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The role of selected mitogen-activated protein kinases in Arabidopsis
LUPTOVČIAK, Ivan; ŠAMAJ, Jozef
2017 - English
Predložená dizertačná práca je zameraná na mitogen-aktivovanú proteín kinázu 6 (MPK6) a proteín strihajúci mikrotubuli KATANIN 1 (KTN1) u modelovej rastliny Arabidopsis thaliana. V práci je prezentovaná genetická komplementácia mpk6-2 mutantného fenotypu chimérnym GFP-MPK6 proteínom exprimovaným pod natívnym promótorom, ktorý bol použitý na in vivo lokalizačné štúdie. Ďalší typ chimérneho proteínu MPK6-VenusN bol použitý na testovanie potenciálnej interakcie MPK6 kinázy a KTN1. Tiež boli vykonané fenotypické analýzy vybraných KTN1 mutantov a tieto mutanty boli geneticky komplementované chimérnym GFP-KTN1 proteínom exprimovaným pod natívnym promótorom. Práca je rozdelení na dve časti, z toho prvá časť stručne sumarizuje súčasné poznatky o signalizácií sprostredkovanej MAP kinázami so zameraním na MPK6, ďalej na molekulárne klonovanie GFP-fúzovaného MPK6 proteínu exprimovaného pod natívnym MPK6 promótorom a jeho stabilnú transformáiu do mpk6-2 nulového mutanta za účelom získania "rescue" fenotypu. Dva konštrukty, pMPK6::GFP:MPK6 a pMPK6::MPK6:GFP, boli schopné zvrátiť alebo zachrániť (rescue) mpk6-2 fenotyp. Fúzny proteín GFP-MPK6 poskytoval lepší signál než MPK6-GFP a bol lokalizovaný v jadre, na plazmatickej membráne a cytosole v interfáznych bunkách. Počas mitózy bol GFP-MPK6 asociovaný s predprofáznym mikrotubulárnym pásom (PPB), fragmoplastom a tiež s novo sa tvoriacou bunkovou prepážkou. Tieto pozorovania spolu s inými výsledkami kde neaktívna forma MPK6 kinázy (kinase-dead) MPK6AEF transformovaná do mpk6-4 mutantného pozadia vykazovala podobný fenotyop koreňa ako yda1 mutant prispeli k záverom, že MPK6 je dôležitý hráč v signalizácií "downstream" od YODA MAPKKK4 kinázy. Oboje YODA aj MPK6 sú zapojené do signalizácie dôležitej pri post-embryonálnom vývine koreňa prostredníctvom orientácie roviny bunkového delenia. Ďaľej boli testované potenciálne interakcie KTN1 s MPK6, ale aj s blízko príbuznými kinázami MPK3 a MPK4, pretože in silico štúdie zamerané na MAPK fosforyláciu odhalili peptid, ktorý obsahujúci SP motív (serín-prolín) v 92. pozícií KTN1 proteínu. Výsledky YFP-split experimentu (BiFC, Bimolecular Fluorescent Complementation) ukázali interakcie oboch MPK4 aj MPK6 s KTN1 a výsledky získané z kvasinkového dvojhybridného testu (yeast-two-hybrid,Y2H) ukázali interakciu MPK3 s KTN1. Tieto proteín-proteín interakčné štúdie podporujú potenciálnu reguláciu KTN1 proteínu MAP kinázami. Druhá časť tejto práce sumarizuje súčasné poznatky o KTN1 proteíne, ktorý strihá mikrotubuly so zameraním na fenotypy jeho mutantov. Teda sa zaoberá fenotypovou analýzou vybraných KTN1 mutantov (fra2, lue1 a ktn1-2) so zameraním na plodnosť, vývin embrya a semien a tiež schopnosťou konštruktu pKTN1::GFP:KTN1 zvrátiť alebo zachrániť (rescue) mutantné fenotypy. Fenotypické analýzy KTN1 mutantov odhalili abnormálny vývin sporopfytických pletív a tiež aj samčích a samičích reprodčných orgánov. Stabilné transformácie mutantov fra2, lue1 a ktn1-2 s pKTN1::GFP:KTN1 konštruktom exprimovaným pod natívnym promótorm umožnili vzniknúť líniám T3 generácie, ktoré vykazovali záchranu "rescue" (genetickú komplementáciu) mutantných fenotypov. Z uvedených pozorovaní môžeme konštatovať, že KTN1 má esenciálnu úlohu v plodnosti a tvorbe semien u Arabidopsis thaliana. The present Ph.D. thesis is focused on Arabidopsis thaliana mitogen-activated protein kinase 6 (MPK6) and the microtubule severing protein KATANIN1 (KTN1). Herein, a chimeric GFP-MPK6 protein expressed under the native MPK6 promoter capable of rescuing the mpk6-2 mutant phenotype was used to perform in vivo localisation studies. Another chimeric protein MPK6-VenusN was used to test potential interactions of MPK6 with KTN1. Moreover phenotypic analyses of selected KTN1 mutants were performed and genetically rescued these mutants by chimeric GFP-KTN1 protein expressed under the native promoter. Therefore this thesis is divided into two parts. The first part of thesis briefly summarises current knowledge on plant MAPK signalling with main focus on MPK6, and describes the molecular cloning of GFP- fused MPK6 expressed under the native MPK6 promoter and the stable transformation of the fusion construct into mpk6-2 null mutant to achieve rescue of the mutant phenotype. Two constructs, pMPK6::GFP:MPK6 and pMPK6::MPK6:GFP, were able to revert mpk6-2 phenotype. GFP-MPK6 provided better signal than MPK6-GFP and it was localised to the nucleus, the plasma membrane and the cytosol in non-dividing cells. During mitosis GFP-MPK6 was associated with the preprophase microtubule band (PPB), phragmoplast and the newly formed cell plates. These observations together with the finding that a kinase-dead form MPK6AEF transformed into mpk6-4 mutant background showed a similar root phenotype like yda1 mutant contributed to the conclusion that MPK6 is an important player downstream of YODA (MAPKKK4). Both YODA and MPK6 are involved in postembryonic root development by regulating cell division plane orientation. Next, we searched for putative interactions between KTN1 and MPK6, its closely-related MPK3 and also MPK4, since in silico studies predicted the existence of a MAPK-targeted proline-directed serine at the 92nd position of KTN1. Results of YFP-split technology (BiFC, Bimolecular Fluorescent Complementation) experiments showed interactions of both MPK4 and MPK6 with KTN1 while preliminary results obtained by yeast-two-hybrid (Y2H) assay also showed interaction of MPK3 with KTN1. These protein-protein interaction data support the potential regulation of KTN1 by MAPKs. The second part of thesis summarises current knowledge on microtubule severing protein KTN1 with main focus on mutant phenotypes. Thus, it is devoted to the phenotypic analysis of the selected KTN1 mutants used herein (fra2, lue1 and ktn1-2) as related to fertility, embryo and seed development, and to the ability of a pKTN1::GFP:KTN1 construct to rescue mutant phenotypes. The phenotypic analyses of KTN1 mutants revealed an abnormal development of sporophytic tissues and of male and female reproductive organs. Stable transformations of fra2, lue1 a ktn1-2 mutants with pKTN1::GFP:KTN1 construct (expressed under control of the native KTN1 promoter) were performed and T3 lines showed rescue (genetic complementation) of mutant phenotypes. Therefore, we propose a new role of KTN1 in plant fertility and seed formation of Arabidopsis thaliana.
