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Voltage sensitive fluorescent protein based on the motor protein prestin
DIVOKÝ, Karel
The aim of this work was to create a series of fluorescently labeled constructs based on the motor protein prestin, express these constructs in mammalian cells, and measure their linear dichroism by two photon polarization microscopy, in order to develop a genetically encoded optical probe of cell membrane voltage. This thesis provides an introduction to current methods of observing electrical activity in neurons, describes the method of two-photon polarization microscopy, describes our results in preparing the abovementioned constructs, and summarizes our microscopic observations.
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Development of Meta-Server for Prediction of Mutations Effects on Protein Function
Lisák, Peter ; Burgetová, Ivana (referee) ; Jaša, Petr (advisor)
This bachelor thesis deals with analysis of genomic data, more specifically prediction of effects of mutations on protein function using a protein sequence or tertiary structure. The theoretical introduction describes the basics of genetics and bioinformatics and is followed by description of selected prediction tools such as SIFT, MAPP and AUTO-MUTE. A unified interface for work with different tools is proposed in the thesis. The meta-server interface allows running a computation and collecting results from one site. meta-server combines results of implemented tools and provides a consensual prediction, which is expected to be more accurate than the results from individual tools. Finally, testing of meta-server on the real data and comparisons of predictions with the experimentally obtained results are presented.
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The role of protein kinase C in the pathogenesis of insulin resistance and its complications
Marková, Irena ; Novák, František (advisor) ; Zeman, Miroslav (referee) ; Zídková, Jarmila (referee)
18 effects of TZDs are probably due to the remodeling of adipose tissue and increased adiponectin secretion. SUMMARY Studying the pathogenesis of insulin resistance and the role of PKC in insulin resistance In HHTg rats, elevated serum triglycerides and FFA were associated with the ectopic accumulation of triglycerides in tissues and reduced insulin sensitivity of peripheral tissues. Impaired glucose utilization in the peripheral tissues was associated with the reduced activity of GS in skeletal muscle. Decreased GS activity and glucose utilization in peripheral tissues indicate a possible defect in insulin signal transduction. In line with this, our results show that skeletal muscle IR was associated with the increased activation and translocation of PKC θ. Nutritionally induced obesity of HHTg rats resulted, in many cases, in the further deterioration of metabolic abnormalities associated with IR. We found that PKC θ, in particular, could contribute to the metabolic abnormalities associated with IR and obesity. The age-related increase in IR and deterioration of some parametres of carbohydrate and lipid metabolism, were not associated, in HHTg rats, with obesity but with increased serum levels of triglycerides and FFA. The age-related worsening of IR in HHTg rats was accompanied by increased...
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Cultivation of tick-borne encephalitis virus in the presence of inhibitor of glycosylation: its effect on nucleotide sequence of genes encoding viral proteins.
FARKOVÁ, Karolína
In this work I compared nucleotide sequences of virus genes and the resulting aminoacid sequences of viral proteins of TBE virus variant Hypr3TM with the parental virus Hypr50/CB. This comparison serves for identification of nucleotide/aminoacid substitutions resulting for virus replication in an enviroment with inhibited protein glycosylation. I identified four nucleotide substitutions; three of them caused also changes in amino acid sequence. When compared to a low-passaged variant Hypr5, three of the nucleotide substituions were identical with this variant. One amino acid substitution is unique for variant Hypr3TM. This substitution caused changes in electrostatic potencial of receptor domain of protein E and thus can affect the protein-protein interactions within homodimers and homotrimers at the surface of the protein and the interaction of the E protein with host-cell receptors for the TBE virus.
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Prediction methods of protein hot spots
Duras, Jan ; Provazník, Ivo (referee) ; Maděránková, Denisa (advisor)
This master's thesis deals with prediction of hot spots in proteins. The theorethical part describes compositions of proteins interfaces and hot spots. There are mentioned basic principles of hot spots prediction. In the practical part is selected method, using the S-Transform, designed to pseudocode and then implmented to Matlab software. Created program is tested on a sample of data and compared with available methods.
