The aim of this diploma thesis is to find and analyze the causes of the higher than average levels of unemployment found in the Ústí nad Labem region. Recently, there has been great pressure to decrease of level unemployment throughout the Czech Republic. However, persistent levels of long term unemployment have plagued several regions of the CR such as the Ústí nad Labem. Despite concerted attempts to address them they have remained unchanged and will thus provide a useful focus for this paper. The theoretical part of this paper provides a description and explanation of the basic terms and principles that surround the problems of unemployment, a definition of its categories, and a description of the causes and consequences of long term unemployment. In addition, the paper describes several approaches of measuring unemployment. The second chapter contains a detailed commentary of each group based on unemployment statistics and a comparison to other similar groups in other European states. Finally, this part provides a synopsis of several groups in the labor market who have traditionally not responded to policies intended to relieve chronic unemployment and looks at how this has damaged their communities. The practical part of the paper is focused on the causes of unemployment in the Ústí nad Labem region. There are many possible causes such as the transformation from a centrally planned economy to a market economy, the limitations of coal mining in the region, a higher number of Romany people, lower education levels, high rate of long-term unemployment and other causes. Data was compiled from the Czech statistics office, programs of development from the Ústí nad Labem region, the Department of Labor, as well as expert analysis focused on labor market, and personal interviews with unemployment specialists. Descriptive, comparative and analytical methods are used in this part. An additional value of this thesis is an analysis of the structurally damaged region and an analytical evaluation that can be used in the process of developing program documents which will solve interventions included in determined competence of European Social Fund.

The aim of this Diploma Thesis is the elaboration of a study concerning the project for the reconstruction of the historical building of a granary in Hluboká nad Vltavou into an amusement park. The theoretical base of the thesis deals with the importance of the tourism at the national and regional levels where the contemporary situation of the Southern Bohemian Region is described in a more detailed way. A part of the thesis includes selected examples of local and foreign case studies dealing with similar projects like the planned project of an amusement park and it also corresponds with the contemporary market trends. The main practical part of the thesis deals with the design of the reconstruction of the historical building of a granary in Hluboká nad Vltavou into an amusement park. The purpose of the thesis is not the procession of a realistic project including all parts thereof but a study offering the solutions how to use the building in connection with the strategic planning document of the town Hluboká nad Vltavou as well as the Southern Bohemian Region. The practical part includes the description of the project of the granary, including the design, basic economic analysis and possibilities of the marketing communication. The technical documentation of the building is a part of attachments.