Keywords:
Arabidopsis thaliana; mitogen-aktivované protein kinázy; MPK3; MPK4; MPK6; BiFC; Y2H; KATANIN 1; fra2; lue1; ktn1-2; genetická komplementácia; mikrotubuly; asymetrické bunkové delenie; plodnosť; tvorba semien; Arabidopsis thaliana; mitogen-activated protein kinases; MPK3; MPK4; MPK6; BiFC; Y2H; KATANIN 1; fra2; lue1; ktn1-2; genetic complementation; microtubules; asymmetric cell division; fertility; seed formation
Available in the UPOL Library.
The role of selected mitogen-activated protein kinases in Arabidopsis
Predložená dizertačná práca je zameraná na mitogen-aktivovanú proteín kinázu 6 (MPK6) a proteín strihajúci mikrotubuli KATANIN 1 (KTN1) u modelovej rastliny Arabidopsis thaliana. V práci je ...
Linking events through the annual cycle in migratory songbirds: causes and consequences of different spatiotemporal strategies
BRIEDIS, Martins; ADAMÍK, Peter
2017 - English
Each year animals move across and between continents to track seasonal changes in resource availability and abundance to improve their survival and reproductive opportunities. Birds represent one of the largest animal group that engage in these global-scale migration events. Afro-Palearctic bird migration system is estimated to facilitate more than 2 billion individual birds that link ecological communities of the two continents. Recent advances in tracking technology, like light-level geolocators, have enabled us to follow even the smallest of individual migrants as they complete their annual cross-continental journeys. This has opened a new era in bird migration research allowing us to gain unique insights into spatiotemporal organization of full annual cycles of individual songbirds. Furthermore, these technological advances allow for estimation of migratory connectivity between breeding and non-breeding populations as well as linking spatially and temporarily distinct phases of the annual cycles of migratory birds. In my dissertation I couple geolocator tracking with stable isotope signatures, breeding data, and environmental variables to unravel spatiotemporal migration strategies and identify the importance of carry-over effects operating in wild populations of long-distance migrants. I reveal how annual events are interlinked with one another and the environment on the level of individual birds, populations, and species. I use three Afro-Palearctic migratory birds Tawny Pipit Anthus campestris, Collared Flycatcher Ficedula albicollis, and Semi-collared Flycatcher F. semitorquata as model species. Besides answering trivial questions of where and when each of the model species migrate, I found that there is a great deal of variation in the migration strategies among individuals and populations. Breeding site phenology is the main driver of migratory schedules on a population level, while individual variation in timing of migratory episodes are mediated via carry-over effects of previous breeding effort and conditions experienced at the non-breeding sites. Crossing of the Sahara Desert, which majority of the successful migrants accomplish in a single endurance flight, serves as a gateway between Europe and Africa for long-distance migratory birds. Only after the cross-desert flight, environmental and phenological cues can be picked up and used for accurate timing of spring migration and timely arrival for breeding. Studies from the perspective of full annual cycles are still underrepresented in animal ecology research. The chapters comprising this dissertation adds only a drop in the vast pool of knowledge in animal migration ecology. However, such information is of topmost priority for understanding the drivers of population dynamics as many migratory species are currently declining. Each year animals move across and between continents to track seasonal changes in resource availability and abundance to improve their survival and reproductive opportunities. Birds represent one of the largest animal group that engage in these global-scale migration events. Afro-Palearctic bird migration system is estimated to facilitate more than 2 billion individual birds that link ecological communities of the two continents. Recent advances in tracking technology, like light-level geolocators, have enabled us to follow even the smallest of individual migrants as they complete their annual cross-continental journeys. This has opened a new era in bird migration research allowing us to gain unique insights into spatiotemporal organization of full annual cycles of individual songbirds. Furthermore, these technological advances allow for estimation of migratory connectivity between breeding and non-breeding populations as well as linking spatially and temporarily distinct phases of the annual cycles of migratory birds. In my dissertation I couple geolocator tracking with stable isotope signatures, breeding data, and environmental variables to unravel spatiotemporal migration strategies and identify the importance of carry-over effects operating in wild populations of long-distance migrants. I reveal how annual events are interlinked with one another and the environment on the level of individual birds, populations, and species. I use three Afro-Palearctic migratory birds Tawny Pipit Anthus campestris, Collared Flycatcher Ficedula albicollis, and Semi-collared Flycatcher F. semitorquata as model species. Besides answering trivial questions of where and when each of the model species migrate, I found that there is a great deal of variation in the migration strategies among individuals and populations. Breeding site phenology is the main driver of migratory schedules on a population level, while individual variation in timing of migratory episodes are mediated via carry-over effects of previous breeding effort and conditions experienced at the non-breeding sites. Crossing of the Sahara Desert, which majority of the successful migrants accomplish in a single endurance flight, serves as a gateway between Europe and Africa for long-distance migratory birds. Only after the cross-desert flight, environmental and phenological cues can be picked up and used for accurate timing of spring migration and timely arrival for breeding. Studies from the perspective of full annual cycles are still underrepresented in animal ecology research. The chapters comprising this dissertation adds only a drop in the vast pool of knowledge in animal migration ecology. However, such information is of topmost priority for understanding the drivers of population dynamics as many migratory species are currently declining.
