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Protein chemistry and mass spectrometry in biochemical research
Pompach, Petr ; Bezouška, Karel (advisor) ; Chmelík, Josef (referee) ; Kovářová, Hana (referee)
Protein chemistry and mass spectrometry in biochemicul research Petr Pompach Ph. D. Thesis Department of Biochemistry Faculty of Science Charles University Supervisor: Doc. RNDr. Karel Bezouška, CSc. Prague 2006 tEia*iiitg;;tiBiÉ (n OO oO O\ O\ C.l C.l .+ : : : : :ÉGlc.l c.: Ý l.) a z t-r (J Fl E) lra J a U z a Fr tl5 a Fr zr\J) alai P.: ai a - Lr o L d a (|i iť r h t) o() r a a q C) ! - H F (n F íi a o) O a X, o >. C) o .l I +i oL o -O o o l.r U) N z oo - O o I L a Li +i vi ol Ei!t ,ql -l E 3a (n z E F a F z F z U pg ť: šé3; Ei ĚE$áE€EÉEáiz E ÉĚ E Ě s E ; u ; ó =-E5Ě:gd:'i = t Ei giĚg;;:1lV)iĚggĚgEsáEĚi* E Ě=;Éía:š;Éť E É!řěEEEFgg : ií€gáŤÉ5 ž:::š;;i : zi:€ i=ts€ĚiĚišĚÉiš;š5ĚiE.:. = = € .T ŽlĚE g;t$jíigggřEE iuEái E :É€šáĚ g;iÉ{Ě;ítÉÉF€Ě E iĚĚĚi ; š;šš!iiE řě E ši gE ig Ý E giĚE šgig;išiš::gígFĚi+g s E *'suĚšěĚ Ěie *Ěá, uE trE s'' -k*-=a.*:.* g: EPE€'šEEř€9 ěÍ.E ;š e::E;;áe;iÉgřšiE řĚt.E*E$*eE=$[:gř.v :IEF€=áfrE;='2E-ď ?! : g"HEu.:i=ĚE€;ÉE€ř€ Eš,*éEě9t.F;EE.Ě., ťiEEEE*ě:iiEĚgĚ:Éžáx;šš ř 5šEtsť uE.E.= e *n á:; € žEř: € ÉE.: i= ř-g p; i ĚĚ;: € i íEĚ = Hi;2E É*Ě u, Ě y ?' : 6 ě ř ; fij .: .c F=E;-sg&,Ě7Éď€3E€€ -i€E.E =:Í E=bÉ::ň !B:,: E t šáč š 3e = Ě l E ř f L Ei:í:9:*q;€gq3:;:.E: ťšÉI: Ě i ?E Ě ; Éi É.EE E EEE :!rEě45BEE EE.= €Eě íEEra-vĚ=3ň €.E3 iillťlgtššgtgi;g;ggiĚš; iáágá9 $*i;Ě:E; -Eás;t;É$ \o Ěi gi...
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Proteomics as a tool for understanding molecular mechanisms of human diseases
Pospíšilová, Jana ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...
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Proteomic approaches in cancer biology
Lorková, Lucie ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...
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"The cancer cell proteome and its changes after anti-cancer drug treatment".
Tylečková, Jiřina ; Kovářová, Hana (advisor) ; Strnad, Miroslav (referee) ; Petrák, Jiří (referee)
Cancers represent a group of unprecedented heterogeneous diseases and currently available anti-cancer therapies provide highly variable efficacy with unsatisfactory cure rates. A wide range of proteomic technologies are being used in quest for newer approaches which could significantly contribute to the discovery and development of selective and specific cancer biomarkers for monitoring the disease state and anti-cancer therapy success. Taking into consideration the above aspects, this research was undertaken to study cancer cell proteomes and their changes after anti-cancer treatment with specific focus on: (a) response to conventional anthracycline/anthracenedione drugs with respect to their different clinical efficacy and (b) identification of novel targets for therapy in cancer cells resistant to biological drugs such as inhibitors of (b1) cyclin-dependent kinases and (b2) Aurora kinases. This study identified several interesting key aspects related to the effects of daunorubicin, doxorubicin and mitoxantrone. With the main focus on early time intervals when the influence of apoptosis is minimised, changes common for all three drugs belonging mainly to metabolic and cellular processes were observed. More importantly, significant changes in proteins involved in the generation of precursor...
