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Molecular logic of the Notch ligands in development and disease
Trampotová, Eliška ; Mašek, Jan (advisor) ; Rozbeský, Daniel (referee)
The Notch signalling pathway is one of the key signal transduction pathways in the embryonic development of all metazoa. In mammals, the "canonical" signalling occurs on four Notch receptors, and it is triggered by five ligands varying in both their structures and functions. The importance of the pathway for embryonic development is reflected by the fact that mutations of either receptors or ligands result in various congenital disorders. A prime example is the Alagille syndrome - a rare multisystemic condition caused by mutations of the Notch ligand Jagged1 in 94 % of cases. Much of the research effort so far was concentrated into elucidating how the Notch receptors operate, however one could argue the importance of ligand activity regulation is even higher. This thesis thus aims to provide a comprehensive comparison of the five mammalian Notch ligands' structures and roles in developmental processes. The main focus of the thesis is the variability of ligand-receptor interactions and biophysical mechanisms of signalling, highlighting the importance of the Notch ligands' intracellular domains' interactions.
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New leukocyte membrane adaptor proteins
Králová, Jarmila ; Brdička, Tomáš (advisor) ; Černý, Jan (referee) ; Šedivá, Anna (referee)
Membrane adaptor proteins are characterized by the lack of enzymatic activity and the presence of various interaction sites for other proteins and cellular membranes. They typically function as scaffolds connecting receptors or other adaptors with proximal signaling molecules at cellular membranes. Their overall effects on signaling can be activating or inhibiting depending on the nature of the effector molecules they recruit. SCIMP is one of the membrane adaptors discussed in this thesis. It is expressed in antigen- presenting cells and it has been previously shown to enhance MHCII signaling in B cells. This thesis covers the analysis of SCIMP functions beyond B cells and describes the first analysis of SCIMP deficient mice. Although the results of this analysis did not show any alterations in immune cell populations, the novel function of SCIMP in dendritic cell signaling downstream of DECTIN- 1 was uncovered. DECTIN-1 is a pattern recognition receptor involved in antifungal immunity. The data presented in this thesis describe the role of SCIMP in sustaining DECTIN-1 signaling over relatively long periods of time and the contribution of SCIMP signaling to maintaining prolonged production of pro-inflammatory cytokines. PSTPIP2 is another interesting adaptor discussed in this thesis. It is...
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Involvement of Asthma-associated Protein ORMLDL3 in Mast Cell Signalling
Eitler, Jiří ; Dráber, Petr (advisor) ; Konvalinka, Jan (referee)
4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...
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New leukocyte membrane adaptor proteins
Králová, Jarmila ; Brdička, Tomáš (advisor) ; Černý, Jan (referee) ; Šedivá, Anna (referee)
Membrane adaptor proteins are characterized by the lack of enzymatic activity and the presence of various interaction sites for other proteins and cellular membranes. They typically function as scaffolds connecting receptors or other adaptors with proximal signaling molecules at cellular membranes. Their overall effects on signaling can be activating or inhibiting depending on the nature of the effector molecules they recruit. SCIMP is one of the membrane adaptors discussed in this thesis. It is expressed in antigen- presenting cells and it has been previously shown to enhance MHCII signaling in B cells. This thesis covers the analysis of SCIMP functions beyond B cells and describes the first analysis of SCIMP deficient mice. Although the results of this analysis did not show any alterations in immune cell populations, the novel function of SCIMP in dendritic cell signaling downstream of DECTIN- 1 was uncovered. DECTIN-1 is a pattern recognition receptor involved in antifungal immunity. The data presented in this thesis describe the role of SCIMP in sustaining DECTIN-1 signaling over relatively long periods of time and the contribution of SCIMP signaling to maintaining prolonged production of pro-inflammatory cytokines. PSTPIP2 is another interesting adaptor discussed in this thesis. It is...