Keywords:
long-distance migrant; migration strategy; annual cycle; carry-over effects; migratory connectivity; long-distance migrant; migration strategy; annual cycle; carry-over effects; migratory connectivity
Available in digital repository of UPOL.
Linking events through the annual cycle in migratory songbirds: causes and consequences of different spatiotemporal strategies
Each year animals move across and between continents to track seasonal changes in resource availability and abundance to improve their survival and reproductive opportunities. Birds represent one of ...
Modelling and Simulation in High Energy Physics
HAMAL, Petr; HRABOVSKÝ, Miroslav; SÝKORA, Tomáš
2017 - English
Cílem předložené disertační práce je prezentovat schopnost autora aplikovat získané znalosti z oblasti modelování a simulace ve fyzice vysokých energií na příkladu fyzikální analýzy využívající experimentálně naměřených dat a následné porovnání získaných výsledků s teorií studovaného procesu. Práce je zaměřena na exluzivní proces produkce pionového páru v proton-protonových srážkách o těžišťové energii sqrt(s) = 7 TeV měřeného experimentem ATLAS na urychlovači LHC v CERNu. Tento proces je nejprve studován po teoretické stránce s využitím výsledků simulací uvedeného procesu. Celý softwarový rámec je pak následně aplikován i na experimentální data za účelem určení viditelného účinného průřezu exkluzivní produkce. Ke studiu procesu a jeho analýze jsou využity standardní softwarové nástroje z oblasti fyziky vysokých energií a analýzy dat jako jsou Geant4, knihovny a nástroje ROOT, příslušné generátory studované produkce (GenEx, DiMe) a softwarový rámec Athena standardně využívaný experimentem ATLAS. The aim of this doctoral thesis is to present author's ability to apply the gained knowledge of modelling and simulation in high energy physics to the physical analysis of a given process using experimental data followed by a comparison of given results with a corresponding theory. The thesis is focused on the exclusive pion production in proton-proton collisions measured by ATLAS experiment at centre-of-mass energy sqrt(s) = 7 TeV. First of all this production is studied on a theoretical level using the results of the appropriate simulations. The obtained results are later applied on experimental data to estimate the cross-section of a given production. A common software tools are used to study the exclusive pion production, namely Geant4, ROOT framework, generators of the given production like GenEx and DiMe or the common tools used by ATLAS experiment known as Athena framework.
Keywords:
Athena; ATLAS; CERN; difrakční fyzika; dopředné detektory; exkluzivní produkce; simulace; modelování; GenEx; DiMe; ROOT; analýza; Athena; ATLAS; CERN; diffractive physics; exclusive production; forward detectors; simulation; modelling; GenEx; DiMe; ROOT; analysis
Available in digital repository of UPOL.
Modelling and Simulation in High Energy Physics
Cílem předložené disertační práce je prezentovat schopnost autora aplikovat získané znalosti z oblasti modelování a simulace ve fyzice vysokých energií na příkladu fyzikální analýzy využívající ...
