Plants as a sessile organisms have evolved complex mechanisms to cope with unfavorable environmental conditions. This diploma thesis summarizes current state of knowledge and focuses on light, temperature and phytohormones in plant abiotic stress responses. The practical part is devoted to proteomics and completes and extends the project started during my Bachelor program. Transgenic Arabidopsis seedlings carrying inducible barley cytokinin oxidase/dehydrogenase (CaMV35S> GR> HvCKX2) were profiled to elucidate proteome-wide responses to down-regulation of cytokinin levels under heat stress at standard (80 umol.m-2.s-1) and low light intensity (20 umol.m-2.s-1). In total, 2-DE analysis and mass spectrometry analysis revealed 63 differentially abundant proteins involved in diverse metabolic processes. These data provide evidence of a link between temperature, light and cytokinin signaling in Arabidopsis and will be used in the modeling of cytokinin-light-temperature interactions.

Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes, but also present a solid obstacle in the proteome analysis. Identification and mapping of PTMs in proteins have improved dramatically, but to comprehend complex mechanisms and biological functions, one must address also a very low abundant proteins. Here, in this thesis entitled 'Analysis of DELLA protein ubiquitination' we demonstrate the use of a recombinant protein standard for the determination of in vivo modified peptides of the DELLA family protein RGA. The candidate peptide sequences were targeted in an SRM-based analysis to detect the ubiquitination site and the results of this analysis and that of an MS/MS data processing indicate that the modification is localized in the conserved N-terminal region of RGA protein.

Histonų potransliacinės modifikacijos sąlygoja chromatino struktūros pakitimus, lemiančius genų, atsakingų už ląstelėje vykstančių įvairių procesų, tokių kaip proliferacija, diferenciacija, apoptozė reguliavimą. Šiame darbe įvertinome chromatino baltymų, histonų H3 ir H4, modifikacijų dinamiką HL-60 ląstelėse, indukuotose granuliocitinei diferenciacijai su retinoine rūgštimi (RA) ir histonų deacetilazių slopikliais, fenilo butiratu (PB) ir vitaminu B3 (vitB3) bei jų kombinacijomis. Aktyvaus chromatino baltymų kompleksai proliferuojančiose ir diferenciacijai indukuotose ląstelėse buvo analizuojami chromatino imunoišsodinimo metodu, naudojant antikūnus prieš histonus - hiperacetilintą H4 ir trimetil-Lys4 H3, esančius aktyvaus chromatino vietose mononukleosomų frakcijoje. Atlikta baltymų, esančių komplekse su modifikuotais histonais H3 ir H4 proteominė analizė vienmatėje (SDS/PAGE) ir dvimatėje (2DE) elektroforezės sistemose. Nustatyti baltymai, sąveikaujantys su hiperacetilintu H4 ir trimetil-Lys4 H3. Tai baltymai, dalyvaujantys genų raiškos iniciavime bei chromatino modifikacijose, t.y. transkripcijos faktorius Sp1, metionino acetiltransferazė, DNR metiltransferazė ir kt. Taip pat patodyta, kad HL-60 ląstelėse p21 WAF1/CIP geno raiška priklauso nuo pasirinkto induktoriaus. Apibendrinant manome, kad poveikio variantas - 3mM PB su 5mM vit.B3 6 val., nuplovus tolesnis poveikis su 1μM RA ir 5mM vit.B3 24 val., galėtų būti tinkamas leukeminių ląstelių diferenciacinei terapijai... [toliau žr. visą tekstą] / Recently, a novel strategy for the treatment of leukemia’s through the modulation of chromatin structure is applicable. In this study, we conducted a detailed analysis of anti-leukemia effects of histone deacetylase (HDAC) inhibitors and their combinations with retinoic acid using human promyelocytic leukemia cell line HL-60. We have shown that HDAC inhibitors - phenyl butyrate and vitamin B3, cause rapid histone H3 and H4 modifications. Further we examined how HDACI and retinoic acid (RA) can modulate gene expression via acetylation and other modifications of histones associated with targeted genes. We performed Chip assay to identify proteins associated with hyperacetylated histone H4. Immunoprecipitated proteins were fractionated by SDS/PAGE and 2DE. Proteomic analysis was performed by using mass spectrometry (MALDI TOF and ESI MS/MS). We identified Sp1 transcriptional activation, DNA methyltransferase, methionine acetyltransferase and other proteins that were associated with modified histones H3 and H4. To evaluate the changes ofp21 gene expression affected by hyperacetylation of histone H4 during HL-60 cell granulocytic differentiation we performed PGR of active chromatin immunoprecipitated with hyperacetylated H4 by using different primers of p21 gene. In this study we have shown that p21 gene expression changes during granulocytic differentiation and depends on inducer. Our results suggest that the chromatin remodeling caused by HDAC inhibitors could be a promising... [to full text]