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Evolution of regulatory mechanisms of EGF receptor activation
Trávníčková, Květa ; Stříšovský, Kvido (advisor) ; Koudelková, Lenka (referee)
Signalling through EGF receptor is crucial both for ontogenesis and for maintaining homeostasis in adult organisms. It is involved in controlling cellular behaviours such as proliferation, migration or differentiation. This thesis provides an insight into evolution of the regulatory mechanisms of EGF receptor activation by discussing their principles in C. elegans, D. melanogaster and H. sapiens sapiens, on the basis of which conclusions about their evolutionary tendencies are made. Attention is focused on the roles of the rhomboid family of proteins, whose activity is tightly associated with EGF receptor signalling. Dysregulation of the EGF receptor unnegligibly contributes to the development of various diseases, mainly many types of cancer, but also schizophrenia, psoriasis and cardiovascular disorders. Experimental results obtained on this field of research therefore have the potential to be applied in drug design.
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A diffusion-based model of signal transduction in the vertebrate olfactory sensory neuron, and its sensitivity analysis
Beneš, Martin ; Zápotocký, Martin (advisor) ; Jelínek, Jan (referee)
The goal of this model is to create and to implement qualitative model of the signal track of olfactory sensory neuron, including the feedback with a focus on diffusion of substances that allows to conduct more simulations for the better understanding of dynamics of the signal track. This model is expected to be used for the simulation of influencing during the activation of two receptors in firstly defined distance. Model was created and therefore implemented in a programming language Python with the use of library STEPS. Then I have conducted sensitivity analysis by a method Morris OAT on the model, together with an optimization with the usage of change of individual parameters with a great importance on the output of the model. Model is conducting good and biologically comparable results when there are from 10 to 100 active receptors at the beginning of the track. Unfortunately with a lower numbers, the results are not valid and therefore not to be used for the simulation of influencing of two activated receptors. Despite this is a main benefit of the work the model of signal transduction for the whole signal track with an included feedback and emphasis on diffusion. Another benefit is a set of scripts for the sensitivity analysis by a method Morris OAT and optimization.
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Phosphoinositides and their effectors in regulation of the Wnt signalling pathway
Knop, Filip ; Macůrková, Marie (advisor) ; Krausová, Michaela (referee)
Phosphoinositides (PIs) make up only a small proportion of overall amount of lipids in cell membranes. However, their function mediated through protein effectors is indispensable for cell signaling, vesicular trafficking, cell movement and other important aspects of cellular life. In this bachelor thesis function of PIs is described in relation to Wnt signaling pathway. Proper execution of several steps of the Wnt signaling pathway requires the presence of PIs. Retrograde transport of Wntless (Wls) from the plasma membrane (PM) back to the Golgi apparatus (GA) in Wnt producing cells or internalization of Wnt receptors in Wnt receiving cells are only two examples. All processes are tightly regulated and malfunction of enzymes processing PIs can cause their deregulation resulting in disruption of the Wnt signaling pathway. As deregulated Wnt signaling is a known cause of serious diseases including cancer, understanding the crosstalk between PIs and Wnt signaling could help in designing novel strategies for therapeutic intervention.
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Molecular mechanisms of signal transduction by the ERK signaling cascade.
Bráborec, Vojtěch ; Rösel, Daniel (referee) ; Vomastek, Tomáš (advisor)
The MAPK (mitogen-activated protein kinase) cascade represents an evolutionary conserved mechanism by which cells sense extracellular signals and convert them into variety of context-dependent responses. The best studied member of the MAPK protein family is protein kinase ERK (extracellular signal-regulated kinase). Together with protein kinases Raf and MEK (MAPK/ERK kinase) comprise a prototypical signaling pathway which regulates broad-spectrum of biological processes such as cellular proliferation, differentiation, cellular migration, adhesion or apoptosis. To modulate such a multitude of distinct responses by a single pathway, cells utilize mechanisms such as signal strength and duration, distinct protein localization, communication with other signaling pathways, differential substrate selection and the selection of interactive partners. All presented means of regulation are influenced by proteins with non-enzymatic functions - scaffold proteins, protein inhibitors and anchoring proteins. These protein modulators channel the signals leading to particular cellular response, and thus represent the key element of signal transduction. Despite increasing importance of protein modulators in cellular signaling, their biological roles remain mostly unknown. The physiological importance of protein modulators is...
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