Konstrukce reportérových buněčných linií pro toxikologické a environmentální aplikace
BARTOŇKOVÁ, Iveta; DVOŘÁK, Zdeněk; DVOŘÁK, Zdeněk
2017 - English
Endokrinní disruptory (EDCs) jsou látky mající schopnost interagovat se signálními dráhami endokrinního systému, jejíž hlavním mechanismem je změna transkripční aktivity jaderných receptorů. Příkladem těchto látek jsou nejrůznější pesticidy, potravinová aditiva a kontaminanty nebo látky znečišťující životní prostředí. Vzhledem ke vlivu endokrinních disruptorů na živé organismy včetně člověka představuje vývoj spolehlivých detekčních systémů jeden z hlavních směrů jejich studia. V předkládané disertační práci jsem se zabývala konstrukcí a charakterizací lidských stabilně transfekovaných reportérových buněčných linií určených pro stanovení transkripční aktivity androgenního receptoru (buněčná linie AIZ-AR) a vitamin D receptoru (buněčné linie IZ-CYP24 a IZ-VDRE). Tyto buněčné linie představují unikátní lidský reportérový systém odvozený od lidských nádorových buněčných linií exprimujících plně funkční androgenní receptor/vitamin D receptor, které byly transfekovány reportérovými plasmidy obsahujícími reportérové sekvence odvozené z lidských AR/VDR responsivních genů. Všechny výše zmíněné buněčné linie byly plně funkční po více než 2 měsíce v buněčné kultuře, což odpovídá více než 25 pasážím, a stejně tak i pro kryoprezervaci. Spolehlivá detekce AR/VDR ligandů je možná již po osmi hodinách po aplikaci látek. Výše uvedené stabilní buněčné linie jsou natolik citlivé, že umožňují testování v 96-jamkovém formátu. Specifita buněčných linií byla ověřena testováním modelových ligandů ostatních steroidních a jaderných receptorů, přičemž nebyly pozorovány žádné signifikantní indukce luciferázové aktivity, které by potenciálně mohly ovlivnit interpretaci experimentálních dat. Závěrem lze konstatovat, že lidské stabilně transfekované reportérové buněčné linie AIZ-AR, IZ-CYP24 a IZ-VDRE představují rychlý, spolehlivý, efektivní, selektivní a reprodukovatelný nástroj pro detekci ligandů a aktivátorů lidského androgenního receptoru, respektive vitamin D receptoru. Využití těchto linií spočívá ve farmakologických, environmentálních a toxikologických aplikacích. Endocrine disrupting chemicals (EDCs) are substances possessing the ability to interfere with endocrine signalling by altering steroid hormone homeostasis and the transcriptional activity of nuclear receptors. Examples of EDCs are agrochemicals, food additives and contaminants, and industrial and environmental pollutants. Given the impact of EDCs on human health and living organisms in general, it is relevant and interesting to develop reliable tools for the detection of endocrine disruptors. In the present thesis, I describe the development and characterization of human stably transfected reporter cell lines for the assessment of androgen receptor (AR; AIZ-AR cell line) and vitamin D receptor (VDR; IZ-CYP24 and IZ-VDRE cell lines) transcriptional activities. These cell lines represent unique entirely human systems derived from human cancer cell lines expressing fully functional endogenous AR and/or VDR, respectively, that were transfected with reporter plasmids containing reporter sequences from promoter regions of human AR and/or VDR-responsive genes. These cell lines remained fully functional for over 2 months in the culture (this period corresponds to more than 25 cell passages) as well as after a freezethaw cycle. The reliable detection of ligands of human AR and VDR, respectively, is possible as soon as after 8 hours of the incubation. The assay was sensitive enough to allow for experiments in 96-well plates. The specificity of luciferase induction was proven by applying model ligands of other nuclear and steroid receptors: no significant off-activation was observed. In conclusion, the human stably transfected reporter cell lines AIZ-AR, IZ-CYP24 and IZ-VDRE present rapid, reliable, effective, sensitive, selective and reproducible tools for identification of ligands and activators of human AR and VDR, respectively. These cell lines can potentially be used in pharmacological, environmental and toxicological studies.
Keywords:
stabilní transfekce; vitamin D receptor; reportérová esej; endokrinní disrupce; stable transfection; vitamin D receptor; androgen receptor; gene reporter assay; endocrine disruption
Available in digital repository of UPOL.
Konstrukce reportérových buněčných linií pro toxikologické a environmentální aplikace
Endokrinní disruptory (EDCs) jsou látky mající schopnost interagovat se signálními dráhami endokrinního systému, jejíž hlavním mechanismem je změna transkripční aktivity jaderných receptorů. Příkladem ...
Vzájemné propojení erytropoézy a metabolismu železa u vybraných vrozených anémií
SAXOVÁ, Zuzana; HORVÁTHOVÁ, Monika; ROHOŇ, Peter; VYORAL, Daniel
2017 - English
Cieľom dizertačnej práce bolo študovať molekulárnu patofyziológiu vybraných vrodených anémií so zameraním na metabolizmus železa. V prvej časti práce sme u DMT1-mutantných myší (kmeň 129S6/SvEvTac) analyzovali dôsledky mutácie v DMT1 proteíne na erytropoézu a homeostázu železa. V druhej časti práce sme charakterizovali homeostázu železa na systémovej úrovni u pacientov s Diamond-Blackfan anémiou, nedostatkom pyruvátkinázy, talasémiou a u detských pacientov s poruchami erytrocytárnej membrány. Pochopenie prepojenia erytropoézy a metabolizmu železa je dôležité nielen pre porozumenie patofyziológie vrodených anémií, ale poskytuje aj základ pre vývoj nových liečebných postupov. The presented thesis aimed to uncover molecular pathophysiology of selected congenital anemias with focus on disrupted iron metabolism. In the first part of the thesis we analyzed the consequences of DMT1 mutation on erythroid cells and iron homeostasis in a mouse model - DMT1-mutant mice (129S6/SvEvTac). In the second part of the thesis we characterized systemic iron homeostasis in patients with Diamond-Blackfan anemia, pyruvate kinase deficiency, thalassemia and in pediatric patients with erythrocyte membrane defects. The knowledge of interconnection between erythropoiesis and iron metabolism is important for understanding of the pathophysiology of congenital anemias and also provides the basis for the development of new treatment strategies.
Keywords:
DMT1; železo; hepcidín; erytropoéza; anémia; DMT1; iron; hepcidin; erythropoiesis; anemia
Available in digital repository of UPOL.