Seed germination is a crucial phase of the plant life cycle. Despite the huge progress that has been made in the last decade, the molecular mechanisms regulating seed germination are far from being resolved. Most of the early events of germination are mediated by molecules stored in seeds during the seed development and thus we employed proteomic analyses to elucidate the mechanism behind this process. This thesis - Proteome dynamics during seed germination - reviews present-day knowledge of this topic. In the experimental part, the results of three related research projects are summarized. First, the effects of a novel class of growth regulators were analyzed in the barley germination assay and the optimal concentrations were determined. The methodology for an improved analysis of barley seed proteins was developed and the optimized targeted analysis will allows the detection of over 4,000 proteins. Finally, Arabidopsis thaliana seed proteome was analyzed and the results indicate that (i) an alternative MS/MS data processing significantly improves the detection limits and (ii) the proteasome-ubiquitin system plays a crucial role in adjusting the seed proteome dynamics during the germination progress.

The biochemical and physiological responses of organisms to xenobiotic chemicals have been investigated for many years as general indicators of organism health. These markers have been used in the development of the synthetic pharmaceuticals and screening of effects of environmental pollutant on biological systems, and in clarifying their modes of action. Recently, use of "OMICS" approaches has received great attentions in exploring the effects of chemical contaminants at the molecular level and is one of the rapidly developing areas in the field of toxicology. Use of molecular response within aquatic organisms has been reviewed and are considered as early changes occurring in response to chemical exposure. ZnO NPs are widely used and possess great potentials in food industry and agriculture. Their subsequent release into environment has raised concerns about their potential effects on aquatic organisms. Although, the dietary exposure perhaps is the main route to expose aquatic animals to nanomaterials, the majority of studies are focused on assessment of waterborne exposure. The molecular mechanisms inducing their toxicity on biological system even less investigated. The present study was conducted to explore weather ZnO NPs can be accumulated in internal organs of carp, as a model fish, as well as to assess fish responses to the presence of ZnO NPs in the feed at protein, biochemical, and histological levels. Our results showed nor apparent accumulation of ZnO NPs neither major changes in haematological parameters, lipid peroxidation level, and histology of internal tissues. Our observations showed that 500 mg ZnO NPs per kg of feed caused changes in pathways and the level of proteins associated with cell motility, immune system response, protein synthesis, cell metabolism, and cell survival in intestine as well as it affected the proteins related to immune system in serum. Treated fish underwent these physiological and molecular changes probably attempted to adjust to ZnO NPs as an external stressor. These changes can be considered as compensatory mechanisms to maintenance homeostasis which have an associated energetic cost. If energetic demands to cope with stress exceeds the capacity of limit, then organism's health will be negatively affected. In the second part of study, peracetic acid in tested therapeutic doses (1 and 3 mg L-1 in grass carp and 10 mg L-1 in signal crayfish) caused some histological alterations in gills as well as changes in the activities of antioxidant enzyme in treated animals. It seems that the observed changes were not large enough to induce mortality in treated animals. According our observation after recovery period, it seems that those changes are reversible if it is followed by a sufficient recovery period allowing animals to restore their disturbed homeostasis. However, species-specific differences should be considered while treating more sensitive species. Toward establishing a safe application guideline, more studies will be required to investigate the given issues.

Advances in DNA sequencing led to a technological breakthrough, that allowed analyzis of complete genomes including those of parasitic protists Trichomonas vaginalis and Giardia intestinalis . These organisms are studied not only for their clinical importance, but also from the evolutionary point of view for their adaptation to anaerobic environment. Genome sequencing and annotations of predicted proteins alone did not bring detail view into functioning of their mitochondrion related organelles ­ in G. intestinalis mitosomes, not­participating in energetic metabolism, in T. vaginalis hydrogenosomes, producing molecular hydrogen and ATP by means of substrate phosphorylation. Traditional methods based on a fractionation by ultracentrifuging in density gradient and subsequent biochemical and enzymological analyzes were extended by one­ and two­dimensional electrophoresis with subsequent identification of proteins by mass spectrometry. Methods of multidimensional separation of peptides produced by specific proteolysis of a complex mixture...

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;...

Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease affecting the central nervous system. Although research on the diagnosis and treatment of this serious illness has made significant advances, the MS etiology remains unknown. Although there is still much to be found in the MS pathogenetic mechanisms, understanding the mechanisms of immune-mediated damage to the central nervous system (CNS) components of MS allows not only the introduction of new drugs that positively modulate inflammatory inflammation but also the understanding of immune- mediated diseases affecting the CNS in a broader sense. Methods The Ph.D. thesis is a general introduction followed by an annotated set of author's publications on proteomics, lymphocytic population in MS and disability. Study 1: Proteomic analysis of cerebrospinal fluid for relapsing-remitting multiple sclerosis and clinically isolated syndrome was conducted to find changes in the low molecular cerebrospinal fluid (CSF) segment and to determine what native peptides and how much they are in cerebrospinal fluid among patients with clinically isolated syndrome (CIS) and relapsing-MS (RR MS) compared to a healthy population. Study 2: Lymphocytes in the treatment with interferon beta-1b targeted individual...

Tato práce se zabývá proteomickými studiemi ječmene v souvislosti s výrobou piva. Ječmen patří mezi nejvýznamnější plodiny na světě a je využíván hlavně pro sladovnické účely, nejčastěji pro pivovarnictví. Studium proteinů ječmene během sladování a výroby piva poskytuje informace o změnách v proteinovém složení nebo jejich posttranslačních modifikacích. Jelikož jsou proteiny v ječmeni a jejich změny zásadní pro kvalitu sladu a piva, proteomické studie ječmene mají potenciál pro zlepšení procesu sladování a pivovarnictví. Hlavním cílem této práce je studium ve vodě rozpustných proteinů ječmene a jejich změn, ke kterým dochází během sladování a výroby piva. Rozdíly v proteinovém složení byly sledovány pomocí gelové elektroforézy, kapalinové chromatografie na reverzní fázi, gelové chromatografie a MALDI-TOF hmotnostní spektrometrie. Během sladování se vlivem klíčení zrna zvyšuje množství některých proteinů a také jsou tvořeny nové proteiny. V průběhu vaření piva se naopak v důsledku vysoké teploty a enzymatické aktivity proteáz mnoho proteinů rozkládá. Těmto drsným podmínkám odolají jen některé proteiny, které přechází až do piva a mohou ovlivnit jeho kvalitu. Dále byly zkoumány různé odrůdy ječmene a jejich rozdíly. Byly porovnány odrůdy povolené pro výrobu certifikovaného Českého piva s jednou osvědčenou sladovnickou odrůdou a jednou nesladovnickou odrůdou ječmene. Kromě toho byly studovány v alkoholu rozpustné proteiny ječmene a jejich změny v průběhu sladování. Zvláštní pozornost byla věnována vybrané skupině posttranslačních modifikací proteinů: glykosylacím. Neenzymaticky glykosylované proteiny ječmene (neboli glykované proteiny) jsou tvořeny v průběhu sladování kvůli přítomnosti velkého množství glukózy uvolněné z rozkladu škrobu. Glykované proteiny ovlivňují stabilitu proteinů a kvalitu piva, obzvlášť pěnotvorný účinek. Enzymatické N-glykosylace představují nejčastěji studované posttranslační modifikace u rostlin, protože glykoproteiny hrají klíčovou roli v různých biologických funkcích. Glykoproteiny jsou často přítomny v malém množství, a proto je pro jejich analýzu potřebné obohacení glykoproteinů z komplexní směsi. Pro studium glykoproteinů byla využita afinitní chromatografie s lektinem concanavalin A. Kromě toho byla také optimalizována analýza sacharidové části glykoproteinů. Tato disertační práce přináší důležité informace o proteinech ječmene, jejich změnách a analýze, které budou užitečné pro další studium.

Cardiovascular diseases account for the majority of deaths both worldwide and in the Czech Republic. Main factors contributing heart disease development, aside age and sex, are obesity, high blood pressure and high blood cholesterol and triglyceride levels. Spontaneously hypertensive rat (SHR) was developed and used for search of genetic determinants of these traits. This commonly used rat model develops hypertension, dyslipidemia, and insulin resistance naturally which is caused by aberrant Cd36 fatty acid translocase gene. Previous studies have shown that rescue of Cd36 performed in the transgenic SHR-Tg19 strain enhances cardiac beta-adrenergic system, slightly increases heart mass and leads to higher susceptibility to arrhythmias. The present thesis had two main aims: 1) To investigate whether and how a transgenic rescue of Cd36 in SHR affects protein composition, mitochondrial function and activity of selected metabolic enzymes of the heart. 2) To study the expression and distribution of selected components of beta-adrenergic signaling system in lipid raft isolated form membranes using the TX-100 detergent. We set to compare two commonly used proteomic approaches, 2D electrophoresis with MALDI-TOF mass spectrometry and label-free LC-MS. The results did not reveal any overlap between...