Malaria vaccine research has been battling with persistent challenges, including polymorphisms of vaccine antigens, difficulties with production processes, and limited immune protection against the disease. Intrinsically unstructured proteins (IUPs) are a fairly newly classified group of proteins that have no stable 3D structure and are generally heat-resistant. They usually contain low complexity regions and repetitive sequences, both of which are distinct characteristics of the malaria proteome. Surprisingly, some of the vaccine candidates that have been extensively studied were later reported to have unstructured regions, some of which serve as targets of protective immunity. In keeping with their interesting immunological profiles and their unique properties, which are exceptionally beneficial for vaccine production, malarial IUP antigens may be good vaccine candidates. This PhD project has the following aims:- 1) to develop a synthetic unstructured protein antigen based on the Block 2 region of MSP-1, named the MSP-1 hybrid 2) to characterize a novel vaccine antigen derived from the MSP-3.3 protein, namely an IUP region of PF10_0347 gene product, for its potential as a vaccine candidate 3) to develop a second-generation vaccine by combining the MSP-1 hybrid, with two allelic variants of MSP-2, to overcome antigenic polymorphism and strain-specific immune responses 4) to validate protocols for IUP identification from proteins extracted from the malaria parasite. This study showed that 1) MSP-1 hybrid production was scalable, yielding high protein yields with comparable immunological properties to small-scale production. MSP-1 hybrid was shown to be compatible with different adjuvants, and elicited specific antibodies covering the whole range of Block 2 allelic diversities. 2) A novel antigen, MSP-3.3C, an IUP based on the 3’ region of the PF10_0347 gene, was cloned, expressed and purified. Anti-MSP3.3C antibodies showed very strong parasite growth inhibitory effects in vitro. 3) The MSP-multihybrid antigen was expressed using simple techniques, but only at low levels. It contains epitopes from all three parasite antigen components, and is recognized by specific naturally acquired antibodies. 4) an unconventional 2D gel technique was tested as a method of malaria parasite IUP identification. Plans for further validation of this technique were discussed.

Práca hodnotí pomoc zo zdrojov Európskeho investičného fondu zameranú na rozvoj malého a stredného podnikania na Slovensku.

Rhomboids are intramembrane serine proteases that cleave transmembrane (TM) protein substrates within the phospholipid bilayer. Since the discovery of the first rhomboid protease, many homologous rhomboids have been identified in all kingdoms illustrating their biological significance. Rhomboids are key players in a variety of biological processes such as, cell signalling, protein degradation, mitochondria health, apoptosis, and pathogenicity. While the mechanism of substrate entry into the rhomboid active site is still not clear, it is thought to involve dynamics around the putative substrate gate, of which appears to be comprised of the fifth transmembrane a-helix. A powerful tool that can be used to investigate conformational dynamics around the substrate gate is solution-state nuclear magnetic resonance (NMR). However, due to the size restriction of solution-state NMR, only detergent micelles have been able to produce well- resolved 1H-15N HSQC spectra of rhomboids. However, the lipid membrane environment has a significant impact on rhomboid structure and function. The use of membrane-scaffolding proteins (MSPs) in the formation of nanodiscs has the potential to allow the study of rhomboid dynamics in lipid bilayers by solution-state NMR. Therefore, this thesis investigates the plausibility of incorporating rhomboid into nanodiscs that would be compatible with solution NMR with a focus on the E. coli rhomboid, ecGlpG. The formation of empty (no ecGlpG) and ecGlpG-encapsulated nanodiscs was attempted using two MSP variants. While some successful nanodisc formation was possible, MSP degradation and low yields were seen for all nanodisc samples. Further optimization or alternate nanodisc systems will be required to incorporate ecGlpG into more membrane-like environments in a state that is compatible with solution-state NMR.

Diplomová práce přináší SWOT analýzu společnosti HTP s.r.o., která se zabývá vyhledáváním vhodných dodavatelů strojírenských dílů a komponentů z České a Slovenské republiky pro obchodní partnery především v oblasti zemí Beneluxu, Anglie a Německa. V další části jsou zmapovány možnosti podpory ze zdrojů ČR a EU. Ze strukturálních fondů je podrobněji rozpracován Operační program Podnikání a inovace, z něhož jsou vybrány vhodné programy podpory pro analyzovanou firmu HTP s.r.o.