Vzájemné propojení erytropoézy a metabolismu železa u vybraných vrozených anémií
Cieľom dizertačnej práce bolo študovať molekulárnu patofyziológiu vybraných vrodených anémií so zameraním na metabolizmus železa. V prvej časti práce sme u DMT1-mutantných myší (kmeň 129S6/SvEvTac) ...
Molekulární patogeneze vzácných anémií - erytroenzymopatie
KOŘALKOVÁ, Pavla; MOJZÍKOVÁ, Renáta; FRIEDECKÝ, David; STOPKA, Tomáš
2017 - English
Tato práce se zabývá studiem molekulární patogeneze enzymopatíí - vzácného onemocnění způsobující vrozenou nesférocytární hemolytickou anémii. Deficit glukosa-6-fosfátdehydrogenasy (GPD) a pyruvátkinasy (PK) byl v české a slovenské populaci popsán již v 80. letech. Nicméně až tato studie představuje zatím chybějící ucelený přehled o výskytu tohoto onemocnění v české a slovenské populaci, zvláště pak jeho vzácnější formy. Od roku 2013 se naše laboratoř zabývá metodou přímého stanovení enzymů s následným genetickým vyšetřením u pacientů se suspektní enzymopatií. V současné době jsme jedinou specializovanou laboratoří zabývající se touto diagnostikou v České republice resp. ve střední Evropě. Doposud byl enzymový deficit diagnostikován u dvaceti čtyř pacientů. Vedle nejčastějších deficitů G6PD a PK (9 a 12 případů), byly identifikovány 2 rodiny s deficitem glukosafosfátisomerasy (GPI) a 1 rodina s velmi vzácným deficitem hexokinasy (HK). Deficit GPI a HK byl v české a slovenské populací diagnostikován poprvé. Celkově bylo identifikováno dvacet dva kauzálních mutací, z nichž pět nebylo doposud v literatuře popsáno - G6PD p.(Phe216Tyr), PK p.(Arg518Leufs * 12), p.(Asp293Val), GPI p.(Ser160Pro) a p.(Arg472Cys). Současně tato práce studuje patogenní mechanismy vybraných mutací vedoucí k deficitu HK, fosfoglycerátkinasy (PGK) a fosfofruktokinasy (PFK). Deficit HK byl pouze výjimečně studován na molekulární úrovni. Na základě zahraniční spolupráce jsme vytvořili a detailněji prostudovali největší soubor pacientů s tímto deficitem. Patogenní mechanismus kauzálních mutací p.(His867Tyr), p.(Thr600Met), c.873-2A>G a c.493-1G>A byl objasněn pomocí molekulárních technik a in silico analýz. Dále jsme identifikovali a charakterizovali novou intronovou mutaci c.756+3A>G v genu PGK1 vedoucí k PGK deficitu. Ve většině případů je deficit PGK spojen s hemolytickou anémií a neurologickými poruchami. Avšak v tomto případě byla svalová slabost a myopatie jediným klinickým příznakem. Dále jsme u pacientky španělského původu identifikovali a charakterizovali novou mutaci p.(Asp309Gly) v genu kódující PFK. Jedná se o první případ PFK deficitu ve Španělsku. Charakterizace erytroenzymopatií pomocí biochemických a molekulárních analýz může přispět k porozumění patofyziologie tohoto vzácného onemocnění. Navíc metoda přímého stanovení erytrocytárních enzymů může vést ke zlepšení diagnostiky jiných onemocnění či je možné ji využít při studiu farmakologických sloučenin. The aim of this research is to make a contribution to the molecular pathogenesis of erythroenzymopathies, rare hereditary disorders causing nonspherocytic hemolytic anemia. This work represents an advanced and up-to-date knowledge in the field of red blood cells enzyme deficiencies in the Czech and Slovak populations since the nineteen-eighties when mutations in Czech and Slovak subjects resulting in glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiencies were described. Both deficiencies are the most common erythroenzymopathies worldwide. Since 2013, our laboratory has focused on the introduction of direct enzyme assays followed by genetic testing in patients with hemolytic anemia with suspected erythroenzymopathy. To date, several different enzyme defects were diagnosed in twenty-four patients. Except G6PD and PK deficiencies (9 and 12 cases), 2 families with glucose phosphate isomerase (GPI) defect and 1 family with very rare hexokinase (HK) deficiency have been identified. The last two deficiencies were diagnosed in the Czech and Slovak populations for the first time. Among the 22 identified mutations, 5 of them, namely G6PD p.(Phe216Tyr), PK p.(Arg518Leufs*12), p.(Asp293Val) and GPI p.(Ser160Pro), p.(Arg472Cys) were novel and have not been previously reported in literature. Furthermore, this thesis describes a pathogenic mechanism of several novel mutations responsible for deficiencies of hexokinase (HK), phosphoglycerate kinase (PGK) and phosphofructokinase (PFK) in more detail. HK deficiency is a very rare disorder that has been rarely studied at molecular level. In collaboration with other research centers, the largest cohort of HK deficient patients was selected and extensively studied. Using molecular techniques and in silico analyses, potential pathogenic mechanisms of novel mutations p.(His867Tyr), p.(Thr600Met), c.873-2A>G and c. 493-1G>A have been elucidated. Further, we contributed to the identification and characterization of a novel intronic mutation c.756+3A>G in PGK1 in two brothers with PGK deficiency. Although PGK deficiency is mostly associated with hemolytic anemia and neurological impairment, muscle weakness and myopathy were the only clinical features in these cases. Finally, we contributed to the detection of a novel mutation p.(Asp309Gly) in PFKM gene, the first case of a rare PFK deficiency diagnosed in a 65-year-old female of Spanish origin. Characterization of erythroenzymopathies by biochemical and molecular analyses can contribute to a better understanding of pathophysiology of this rare disorder. Furthermore, the direct enzyme assays may have a wide variety of scientific/diagnostic applications; it may improve diagnostic testing of other disorders or it may be useful to study the mechanism of drug's actions.
