Studium signalizačních molekul imunoreceptorů

A delicate balance in the number, specific type and function of leukocytes is required for proper functionality of the mammalian immune system. Innate immunity, which quickly recognizes pathogens, represents the first line of defense. Later, a more specific response is generated via adaptive immunity. Deregulation of the immune system is manifested by the inability to control infection, development of allergic, autoimmune disorders or even cancer, and ultimately can lead to death. To fulfill their functions, cells develop an intricate network of intra- as well as extra-cellular molecules organized into signaling cascades, which allows them to communicate between each other. Better understanding of the molecular mechanisms of signaling pathways in leukocytes is critical for design of efficient therapies. In this thesis, leukocyte signaling was studied in several aspects. First, the role of adhesion molecules in pathogenesis of cervical cancer and the regulation of their expression was investigated. The second publication describes a new transmembrane adaptor protein (TRAP), called prolin rich 7 (PRR7), as a potentially interesting regulator of signaling and apoptosis in activated T cells. The final publication characterized the role of the Btk kinase downstream of the triggering receptor expressed...Správná funkce imunitního systému savců je zajišťována křehkou rovnováhou mezi množstvím a nejrůznějšími typy leukocytů, které vykonávají velmi specifické funkce. Přirozená imunita, která rychle reaguje na přítomnost patogenů představuje první obrannou linii organismu. Teprve později se zahajuje specifičtější odpověď tím, jak se aktivuje adaptivní imunita reprezentovaná T a B lymfocyty a produkcí protilátek. Deregulace imunitního systému se projevuje neschopností potlačit infekci, rozvojem alergických a autoimunitních onemocnění nebo dokonce rakovinného bujení, takže v konečném důsledku může vést až k smrti. K tomu, aby leukocyty mohly vykonávat své funkce, vyvinuly spletitou síť intra- i extracelulárních molekul uspořádaných do signálních kaskád, které jim umožňují vzájemnou komunikaci. Pochopení molekulárních mechanismů signálních drah leukocytů je zásadním předpokladem pro vyvinutí účinných terapií. V této disertační práci byla leukocytární signalizace studována v několika aspektech. Nejprve bylo zjišťováno, jakou roli hraje zvýšená exprese adhezivních molekul v patogenezi rakoviny děložního čípku a jak je tato exprese regulována na molekulární úrovni. Druhá publikace představuje nový transmembránový adaptorový protein, nazvaný PRR7 (prolin rich 7), jako potenciálně zajímavý regulátor...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Control of development, secondary metabolism and light-dependent carotenoid biosynthesis by the velvet complex of Neurospora crassa

Neurospora crassa is an established reference organism to investigate carotene biosynthesis and light regulation. However, there is little evidence of its capacity to produce secondary metabolites. Here, we report the role of the fungal-specific regulatory velvet complexes in development and secondary metabolism (SM) in N. crassa. Three velvet proteins VE-1, VE-2, VOS-1, and a putative methyltransferase LAE-1 show light-independent nucleocytoplasmic localization. Two distinct velvet complexes, a heterotrimeric VE-1/VE-2/LAE-1 and a heterodimeric VE-2/VOS-1 are found in vivo. The heterotrimer-complex, which positively regulates sexual development and represses asexual sporulation, suppresses siderophore coprogen production under iron starvation conditions. The VE-1/VE-2 heterodimer controls carotene production. VE-1 regulates the expression of .15% of the whole genome, comprising mainly regulatory and developmental features. We also studied intergenera functions of the velvet complex through complementation of Aspergillus nidulans veA, velB, laeA, vosA mutants with their N. crassa orthologs ve-1, ve-2, lae-1, and vos-1, respectively. Expression of VE-1 and VE-2 in A. nidulans successfully substitutes the developmental and SM functions of VeA and VelB by forming two functional chimeric velvet complexes in vivo, VelB/VE-1/LaeA and VE-2/VeA/LaeA, respectively. Reciprocally, expression of veA restores the phenotypes of the N. crassa ve-1 mutant. All N. crassa velvet proteins heterologously expressed in A. nidulans are localized to the nuclear fraction independent of light. These data highlight the conservation of the complex formation in N. crassa and A. nidulans. However, they also underline the intergenera similarities and differences of velvet roles according to different life styles, niches and ontogenetic processes

Studies on immunoreceptor signaling molecules

A delicate balance in the number, specific type and function of leukocytes is required for proper functionality of the mammalian immune system. Innate immunity, which quickly recognizes pathogens, represents the first line of defense. Later, a more specific response is generated via adaptive immunity. Deregulation of the immune system is manifested by the inability to control infection, development of allergic, autoimmune disorders or even cancer, and ultimately can lead to death. To fulfill their functions, cells develop an intricate network of intra- as well as extra-cellular molecules organized into signaling cascades, which allows them to communicate between each other. Better understanding of the molecular mechanisms of signaling pathways in leukocytes is critical for design of efficient therapies. In this thesis, leukocyte signaling was studied in several aspects. First, the role of adhesion molecules in pathogenesis of cervical cancer and the regulation of their expression was investigated. The second publication describes a new transmembrane adaptor protein (TRAP), called prolin rich 7 (PRR7), as a potentially interesting regulator of signaling and apoptosis in activated T cells. The final publication characterized the role of the Btk kinase downstream of the triggering receptor expressed..

Marked Neurospora crassa Strains for Competition Experiments and Bayesian Methods for Fitness Estimates

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A. The csr-1∗ marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.peerReviewe