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Proteome analysis of anti-cancer drug effects and characterisation of drug resistance
Hrabáková, Rita ; Kovářová, Hana (advisor) ; Hernychová, Lenka (referee) ; Šulc, Miroslav (referee)
Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...
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Proteomics of biological fluids
Jarkovská, Karla ; Kovářová, Hana (advisor) ; Petrák, Jiří (referee) ; Ulčová-Gallová, Zdeňka (referee)
Reproductive diseases, mainly those resulting in the infertility affect the chances of human being to reproduce. On the contrary, the heart disease, cancer and degenerative diseases currently account for majority of deaths in the world. Usually, these lifestyle diseases need longer lifespan to become the cause of death. The proteins secreted by cells carry important information about the cell's well-being, as well as about the condition of the tissues formed by these cells. Once secreted, these proteins may also be transferred throughout the body by means of body fluids, many of which are easily accessible for further 'in-depth' studies. Cellular and secreted proteins are often a focus of studies using proteomic means and the revelation of protein alterations can lead us to new ideas about the molecular mechanisms of diseases as well as possible identification of proteins that may be used as new targets for pharmaceutical intervention or molecules that could be used for diagnostic or prognostic purposes. Taking into consideration the above aspects, this research was undertaken to find proteins that could: (a) characterise the human follicular fluid as microenvironment of the maturing oocyte, to increase understanding of reproductive processes to improve the techniques of assisted repro- duction;...
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Proteomic analysis of liver iron overload
Myslivcová, Denisa ; Petrák, Jiří (advisor) ; Kovářová, Hana (referee) ; Hrkal, Zbyněk (referee)
V našich experimentech jsme prokázali, že lidské jaterní buňky mění expresi proteinů v důsledku akutního toxického přetížení železem a následně v důsledku oxidativního stresu a že v játrech přetížených železem dochází ke změnám v močovinovém cyklu, metylačním cyklu, oxidaci mastných kyselin a metabolizmu cukrů. Dále jsme zjistili, že pro dědičnou hemochromatózu je typická odlišná jaterní exprese sarkozin dehydrogenázy, selenium binding proteinu 2, glutathion S-transferázy P1, karboxylesterázy 1 a thioredoxin-like proteinu 2. Identifikované proteiny v první a druhé studii přímo nebo nepřímo souvisejí s přetížením modelového organizmu železem. Jiné jsou spíše obecnou odpovědí buňky na stresové podmínky. Jsou to především PDI, enoláza, keratiny, HSP70, GST, GRP78/Bip a podjednotky proteazómu. Tyto proteiny patří mezi nejčastěji identifikované diferenciálně exprimované proteiny v proteomických studiích využívajících 2-DE (Petrak et al., 2008) a jejich expresní změny tedy nelze považovat za změny specifické pro metabolizmus železa či hemochromatózu. Výsledky naší studie nám poskytly nové informace nezbytné pro detailní molekulárních pochopení mechanizmů uplatňujících se v metabolizmu železa, které je podmínkou rozvoje nových postupů v prevenci i terapii onemocnění souvisejících s metabolizmem železa, především...
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Family house with housing units
Vejmělek, Lukáš ; Kovářová, Hana (referee) ; Sobotka, Jindřich (advisor)
The subject of this thesis work is the two floors house with housing units in the city of Brno - Lisen. The proposal is processed at the level of project documentation for building construction stage. Family house is located on plot no. 3278/92, cadastral area of Brno – Lisen. The building is without a basement on two floors of a wall system of brick blocks POROTHERM, based on the foundation walls and covered with a flat roof vegetation. There are three residential units, two of which used to rent.
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