Keywords:
erytrocyty; anémie; enzymopatie; red blood cells; anemia; enzymopathies;
Available in digital repository of UPOL.
Molekulární patogeneze vzácných anémií - erytroenzymopatie
Tato práce se zabývá studiem molekulární patogeneze enzymopatíí - vzácného onemocnění způsobující vrozenou nesférocytární hemolytickou anémii. Deficit glukosa-6-fosfátdehydrogenasy (GPD) a ...
Aplikace ultrasonografie v diagnostice a monitoraci léčebné odpovědi u revmatoidní artritidy
SKÁCELOVÁ, Martina; HORÁK, Pavel; ŠENOLT, Ladislav; BRADNA, Petr; WEIGL, Evžen
2017 - English
Cílem práce bylo posouzení přínosu ultrasonografického vyšetření pomocí standardizovaného nástroje (US7 skóre) a zhodnocení korelace jeho výsledků k aktivitě choroby a stupni radiografického postižení a dále ke kvalitě života nemocných, jejich funkčním schopnostem a schopnosti pracovat dle hodnocení standardizovaných dotazníkových metod. Vyšetření sérových hladin metaloproteinázy 3 (MMP-3) jako potenciálního markeru aktivity choroby a kloubního postižení, stanovení dalších možných kandidátních molekul vhodných ke sledování aktivity RA. Zhodnocení výskytu komorbidit ve sledované populaci pacientů s RA a vztahu aktivity choroby k lipidovému profilu nemocných. Vyhodnocení účinnosti léčby abataceptem u pacientů s RA. This study was aimed: -to assess the benefit of ultrasound examination with a standardized instrument (US7) and correlate it´s results with disease activity, radiographic degree of structural damage and the quality of life of patients and their functional abilities and ability to work as assessed by standardized questionnaire methods -to evaluate the serum levels of matrix metalloproteinase-3 (MMP-3) and some other candidate molecules as a potential markers of disease activity and joint damage -to describe comorbidities in RA patients and the relationship between disease activity and lipid profile to explore the efficacy of abatacept treatment in group of RA patients.
Keywords:
Revmatoidní artritida; ultrasonografie; US7 skóre; kvalita života; metaloproteináza 3; komorbidity; Rheumatoid arthritis; ultrasound; US7 score; quality of life; metalloproteinasis 3; comorbidities
Available in digital repository of UPOL.
Aplikace ultrasonografie v diagnostice a monitoraci léčebné odpovědi u revmatoidní artritidy
Cílem práce bylo posouzení přínosu ultrasonografického vyšetření pomocí standardizovaného nástroje (US7 skóre) a zhodnocení korelace jeho výsledků k aktivitě choroby a stupni radiografického postižení ...
RESEARCH ON THE CONSTRUCTION OF SUPPORTING SYSTEMS FOR INCLUDING CHILDREN WITH AUTISM SPECTRUM DISORDERS IN PRESCHOOL CLASSROOMS
PENG, Yuntong; LANGER, Jiří
2017 - English
Inclusive education, characterized by its emphasis on human rights and educational equity, has become a trend in many countries. Great efforts have been made to ensure the educational rights of children with special educational needs and to promote the implementation of inclusive education. Supports, which are tailored to meet the needs of every individual child, are indispensible elements in inclusive practice. A well-constructed system of supports can help students to reach their potential and to succeed in regular schools. Focused on supporting systems for preschool inclusion of children with ASD, the research is aimed at exploring the supporting systems for preschool inclusion of children with ASD in China, and therefore to contribute to the improvement of the quality of preschool inclusive education. The researcher adopted a qualitative paradigm, and collected data through interview, observation, and examination of physical items. Grounded theory approach was applied to analyze the qualitative data, and to develop theories that can further interpret the reality and offer new insights into the practice of supporting systems construction. Based on the data collected, the researcher summarized the current situation of children with ASD in regular classrooms, and outlined the structure of the current supporting systems. The socio-ecosystem of the children with ASD in the inclusive context consisted of the inclusive classroom, the kindergarten, the family, institutions, the resource center, hospitals, government and the society; persons involved in the systems provided direct or indirect supports for the children. The supports providers included preschool teachers, childcare workers, parents, peers, directors of the kindergartens, resource center teachers, other professionals, doctors and so on. The roles played by different support providers were presented. The interaction between different support providers forms a network within the support systems. The collaboration between support providers varies in term of ways of communication, frequencies of collaboration, content/ways of collaboration, and effectiveness of the collaboration. The collaboration between main support providers was further elaborated. Having illustrated the current supporting systems for including children with ASD, barriers and facilitators which were found in the current supporting systems were discussed. The barriers included: (1) loose internal structure of the supporting systems, (2) insufficient collaboration between different supports providers, (3) lack of services integrated within routines in kindergartens, (4) insufficient preparation for inclusion from the kindergartens, and the (5) lack of mechanisms to underpin the implementation of inclusion. The facilitators comprised: (1) straightforward and open attitudes during collaboration, (2) personal relationship between support providers, (3) successful experience of including children with ASD, and (4) well-organized internal structure of family systems. In the final part, recommendations have been proposed, including: (1) establishment of referral systems, (2) regularization of the functioning of resource centers, (3) pre-service and in-service teachers training, (4) intervention for parents of children with ASD, (5) further development of regulations and policies, and (6) promotion of inclusive education in the public. These strategies were aimed at optimizing the construction of supporting systems so as to improve the inclusive practice for children with ASD. Inclusive education, characterized by its emphasis on human rights and educational equity, has become