|
|
Study of protein-protein interactions of human E3 ubiquitin ligase Nedd4-2.
Joshi, Rohit Ashok ; Obšilová, Veronika (advisor) ; Krůšek, Jan (referee) ; Holoubek, Aleš (referee)
Posttranslační modifikace prostřednictvím ubikvitinace hrají klíčovou roli v regulaci membránových proteinů. Nedd4-2, lidská HECT E3 ubikvitin ligáza, je poslední komponentou ubikvitinační kaskády, která přenáší molekuly ubikvitinu a spouští endocytózu svých následných cílových molekul. Dysregulace Nedd4-2 může způsobovat různé poruchy, včetně epilepsie, respirační úzkosti a Liddleova syndromu. Přestože se na regulaci Nedd4-2 podílejí různé adaptorové proteiny, v tomto výzkumu jsme se zaměřili na konzervované proteiny 14-3- 3, známé negativní regulátory Nedd4-2. V této studii jsme provedli biofyzikální charakterizaci konstruktů Nedd4-2190-581 a Nedd4-2186-975 v komplexu s 14-3-3, abychom získali další náhled do dynamiky této interakce. Naše výsledky časově rozlišené fluorescenční spektroskopie odhalily, že vazba 14-3-3 ovlivňuje emisní vlastnosti a pohyblivost specifických WW domén (WW3 a WW4) Nedd4-2, zatímco ostatní (WW1) šetří. Zajímavé je, že katalytická doména HECT prochází při tvorbě komplexu konformačními změnami a zvýšenou exponovaností rozpouštědlu. Předpokládáme, že sterická inhibice domén WW3 a WW4 v kombinaci s konformačními změnami v katalytické doméně může být základem regulačního mechanismu zprostředkovaného vazbou proteinem 14-3-3. Chemické zesítění spolu s hmotnostní spektrometrií...
|
|
|
New approaches in drop coating deposition Raman spectroscopy for sensitive molecular detection
Přikryl, Mikuláš ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
in english: Drop coating deposition Raman spectroscopy is a vibrational spectroscopic method based on the principle of Raman scattering. Deposition of a small volume of solution (μl) of a low concentration (μM) on a special hydrophobic substrate and subsequent drying leads to a formation of a deposit from which we can accumulate a higher quality Raman spectrum than in the case of classical Raman spectroscopy. In this thesis, four contaminants (melamine, picloram, thiram, bentazone) were studied on new nanoparticle-based substrates prepared at our faculty. These hydrophobic substrates were compared to commercial substrates and proven to be suitable for further development. Next, artificially prepared mixtures of pairs of contaminants were studied using the commercial substrate. Every contaminant was detected and differentiated in each of these mixtures using factor analysis at relatively low concentrations. Furthermore, the contaminants were separated in some deposits. However, unknown spectra were detected, probably due to chemical reactions between the contaminants. Possibilities of contaminations or interactions with the substrate were eliminated. Finally, a real sample of infant formula artificially contaminated by melamine was studied. The contaminant was detected at concentrations equal to...
|
|
|
Molecular events associated with resistance to tyrosine kinase inhibitors in leukemia cells.
Hrdinová, Tereza ; Vyoral, Daniel (advisor) ; Klener, Pavel (referee) ; Holoubek, Aleš (referee)
Chronic myeloid leukemia (CML) is a myeloproliferative stem cell disease characterized by the expression of BCR-ABL oncoprotein with constitutive tyrosine kinase activity. Although the development of tyrosine kinase inhibitors (TKI) such as imatinib dramatically improved the treatment of CML, a certain subset of patients develops resistance to TKI drugs. The most common cause of TKI resistance are point mutations in the BCR-ABL1 gene, followed by other mutation-independent mechanisms. Survival and proliferation of CML cells in the presence of TKI drugs are accompanied by adaptive changes in their metabolism. Drug resistance can be maintained by extrinsic signals, among which exosomes, small vesicles released by (drug-resistant) cells, have been shown to play an important role. The aim of this thesis was to characterize two CML cell lines sensitive and resistant to imatinib, as well as the exosomes derived from imatinib-resistant CML cells by proteomic approaches. Identification of metabolic vulnerabilities in drug-resistant cells enables their targeting by clinically available drugs, thus offering potential therapeutic targets for their selective elimination. Analysis of exosomes derived from imatinib-resistant cells can identify specific membrane surface proteins exploitable as clinically relevant...
