems. I used pharmacological manipulations of the Wnt/β-catenin pathway activity in the planktonic larvae of the marine polychaete annelid Platynereis dumerilii, the representative of the clade Spiralia, to investigate the role of Wnt/β- catenin signalling in the development and evolution of three hallmarks of Bilateria: the central nervous system, the body segmentation and the digestive tube. Wnt proteins are produced in all three aforementioned systems in Platynereis where they trigger the Wnt/β-catenin pathway in neighbouring cells. I describe here, for the first time in Platynereis, a homologue of the endpoint transcription factor of the entire pathway, Pdu-Tcf, which is subjected to an alternative splicing and along with a Wnt target gene Pdu-Axin is expressed in tissues with the active Wnt signalling - in the brain ganglia, in the neuroectoderm along the ventral midline, in segments, in the posterior growth zone and in the gut. Pharmacological manipulations...
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Determination of enantiomers of amino acids by HPLC
Michalčíková, Regina ; Tesařová, Eva (advisor) ; Janečková, Lucie (referee)
This Bachelor's thesis deals with determination of amino acids enantiomers, namely alanine in the experimental part, by high performance liquid chromatography using chiral stationary phases based on teicoplanin. The theoretical part is focused on summarizing the fundamental issue of chirality and chiral amino acids in foods, the characteristics of high-performance liquid chromatography, as one of the most appropriate methods to enantioseparation amino acids, and description of the methods of derivatization, which are used in determination of enantiomers of amino acids. The experimental part deals with the separation of alanine enantiomers, with the choice of the best separation system for the separation and determination of D-and L-alanine in fruit juice.
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