a trend in many countries. Great efforts have been made to ensure the educational rights of children with special educational needs and to promote the implementation of inclusive education. Supports, which are tailored to meet the needs of every individual child, are indispensible elements in inclusive practice. A well-constructed system of supports can help students to reach their potential and to succeed in regular schools. Focused on supporting systems for preschool inclusion of children with ASD, the research is aimed at exploring the supporting systems for preschool inclusion of children with ASD in China, and therefore to contribute to the improvement of the quality of preschool inclusive education. The researcher adopted a qualitative paradigm, and collected data through interview, observation, and examination of physical items. Grounded theory approach was applied to analyze the qualitative data, and to develop theories that can further interpret the reality and offer new insights into the practice of supporting systems construction. Based on the data collected, the researcher summarized the current situation of children with ASD in regular classrooms, and outlined the structure of the current supporting systems. The socio-ecosystem of the children with ASD in the inclusive context consisted of the inclusive classroom, the kindergarten, the family, institutions, the resource center, hospitals, government and the society; persons involved in the systems provided direct or indirect supports for the children. The supports providers included preschool teachers, childcare workers, parents, peers, directors of the kindergartens, resource center teachers, other professionals, doctors and so on. The roles played by different support providers were presented. The interaction between different support providers forms a network within the support systems. The collaboration between support providers varies in term of ways of communication, frequencies of collaboration, content/ways of collaboration, and effectiveness of the collaboration. The collaboration between main support providers was further elaborated. Having illustrated the current supporting systems for including children with ASD, barriers and facilitators which were found in the current supporting systems were discussed. The barriers included: (1) loose internal structure of the supporting systems, (2) insufficient collaboration between different supports providers, (3) lack of services integrated within routines in kindergartens, (4) insufficient preparation for inclusion from the kindergartens, and the (5) lack of mechanisms to underpin the implementation of inclusion. The facilitators comprised: (1) straightforward and open attitudes during collaboration, (2) personal relationship between support providers, (3) successful experience of including children with ASD, and (4) well-organized internal structure of family systems. In the final part, recommendations have been proposed, including: (1) establishment of referral systems, (2) regularization of the functioning of resource centers, (3) pre-service and in-service teachers training, (4) intervention for parents of children with ASD, (5) further development of regulations and policies, and (6) promotion of inclusive education in the public. These strategies were aimed at optimizing the construction of supporting systems so as to improve the inclusive practice for children with ASD.
Keywords:
preschool inclusion; inclusive education; support; supporting system; autism spectrum disorders; preschool inclusion; inclusive education; support; supporting system; autism spectrum disorders
Available in digital repository of UPOL.
RESEARCH ON THE CONSTRUCTION OF SUPPORTING SYSTEMS FOR INCLUDING CHILDREN WITH AUTISM SPECTRUM DISORDERS IN PRESCHOOL CLASSROOMS
Inclusive education, characterized by its emphasis on human rights and educational equity, has become a trend in many countries. Great efforts have been made to ensure the educational rights of ...
Patogenní variabilita a studium interakcí v patosystému Helianthus spp. - Plasmopara halstedii
DRÁBKOVÁ TROJANOVÁ, Zuzana; SEDLÁŘOVÁ, Michaela
2017 - English
Předkládaná disertační práce se zabývá biotrofní parazitickou oomycetou P. halstedii, která je původcem karanténní choroby zvané plísňovitost slunečnice. Po úvodním kritickém přehledu metod, používaných při studiu P. halstedii, přináší tato práce první informace o patogenní variabilitě populací P. halstedii na pěstovaných slunečnicích v České republice. Kromě celosvětově rozšířených ras 700 a 710 byly zaznamenány také v poslední době časté rasy 704 a 714 a především poprvé na světě vůbec odhaleny rasy 705 a 715. Informace získané během posledního desetiletí jsou klíčové zejména pro šlechtitele a pěstitele slunečnic ve střední Evropě, vyplňují prázdné místo na mapě poznání a rozšiřují vědomosti o P. halstedii v Evropě. This Ph.D. thesis focuses on biotrophic parasitic oomycete Plasmopara halstedii, the cause of sunflower downy mildew, a quarantine disease spread globally. Following a critical review of methodology used in P. halstedii research, it brings the first overview of pathogenic variability in P. halstedii populations on cultivated sunflower in the Czech Republic. Since 2007, when detailed research was initiated, a limited but repeated incidence of sunflower downy mildew was recorded. Besides widely distributed races 700 and 710, also recently frequent races 704 and 714 were found, together with the first ever records of races 705 and 715. Data, obtained during last decade, are crucial for sunflower breeders and growers and fill in the white spot in the knowledge about P. halstedii variability in sunflower-producing countries in Europe.
Keywords:
difrenciační soubor; Helianthus annuus L.; karanténní choroba; Plasmopara halstedii; plísňovitost slunečnice; virulence; differential set; Helianthus annuus L.; Plasmopara halstedii; quarantine disease; sunflower downy mildew; virulence formula
Available in digital repository of UPOL.
Patogenní variabilita a studium interakcí v patosystému Helianthus spp. - Plasmopara halstedii
Předkládaná disertační práce se zabývá biotrofní parazitickou oomycetou P. halstedii, která je původcem karanténní choroby zvané plísňovitost slunečnice. Po úvodním kritickém přehledu metod, ...