|
|
| |
|
|
Drop coating deposition Raman spectroscopy of liposomes with different compositions
Antalík, Andrej ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Method of drop-coating deposition Raman spectroscopy is a special tech- nique that employs deposition of a small droplet of the sample on a hydro- phobic surface with subsequent solvent removal by evaporation. As a result the concentrated material forms a ring at the edge of the droplet. This techni- que has proven to be efficient for liposome studies at low concentrations. We focused on a study of spectral changes and ring formation in phosphatidylcho- line liposomes due to increasing amount of cholesterol. Higher concentration of cholesterol didn't cause significant changes in membrane structure, such as phase transition, however the ring wasn't formed. Second part of this bache- lor thesis concerns with the search for some alternative surface to previously used SpectRIM TM DCDR plates made by Tienta Sciences, Inc. which are no longer available. The polished calcium fluoride glass (CaF2) seems to be the most suitable for the further use from all tested surfaces. Raman signal at the other surfaces was either weak or spectral bands that don't belong to lipid were observed.
|
|
|
Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra ; Konopásek, Ivo (advisor) ; Holoubek, Aleš (referee) ; Hof, Martin (referee)
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
|
|
|
Study of transport proteins of the Nramp family
Surá, Lucie ; Chaloupka, Roman (advisor) ; Holoubek, Aleš (referee)
The Natural Resistance-Associated Macrophage Proteins (Nramp) form functionally conserved family of proton-dependent divalent metal ion transporters. In the present study, we investigated transport properties of a prokaryotic Nramp homolog - MntH transporter from Escherichia coli. H+ transport mediated by MntH was monitored in a bacterial model system using pH-sensitive green fluorescent protein (pHluorin). Our experimental conditions enabled us to observe an uncoupled H+ transport mediated by MntH. Uncoupled H+ flux had been previously described in eukaryotic Nramp proteins, nevertheless this is the first observation of this phenomenon in a prokaryotic homolog. We demonstrated that the uncoupled H+ transport is pH- and temperature- dependent. The uncoupled transport H+ is also affected by specific single-point mutations at functionally important residues Asp34, His211 and Asn401. The second part of the work focused on effect of different ions, which are not MntH substrates, on transport properties of MntH. It was shown that addition of excess calcium or magnesium resulted in increase of H+ transport induced by divalent metal ions, but on the other hand our data suggest that calcium inhibits uncoupled H+ transport.
|
|
|
Study of angular and time characteristics of suspensions by using of elastic scattering
Peikertová, Pavlína ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Elastic light scattering represents relatively simple and noninvasive technique to study angular dependencies of the scattered light intensity that contains important information about scattering objects. In the frame of this thesis the data from adapted computer controlled apparatus Photo-gonio-diffusometre type 42.000 were collected. Experimental set-up was upgraded to use it for small sample volumes. Two model systems were studied: polymer and latex suspensions and real systems: liposomal suspension of various sizes, as well. The size estimation by using of Guinier plot was performed from experimental data. This estimation is in good agreement with the defined sizes in case of model systems. For suspensions of liposomes the results are also in relatively good agreement with expected values. Aggregation process and subsequent sedimentation after addition of porphyrin CuTMPyP4 was observed for silver borohydrid colloid. Acquired time and angular dependencies were processed by factor analysis that is able to make a qualitative and temporal separation of both processes
|
|
|
Study of oligonucleotide penetration through liposomal membrane
Nováková, Jana ; Kočišová, Eva (advisor) ; Holoubek, Aleš (referee)
Modified oligonucleotides, short pieces of nucleic acids, have become potential candidates for treatment of viral, malignant or genetic diseases. Efficient application of modified oligonucleotides requires their sufficient penetration inside the cell through the lipid membrane. Diploma thesis employs liposomes, synthetic vesicles formed by the lipid bilayer, as a model system for the study of interaction of lipid and complex of homooligonucleotides dT15 with cationic porphyrin CuTMPyP4 using Raman spectroscopy. Raman spectra were measured in solution on the slide and using special technique of drop coating deposition Raman spectroscopy (DCDR) on integrated Raman confocal microspectrophotometer. Processes of liposome formation and sample preparation were optimized. Experimental data were analysed using factor analysis based on singular value decomposition (SVD) algorithm. It was observed that interaction of oligonucleotide with porphyrin induces fluorescent background increase. Raman spectra of complex of oligonucleotide with porphyrin exhibit the domination of porphyrin vibrational bands. Stretching vibrations of lipid assigned to hydrogens of fatty acids are observed in the spectra of complex with liposomes. The observed spectral changes show the interaction of lipid with complex of oligonucleotide and...
|
|
|
Cytoplasmic membrane of Bacillus subtilis Regulation of the physical parameters
Beranová, Jana ; Konopásek, Ivo (advisor) ; Branny, Pavel (referee) ; Holoubek, Aleš (referee)
Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...
|
guest ::
