A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...

Capillary zone electrophoresis (CZE) is a widely used analytical method. CZE is described theoretically very well and there are many simulation programs, which enable one to predict results of electrophoretic separations, and alternatively to study phenomena taking place during the electrophoretic separation in detail. The CZE method is not only an analytical method, but is often used for determination of many physical parameters of compounds, such as stability constants or complex mobilities. Among methods most often used for determination of complexation parameters belongs the affinity capillary electrophoresis (ACE). Its alternative is the vacancy affinity capillary electrophoresis (VACE). Whereas by the ACE method the stability constant is determined from the dependence of the analyte effiective electrophoretic mobility on the background electrolyte (BGE) composition, in the case of the VACE system peaks are used for this purpose. In this work the legitimacy of using system peaks in the VACE method for determination of stability constants was investigated. Several approaches dealing with the concentrating of complexing agent in the peak area were compared, both for the ACE and the VACE method. Two different kinds of electrophoretic systems were studied. In the first one, neutral cyclodextrin was used as...

I characterized several potential glycoproteins in salivary gland extracts from unfed and partially fed females of ticks Ixodes ricinus using enzyme deglycosylation and lectin labeling. Affinity-based (chromatografic) analysis was applied for isolations of glycoproteins with specificity for GNA (mannose), HPA (N-acetylgalactosamine) and MAA II (sialic acid) lectins. GNA specific 120 kDa glycoprotein was isolated from partially fed females and is modified with N-linked glycans containing {$\alpha$}1,3-mannose. Mass spectrometry analyses confirmed the presence carboxypeptidase M in elution fraction gain with GNA affinity chromatography. GNA specific proteins were purified from unfed female salivary gland extracts. MS analyses identified them as proteins similar to arylsulfatase B and cytoskeletal Sojo protein. Proteins (85 and 56 kDa) isolated with HPA affinity chromatography were characterized as Trappin 12, which is a host protein. MAA II lectin was used for labelling and isolation of 100 kDa protein. N-terminal sequence of the MAA II specific protein predicted similarity with a host protein, Siglec 1. Fucose in salivary gland extract was detected with the labelling of AAA, AAL, UEA I and LTL lectins. Results showed that salivary gland extracts contain {$\alpha$}1,2-; {$\alpha$}1,3- and {$\alpha$}1,6- N-linked fucose and O-linked fucose probably as well. GNA specific proteins were detected in partially fed salivary glands acini type II and III using electron transmission microscopy. Fucose was detected on gut and salivary gland structures using fucose-specific lectin AAL.

The aim of this work was to characterize a member of the Drosophila Idgf family. IDGF2 was expresed in Drosophila S2 cells and purified by affinity chromatography. Function of wild-type and mutant IDGF2 was compared.

The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...

It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.

Sensitive layers for optical biosensors and protein chips The goal of this thesis was the development of sensitive surfaces for optical affinity biosensors detecting in complex biological media. The practical application of these surface-based technologies has been hampered by protein fouling from biological media, in particular blood plasma, where the vast majority of relevant analytes are present. The work of the thesis was centred in three main foci:  Design and preparation of antifouling and non-fouling surfaces  Evaluation and conceptualisation of their resistance to fouling from blood plasma and serum as well as other biological fluids  Preparation of sensitive layers for detection in complex biological media Three approaches were used to prepare protein resistance surfaces, i) ω-functional self-assembled monolayers (SAM), ii) end-tethered polymers and iii) polymer brushes prepared by surface initiated controlled radical polymerisation. Investigation of proteins in the blood plasma deposits on PEG- based surfaces revealed that some fouling is unavoidable in PEG-based surface modifications. A novel type of non-fouling polymer brushes based on poly[N-(2-hydroxypropyl) methacrylamide] challenged the accepted ideas for the design of protein resistant surfaces. For the first time a...

Capillary electrophoresis is a widely used separation method of analytical chemistry. Addition of a selector into the background electrolyte extends its applicability to separation of enantiomers or of compounds of similar physicochemical properties. In analytical practice, mixtures of selectors are also commonly used - either prepared intentionally to achieve better separation or because commercially available selectors may be mixtures of compounds differing in the degree of substitution and substituent positions. Mathematical description of these systems, which are highly relevant in analytical practice, can simplify search for optimal separation conditions. Also, it provides a useful insight into the separation mechanism. In this work, a model of electromigration of an analyte interacting with a mixture of two selectors is proposed and experimentally verified. This model results from a more general description of systems with an arbitrary number of selectors. The model shows that a selector mixture can be treated as a single selector if the ratio of the respective selector concentrations is kept constant. When the mixture is prepared intentionally, this description predicts, how separation potential of the mixture changes with its composition. Thus it allows the optimal composition and total...

Capillary electrophoresis is one of the prominent analytical separation methods. Currently, many computer programs exist which are able to predict the result of an electrophoretic experiment. Firstly, there are programs based on numerical solving of corresponding continuity equations and equations of chemical equilibria. Secondly, there are programs based on approximative models of capillary electrophoresis. Programs belonging to the first group are applicable to a wide range of modes of capillary electrophoresis and provide a precise solution. Their disadvantage is though a considerable time demand. On the other hand, the approximative models give the results almost immediately and, in addition, provide some theoretical relationships which are useful for optimization of the separation process. This dissertation thesis is focused on improvement and extension of validity of existing approximative models of capillary electrophoresis. As a part of this thesis, a model capable of a full-blown description of capillary electrokinetic chromatography is introduced. This model is implemented into program PeakMaster 6. The attention is also paid to a nonlinear model of electromigration without diffusion. This model enables a very good description of electromigration dispersion including effects related to...

Motion of ions under the influence of electric field has been a subject of scientific interest for many decades. Capillary electrophoresis in particular benefited greatly from this research and mathematical models of electromigration applicable to capillary electrophoresis have been developed. As the sophistication of the models grew, so did the computational demands to evaluate them. In order to fully exploit the possibilities of advanced mathematical models a computer implementation capable of solving non-trivial problems at sufficient speed is necessary. This dissertation thesis explores applications of computer implementations of mathematical models re- lated to electromigration in two different areas. The main focus of this thesis is on the topic of linear theory of electromigration. We discuss the extension of the linear theory of electromigration beyond of just acid-base equilibria and computer implementation of this extented theory which is specialized to include complex-forming equilibria in order to be able to deal with affinity capillary electrophoresis prob- lems. Some technical aspects of the computer implementation are also discussed. This is followed upon by investigating certain selected affinity capillary electrophoresis systems. The purpose of this investi- gation is to re-derive...