O crescimento de uma camada neoíntima é o marcador central do processo aterosclerótico, bem como do remodelamento vascular associado à reestenose após intervenções vasculares terapêuticas, tais como angioplastia ou colocação de stents. A célula neointimal é essencialmente uma célula com fenótipo muscular liso indiferenciado, cuja origem pode ser múltipla e com característica ação secretora de matriz extracelular. Dentre os fatores que coordenam a (des)diferenciação, proliferação e migração destas células, há evidências de que processos redox tenham papel preponderante, porém os mecanismos de tais processos não estão claros. A compreensão mais profunda de tais mecanismos redox tem sido dificultada pela falta de modelos de células neointimais cultivadas. Os objetivos específicos deste trabalho são: 1) Desenvolver um modelo de cultura de células neointimais de artéria ilíaca de coelho obtidas após lesão por catéter-balão. 2) Avaliar em tais células índices do estado redox e potenciais fontes enzimáticas de ERO, com ênfase no complexo NAD(P)H oxidase. 3)Avaliar marcadores de estresse do retículo endoplasmático e sua correlação com os índices do estado redox. 4) Estudar o efeito de estímulos proapoptóticos como deprivação de soro, estressores do RE e particularmente administração exógena de NO na viabilidade e estado redox de células neointimais. Os resultados obtidos demonstram que: é possível obter células neointimais em cultura de modo reproduzível e estável. Células provenientes da artéria lesada mantém um estado persistente de aumento do estresse oxidativo. O estresse oxidativo nessas células decorre de produção aumentada de radical superóxido e ativação do complexo da NADPH oxidase vascular. Diferentemente do observado em vasos lesados, os marcadores do estresse do reticulo endoplasmático não se apresentam alterados se comparados a células musculares lisas normais. Células neointimais têm resposta aumentada a agonistas da NADPH oxidase e estressores celulares. A exposição a óxido nitrico promove aumento da produção de superóxido, particularmente acentuado em células neointimais. As curvas de viabilidade celular indicam uma sensibilidade aumentada a estressores do RE, doadores de NO e particularmente a oxidantes exógenos. Em conjunto, estes resultados permitem concluir que o fenótipo neointimal é um fenótipo de estresse oxidativo acentuado e persistente, mesmo em condições de cultura celular, e que este estresse oxidativo decorre pelo menos em parte da ativação do complexo NADPH oxidase no contexto de uma adaptação à resposta celular integrada ao estresse. Estes dados indicam novas perspectivas no entendimento dos mecanismos envolvidos na fisiopatologia redox da neoíntima, podendo suscitar o desenho de intervenções terapêuticas racionais. / Formation of a neointimal layer is the hallmark of most vascular diseases, such as atherosclerosis and restenosis after angioplasty. Neointimal cells display an undifferentiated noncontractile smooth muscle phenotype with marked extracellular matrix secretion. Their origin can be multiple. Among factors that govern the (de)differentiation, proliferation and migration of neointimal cells, there is evidence for a key role of redox processes, but the underlying mechanisms are unclear. Advancing the knowledge about such redox mechanisms has been difficulted by the absence of a reproducible method of neointimal cell culture. The objectives of this work are: 1) To develop a model of neointimal cell culture, in which cells are harvested from rabbit iliac arteries 14 days after overdistention balloon injury. 2) To assess the redox status in such neointimal cells and the possible enzymatic source of reactive oxygen, with emphasis in the NAD(P)H oxidase complex. 3) To investigate the expression of endoplasmic reticulum stress markers and their corralation with redox status. 4) To investigate the effects of proapoptotic stimuli such as serum deprivation, endoplasmic reticulum stressors and particularly the exogenous administration of nitric oxide in viability and redox status of neointimal cells. Our results show that it is possible to harvest and cultivate neointimal cells after balloon injury. The neointimal cells in culture, even after several passages, exhibit increased indexes of oxidative stress. Oxidative stress in such cells is associated with increased activation of the vascular NAD(P)H oxidase complex. Contrarily to what was observed in healing arteries harvested from in vivo rabbits, markers of ER stress did not show any change when compared with primary smooth muscle cells kept in similar conditions. Oxidative stress response was increased after NADPH oxidase agonists; in particular, exposure to exogenous nitric oxide markedly increased superoxide radical production in neointimal cells. Cell viability curves showed increased sensitivity to ER stressors, NO donors and, particularly, exogenous oxidants. Therefore, the neointimal phenotype is a phenotype of intrinsic sustained oxidative stress even after several passages in culture. Such oxidative stress is due at least in part to activation of the NAD(P)H oxidase complex in the context of adaptation to an integrated stress response. This data provide new perspectives to understand redox mechanisms associated with neointimal pathophysiology and can lead to development of rational therapeutic interventions.

The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.