Leishmania chagasi is the causative agent of visceral leishmaniasis in South America. The most abundant glycoprotein on the surface of L. chagasi promastigotes is the glycosylphosphatidylinositol (GPI) anchored protease MSP (major surface protease), also called GP63. MSP is encoded by more than 18 tandem MSP genes on a single chromosome. MSP genes are classified according to unique sequences at their 3' ends and distinct expression patterns. The five MSPS genes (MSPS1, MSPS2, etc.) express 3.0 kb RNAs in stationary phase of promastigote growth in vitro in culture. The > twelve MSPL genes express 2.7 kb RNAs in logarithmic phase of promastigote growth, and the single MSPC RNA is constitutively expressed as two RNA species (2.6 and 3.1 kb) throughout promastigote growth. The progression from logarithmic to stationary phase is accompanied by an increase in parasite virulence for a mammalian host, and a 16-fold increase in the total MSP protein associated with the cell. As such, MSP has been called a virulence factor of leishmania. Little is known about the differences between isoforms of MSP proteins encoded by the three MSP gene classes, because they have a very similar amino acid sequences. The purpose of this thesis was to study the protein expression and localization of MSPS, MSPL, and MSPC in the promastigote and amastigote stages of the L. chagasi. We took three approaches to this problem. First, we produced constructs in which the fluorescent marker GFP was flanked by putative targeting sequences of the MSPs. Second, we generated Leishmania transfectants expressing Myc-tagged full-length MSPs and studied their localization in promastigote cells. Third, we generated antibodies to immunogenic peptides in the few regions with unique sequences that allowed us to distinguish between some of the MSP classes. One monoclonal anti-peptide antibody, named C51, recognized only MSPS1 and MSPL1. Data indicated that the product of the MSPC gene runs at a higher molecular size than products of the MSPL and MSPS genes, both of which localize to the promastigote surface. Overall the data set the stage for future studies of the properties and functions of specific MSP gene products.

Diplomová práce se zaměřuje na situaci MSP v kontextu Evropské unie, tvorbu Mezinárodních standardů účetního výkaznictví určených pro MSP a úlevy při vedení účetnictví pro MSP v České republice

The research implements interactive music processes involving sound synthesis and symbolic treatments within a single environment. The algorithms are driven by classical instrumental performance through hybrid systems called hyperinstruments, in which the sensing of the performance gestures leads to open and goal-oriented generative music forms. The interactions are composed with MAX/Msp, designing contexts and relationships between real-time instrumental timbre analysis (sometimes with added inertial motion tracking) with a gesture-based idea of form shaping. Physical classical instruments are treated as interfaces, giving rise to the need to develop unconventional mapping strategies on account of the multi-dimensional and interconnecting quality of timbre. Performance and sound gestures are viewed as salient energies, phrasings and articulations carrying information about human intentions, in this way becoming able to change the musical behaviour of a composition inside a coded dramaturgy. The interactive networks are designed in order to integrate traditional music practices and “languages” with computational systems designed to be self-regulating, through the mediation of timbre space and performance gestural descriptions. Following its classic definition, technology aims to be mainly related not to mechanical practices but rather to rhetorical approaches: for this reason the software often foresees interactive scores, and must be performed in accordance with a set of external verbal (and video) explanations, whose technical detail should nevertheless not impair the most intuitive approach to music making.

Hlavním cílem je navržení vnitropodnikových směrnic pro účetnictví ve firmě Data Credit Jany Sazimové podnikající jako fyzická osoba. A to z legislativního i formálního hlediska a zpracovat jejich návaznost z hlediska systémové koncepce a komplexního chápání probíhajících procesů ve vnitřní organizační struktuře a vazeb mezi nimi. Práce je zaměřená především na oblasti, kde může docházet k různým výkladům zákona o účetnictví a na oblasti, kde je možno použít několik způsobů účtování.

Set in Motion is a composition in two movements for an ensemble of 10 instruments that interacts with a computer in real-time performance using Max/MSP software. The instrumental writing in the piece focuses on incorporating electronic composition techniques into acoustic composition. Techniques such as constructing envelopes, shading, and sound masses, which are common in electronic works are applied to the ensemble. Also, the intervals of a major and minor second are important in the instrumental writing. These intervals provided the basis for the material, which was then transformed algorithmically, both in individual lines and whole sections of music. In terms of the electronics, the first movement takes advantage of the interactive possibilities of the software. The electronic part is created through the computer extending, processing, and responding to the instruments. The techniques include extracting small grains of sound from the live instruments and repeating them, analyzing the pitch of the signal and amplifying overtones, and rearranging fragments in blocks of recorded sound. The second movement changes the focus of the electronic sound. The electronics are made of precomposed gestures that compliment the live instruments. These gestures are categorized and selected randomly according to specific characteristics. Filtering and other effects applied to the sounds are chosen randomly as well.

The research involves the creation of an interactive installation showcasing the dynamic nature of human visual observation of a still photograph. Using an eye tracker as an input device, the data collected is used to create a photographic collage in stereoscopic 3D. The installation is artistically inspired by selected photographic works of artists David Hockney, Maurizio Galimberti, Joyce Neimanas and Cubist painters especially Picasso. One of the key factors in their work that is adapted in this research, is the representation of the way eyes search points of interest demonstrated in what they painted/photographed. The installation will demonstrate an expressive representation of the viewers' experience of looking at a photograph. This will be achieved by applying certain manipulations of the photograph based on the input obtained from the viewer using an eye tracker. The eye tracker collects information about the location and number of instances of where the viewer is when observing a photograph. This is fed into software that processes the data and determines the location and the size of the area of the photograph and amount of the manipulation to be applied to that area. These two constitute the artistic rules that are used to create the end product the photo collage. The individual pieces of the collage will be arranged in a virtual 3D model by the artist and will be projected in stereoscopic 3D. The development of this installation progressed through multiple case studies and optimization based on ease of use, cost and availability of resources. This process is intended to be a framework for artists working in interactive visual media.