Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition to its principal virulent factors, Toxin A and Toxin B, some C. difficile strains produce a binary toxin (CDT) composed of two subunits namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut, these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. This thesis is a step towards understanding structural and functional aspects of the binary toxin produced by C. difficile. The first half of this thesis (chapter I and II) provides a brief introduction to the method of structure determination of proteins molecules, i. e. X-ray crystallography and a detailed overview of C. difficile and the three known toxins from C. difficile namely – Toxin A, Toxin B and the binary toxin. Chapter II further focuses on C. difficile binary toxin and other related toxins. These toxins, known as the ADP-ribosylating toxins (ADPRTs) form a big family of potent toxins which includes Cholera, Pertussis and Diphtheria toxins and are capable of transferring the ADP-ribose part of NAD/NADPH to a varity of substrates in the target cell which ultimately results in cell death. The second half of the thesis comprises of experimental procedures that were carried out during the course of this study and their results. Cloning and expression methods for recombinant CDTa and CDTb in bacterial system followed by their purification are described with the abnormal behaviour exhibited by CDTb (chapter III). We show for the first time that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells (Chapter IV). The purification processes described yielded milligram quantities of binary toxin fragments of high purity that led to the successful crystallisation of the proteins (chapter IV) for further functional and structural studies. High resolution crystal structures of CDTa in its native form (at pH 4.0, 8.5 and 9.0) and in complex with the ADP ribose donors -NAD and NADPH (at pH 9.0) have been determined (chapter V). The crystal structures of the native protein show ‘pronounced conformational flexibility’ confined to the active site region of the protein and ‘enhanced’ disorder at low pH while the complex structures highlight significant differences in ‘ligand specificity’ compared with the enzymatic subunit of a close homologue, Clostridium perfringens Iota toxin (Ia). These structural data provide the first detailed information on protein-donor substrate complex stabilisation in CDTa which may have implications in understanding CDT recognition. Crystallisation of CDTb yielded preliminary crystals. The optimisation of these crystallisation conditions is underway. The thesis concludes with some thoughts and discussion on future directions of this research.

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Thesis deals about active component in current mode, CDTA (current differencing transconductance amplifier) and CFTA (current follower transconductance amplifier). CDTA compares two input currents. Outcome current comes trough added terminal Z to load. Caused voltage is transfered to output current by transconductance g. Output surrents shall be possitive (terminal X+) or negative (terminal X-). In contrast to CDTA, CFTA has one current input. Components were used in filters and oscillators. Some filters of second order realized simultaneously low, hight and nad pass were designed. Then, the transfer of filters with diverse topology with opperational amplifier to filter with CDTA and CFTA was shown. Some oscillators were designed. Designed circuits were simulated. Transistor models of CDTA and CFTA were created to purpose of simulation. This components can be realized by AD844 and MAX435. There are shown the conclusions of AC analysis of filters and transission analysis of oscillators. The thesis contains practical realization of second order frequency filter also. Device was made like a PCB by smd. CFTA was realized by components AD844 and MAX435.

This master´s thesis on theme Modeling of the unconventional functional blocks in Pspice deals with the design procedures of electronic elements models and should to help with their create. At the beginning the thesis theoretically describes how the models are created and what possibilities for modeling the program Pspice offers. There are drawn near the individual levels of model and their properties. The second main part of thesis shows practically step by step the design procedures of models of the operational amplifier, current and voltage conveyors, OTA, CDTA, immittance inverter and converter and logarithmic amplifier. There are created the models up to 4.th level, the ideal, resistive, frequency dependent and nonlinear. The basic transmission properties, input and output impedance are modeled this way. The created models with sufficient accuracy correspond to the parameters of the commercial elements. This thesis such shows the possibilities of the program for finding of values for model components like stepping or Optimizer tool.

The presented dissertation thesis deals with modular design of analog circuits in CMOS technology. The goal of the work is to design a set of modular microelectronic building blocks and realize the selected modern active circuits, working primarily in current mode. Nevertheless, the modular approach can be used for design of generally known classical elements, e.g. opamps, as well. As a part of the work, the development of the totally new highly versatile active circuit CCTA has been done, including detailed analysis of utilization and introduction of the most interesting applications. This circuit CCTA, together with relative, already theoretically treated circuit CDTA, has been realized here for the first time, in two different topologies. Final circuits were tested. On the basis of measurement results the library of behavioral models for PSpice was created, including exemplary simulations of the selected applications. Based on the obtained knowledge the brief comparison of voltage mode circuits and current mode circuits was done.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.

This work deals with using modern active functional blocks in electronic circuits. These blocks have better features than classical operational amplifiers and are characterized especially by working in current mode. Therefore these blocks can be used in higher frequencies. Using those blocks it is possible to design e.g. electronically variable-frequency filters of types low-pass filter, high-pass filter, band-pass filter, etc. Then it is also possible to easily realize electronically variable-frequency oscillators. This work is focused on some of these blocks, their basic characteristics and principles. Also three oscillators with current conveyors of second generation are designed. Its made simulation, sensitivity and tolerance analysis and magnitude stabilization. Everything is performed in OrCAD PSpice program. These made oscillators are electronic tunable in the band frequency ones of MHz.

Kmitočtové filtry jsou lineární elektrické obvody, které jsou využívány v různých oblastech elektroniky. Současně tvoří základní stavební bloky pro analogové zpracování signálů. V poslední dekádě bylo zavedeno množství aktivních stavebních bloků pro analogové zpracování signálů. Stále však existuje potřeba vývoje nových aktivních součástek, které by poskytovaly nové možnosti a lepší parametry. V práci jsou diskutovány různé aspekty obvodů pracujících v napěťovém, proudovém a smíšném módu. Práce reaguje na dnešní potřebu nízkovýkonových a nízkonapěťových aplikací pro přenosné přístroje a mobilní komunikační systémy a na problémy jejich návrhu. Potřeba těchto výkonných nízkonapěťových zařízení je výzvou návrhářů k hledání nových obvodových topologií a nových nízkonapěťových technik. V práci je popsána řada aktivních prvků, jako například operační transkonduktanční zesilovač (OTA), proudový konvejor II. generace (CCII) a CDTA (Current Differencing Transconductance Amplifier). Dále jsou navrženy nové prvky, jako jsou VDTA (Voltage Differencing Transconductance Amplifier) a VDVTA (Voltage Differencing Voltage Transconductance Amplifier). Všechny tyto prvky byly rovněž implementovány pomocí "bulk-driven" techniky CMOS s cílem realizace nízkonapěťových aplikací. Tato práce je rovněž zaměřena na náhrady klasických induktorů syntetickými induktory v pasivních LC příčkových filtrech. Tyto náhrady pak mohou vést k syntéze aktivních filtrů se zajímavými vlastnostmi.