The subject of the Master's Thesis is the design activity of architect Dušan Samo Jurkovič for Josef and Cyril Bartoň from Dobenín in Nové Město nad Metují. In a separate chapter, she first introduces the chateau in Nové Město nad Metují in its construction development and also introduces the urban and cultural context of the city in its individual historical periods. In other chapters she exhibits the personality of Dušan Samo Jurkovič and his work before the rebuilding of the chateau in Nové Město nad Metují, as well as the assignment given to Jurkovič in Nové Město. The core of the work is an analysis of Jurkovič's interventions in the chateau. Their further interpretation is used for the independent processing of Jurkovič's buildings in Peklo, a restaurant and tourist cottages. The concluding chapter deals with the evaluation of Jurkovič's work in Nové Město nad Metují and an overview of further development of his architectural work and his work in the preservation of monuments. The work includes a pictorial supplement. Key words: Dušan Samo Jurkovič; Vernacular architecture; Nové Město nad Metují; Arts and Crafts; Art Nouveau; Modern Architecture

The study deals with the reconstruction and appropriate functional use of the castle in Hrušovany nad Jevišovkou. The castle is conveniently located in the city center but is in a state of emergency and can not be used at present. The design offers a new use of the historical premises of the castle and the whole area. The goal is to make the castle available to local residents and tourists.

Traumatic brain injury, peripheral neuropathies, and other neurodegenerative diseases exhibit diverse clinical manifestations but are connected by their underlying trigger, axonal degeneration. These diseases cause extensive morbidity and mortality worldwide, as treatments are palliative and no curative treatments exist. SARM1 has recently emerged as a therapeutic target for these diseases as knockdown prevents axonal degeneration and ameliorates disease prognosis. Later, it was shown that SARM1 hydrolyzes NAD+ in response to degenerative stressors. Given that NAD+ supplementation delays axonal degeneration, we expect therapeutically targeting SARM1 will be efficacious for neurodegenerative diseases. However, the design of SARM1 therapeutics is limited by the dearth of knowledge surrounding its NAD+ hydrolase activity and active structural state. Illuminating this black box has been hindered by technical difficulties in obtaining pure active protein. To circumvent these issues, I began by studying SARM1 in lysates. I synthesized truncated constructs and developed three different assays, which enabled me to characterize the kinetic activity. I also established a high–throughput screening pipeline to identify inhibitors and screened >4,000 compounds. Recently, I identified additives (i.e., PEG and citrate) that activate SARM1 by ~2,000–fold, making it feasible to study the purified protein. I found that the additives enhance activity by inducing SARM1 to form a multimeric precipitate. To further interrogate the role multimerization plays in activity, I performed detailed mutagenesis and cell culture studies. The insights from this thesis have aided in our understanding of this elusive enzyme and provided strategic direction for future SARM1 investigation and drug development.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme of the NAD salvage pathway starting from nicotinamide. Cancer cells have an increased demand for NAD due to their high proliferation and DNA repair rate. Consequently, NAMPT is considered as a putative target for anti-cancer therapies. There is evidence that AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) become dysregulated during the development of hepatocellular carcinoma (HCC). Here, we investigated the effects of NAMPT inhibition by its specific inhibitor FK866 on the viability of hepatocarcinoma cells and analyzed the effects of FK866 on the nutrient sensor AMPK and mTOR complex1 (mTORC1) signaling. Results: FK866 markedly decreased NAMPT activity and NAD content in hepatocarcinoma cells (Huh7 cells, Hep3B cells) and led to delayed ATP reduction which was associated with increased cell death. These effects could be abrogated by administration of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. Our results demonstrated a dysregulation of the AMPK/mTOR pathway in hep- atocarcinoma cells compared to non-cancerous hepatocytes with a higher expression of mTOR and a lower AMPKa activation in hepatocarcinoma cells. We found that NAMPT inhibition by FK866 signifi- cantly activated AMPKa and inhibited the activation of mTOR and its downstream targets p70S6 kinase and 4E-BP1 in hepatocarcinoma cells. Non-cancerous hepatocytes were less sensitive to FK866 and did not show changes in AMPK/mTOR signaling after FK866 treatment. Conclusion: Taken together, these findings reveal an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of hepatocarcinoma cells and suggest NAMPT inhibition as a po- tential treatment option for HCC.

Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation.

Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a secondary 13-C effect in the hydride transfer step. This phenomenon is also applicable to the modified enzyme with NAD as the substrate.