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Analysis of recombinant clones of apoptotic nucleases in "leaf factory" system upon coinfiltration with modifying genes
LOMNICKÁ, Anna
TBN1 is a nuclease with antitumor activity. The main goal of this work was to estimate how TBN1 and its modificated variants are stable in the ?leaf factory? system used for its production and whether it can be enhanced or influenced by chosen potential ?modificators? i. e. silencing supressors, transcription factors, glycosyltransferases and kinases. Nicotiana benthamina plants were infiltrated with the mixture of Agrobacterium tumefaciens strains bearing the nuclease plant expression vectors and co-infiltrated with the ?modifying? vectors. The nuclease and protein analyses revealed that nuclease TBN1 wt and its modificated variants are stable in the used ?leaf factory? system as to their molecular mass, only quantitative changes were detected. Expreximents showed that activity and production of the nucleases increased upon coinfiltration with silencing supressor and decreased upon coexpression with chosen transcription factor. Glycosyltransferases and kinases influenced activity and production only insignificantly. The experiments also revealed that modificated variants of TBN1 have different molecular weight suggesting that different N-glycosylation domains have different length of sugar chain and influence on nuclear activity. Our data show that this expression in planta seems to be suitable for production for study of antitumor activity of these nucleases.
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Biological importance of CAS SH3 domain tyrosine phosphorylation
Janoštiak, Radoslav
Protein CAS is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes. It is a multidomain adaptor protein, which serves as a scaffold for assembly of signalling complexes which are important for migration and invasiveness of Src-transformed cells. A novel phosphorylation site in N-terminal SH3 domain was identified - tyrosine 12 located on binding surface of CAS SH3 domain. To study biological importance of tyrosine 12 phosphorylation, non-phosphorylable (Y12F) and phosphomimicking ( Y12E) mutant of CAS were prepared. We found that phosphomimicking mutation Y12E leads to decreased interaction of CAS SH domain with kinase FAK a phosphatase PTP-PEST and also reduce tyrosine phosphorylation of FAK. Using GFP-tagged CAS protein, we show that Y12E mutation caused delocalization of CAS from focal adhesion but has no effect on localization of CAS to podosome-type adhesion. Non-phosphorylable mutation Y12F cause hyperphosphorylation of CAS substrate domain and decrease turnover of focal adhesion and associated cell migration of mouse embryonal fibroblasts (MEFs) independent to integrin singalling. Analogically to migration, CAS Y12F decrease invasiveness of Src-transformed MEF. The results of this diploma thesis show that phosphorylation of Tyr12 in CAS SH3 domain is...
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Produkce polyklonálních protilátek proti rekombinantnímu proteinu X viru bramboru
Čeřovská, Noemi ; Plchová, Helena ; Moravec, Tomáš ; Filigarová, Marie ; Dědič, P.
The gene encoding the coat protein (CP) of Potato virus X was cloned into expression vector pMPM-4A Omega. PVX CP was expressed in Escherichia coli and was used for raising rabbit polyclonal antibodies. For immunization we purified CP fractions from bacterial lysate either by simple fractionation or by excision from SDS gel. The obtained sera and antibodies were tested for the detection of PVX in laboratory hosts Nicotiana tabacum, cv. Petit Havana SR 1 and N.benthamiana and in various cultivars of natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies were specific for PVX and are usable for the detection of the broad isolates spectrum of the Potato virus X by ELISA and Western blot.
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Role of DD- and DED-containing adaptor proteins in apoptotic signaling
Čaja, Fabián ; Janštová, Vanda (referee) ; Anděra, Ladislav (advisor)
Proteins containing a bundle of six anti-paralel α-helices in so-called "death domain" (DD) and similar structures (DED, CARD) represent important players in apoptotic signaling. To DD/DED/CARD domains-containing proteins belong pro- apoptotic membrane receptors from the TNFR superfamily, then adaptor proteins and enzymes as proteases or kinases. These pro-apoptotic "death receptors" interact with adaptor proteins and initiator caspases containing DDs or DEDs and activate apoptotic signaling cascade. DEDs and DDs are in addition found in many proteins participating in activation of caspases or other non-apoptotic signaling. Many experimental models document that defects in and deregulations of proteins containing DDs and DEDs can have severe if not lethal consequences for an organism. Abberations in these proteins in many cases could lead to cancerogenesis, immunodeficiencies or developmental defects.
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