Group Music Therapy for Children with Autism Spectrum Disorder
LU, Yue; LUSKA, Jiří
2017 - English
With the increasing number of children diagnosed with Autism Spectrum Disorder (ASD), the education and development of children with ASD have become topics under heated discussion. Music therapy for children with special needs is lacking of influential theoretical outcome in China due to limited resources. In either public or private schools, given the limited funding, group therapy practice would be an optimal choice. Especially in China, a developing country, effective group therapy practice would be beneficial for autistic children. This research was designed to demonstrate a practical model of group music therapy for children with Autism Spectrum disorder (ASD). Mixed methods were employed including an action research and a single-subject research. There were 5 phases of the research implementation including measurement and planning, acting, reflecting, modification and acting, and evaluation and discussion. Objects of this research were five children who were diagnosed with ASD. Their ages were from 7.5 years to 9.5 years. Other participants involved in this research included a researcher, a therapist, four class teachers, five guardians of children and an assistant. Having investigated the developmental needs of children and planed therapy sessions within two months, the researcher conducted twelve sessions of group music therapy within six weeks. Detailed process of implementing group music therapy including deciding goals and objectives, choosing and revising materials, applying methods and evaluating the group progression has been presented in the thesis. The results with ATEC score of the group indicate (1) the group got a little improvement after group music therapy; (2) there was a significant difference between male and female participants in the therapy effect; (3) there was a significant difference in the rating between class teachers and guardians. The results of every single group member with ATEC score indicate that two girls in this group gained significant improvement and other three boys achieved improvements only in several developmental areas. In summary, except for the worsening in health/physical/behavior, the improvements were evident in areas of speech, sensory/cognitive awareness and sociability. The results of interviews from the therapist, class teachers and guardians indicate every child gained different improvements at various degrees such as improvement in attending attitudes, emotional control, accepting new things, expressing actively, imitation ability and coordination. The results both of single-subject research and action research demonstrated the unique functions that group music therapies performed for children with ASD. It includes specific improvements in joint attention, communication, interpersonal interaction, cooperation, and imitation. Apart from progress as a whole group, improvements in relaxation, self-confidence, and cognition were also detected in different individuals. With the analysis of measurement results and interviews results, the researcher discussed the implications of group and individual achievements, and compared the evaluating results from class teachers and that from guardians. The result demonstrated the unique functions that group music therapies performed for children with ASD. It includes improvements in joint attention, communication, interpersonal interaction, cooperation, and imitation. Apart from progress as a whole group, improvements in relaxation, self-confidence, and cognition were also detected in different individuals. To conclude, suggestions on the practice of group music therapy for children with ASD have been proposed, including principles of session planning, goals and objectives setting, format, materials, methods and evaluations. With the increasing number of children diagnosed with Autism Spectrum Disorder (ASD), the education and development of children with ASD have become topics under heated discussion. Music therapy for children with special needs is lacking of influential theoretical outcome in China due to limited resources. In either public or private schools, given the limited funding, group therapy practice would be an optimal choice. Especially in China, a developing country, effective group therapy practice would be beneficial for autistic children. This research was designed to demonstrate a practical model of group music therapy for children with Autism Spectrum disorder (ASD). Mixed methods were employed including an action research and a single-subject research. There were 5 phases of the research implementation including measurement and planning, acting, reflecting, modification and acting, and evaluation and discussion. Objects of this research were five children who were diagnosed with ASD. Their ages were from 7.5 years to 9.5 years. Other participants involved in this research included a researcher, a therapist, four class teachers, five guardians of children and an assistant. Having investigated the developmental needs of children and planed therapy sessions within two months, the researcher conducted twelve sessions of group music therapy within six weeks. Detailed process of implementing group music therapy including deciding goals and objectives, choosing and revising materials, applying methods and evaluating the group progression has been presented in the thesis. The results with ATEC score of the group indicate (1) the group got a little improvement after group music therapy; (2) there was a significant difference between male and female participants in the therapy effect; (3) there was a significant difference in the rating between class teachers and guardians. The results of every single group member with ATEC score indicate that two girls in this group gained significant improvement and other three boys achieved improvements only in several developmental areas. In summary, except for the worsening in health/physical/behavior, the improvements were evident in areas of speech, sensory/cognitive awareness and sociability. The results of interviews from the therapist, class teachers and guardians indicate every child gained different improvements at various degrees such as improvement in attending attitudes, emotional control, accepting new things, expressing actively, imitation ability and coordination. The results both of single-subject research and action research demonstrated the unique functions that group music therapies performed for children with ASD. It includes specific improvements in joint attention, communication, interpersonal interaction, cooperation, and imitation. Apart from progress as a whole group, improvements in relaxation, self-confidence, and cognition were also detected in different individuals. With the analysis of measurement results and interviews results, the researcher discussed the implications of group and individual achievements, and compared the evaluating results from class teachers and that from guardians. The result demonstrated the unique functions that group music therapies performed for children with ASD. It includes improvements in joint attention, communication, interpersonal interaction, cooperation, and imitation. Apart from progress as a whole group, improvements in relaxation, self-confidence, and cognition were also detected in different individuals. To conclude, suggestions on the practice of group music therapy for children with ASD have been proposed, including principles of session planning, goals and objectives setting, format, materials, methods and evaluations.
Keywords:
children with ASD; music therapy; group therapy; practice; children with ASD; music therapy; group therapy; practice
Available in digital repository of UPOL.
Group Music Therapy for Children with Autism Spectrum Disorder
With the increasing number of children diagnosed with Autism Spectrum Disorder (ASD), the education and development of children with ASD have become topics under heated discussion. Music therapy for ...
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