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Stanovení hodnoty podniku ZAPA beton a.s. / Valuation of ZAPA beton, a.s.Jiroutová, Monika January 2012 (has links)
This Master thesis deals with the valuation of ZAPA beton, a. s. to the date August 16, 2012. As a valuation method was used the earnings valuation method based on discounted free cash flow to the firm (FCFF). Important parts of Master thesis are analysis of external environment, analysis of building industry and financial analysis of the company. Process of valuation was implemented on the base of well-prepared financial plan and quantification of necessary input variables such as discount rate and rate of growth. Final part includes sensitivity analysis, for which was used Monte Carlo simulation. This simulation helps to determine main risk factors which do have the most significant influence on the final value of company.
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Estudo genético da interação entre FtsZ e o modulador de divisão ZapA em Bacillus subtilis / Genetic Study of the interaction between FtsZ and the division modulator ZapA in Bacillus subtilisBisson Filho, Alexandre Wilson 01 April 2009 (has links)
A citocinese bacteriana é controlada por diversas proteínas que se agrupam em um complexo chamado divisomo. O cerne do divisomo é constituído por FtsZ, uma proteína homóloga à tubulina eucariótica, que se auto-associa formando uma estrutura chamada anel Z. O anel Z serve como arcabouço e recruta diversas outras proteínas componentes do divisomo para o sítio onde o septo será sintetizado na célula. A formação do anel Z é modulada por proteínas que se ligam diretamente a FtsZ e regulam a sua auto-associação, tanto induzindo como inibindo a sua polimerização. Apesar de muitos destes moduladores de FtsZ já serem conhecidos, muito pouco se sabe sobre o mecanismo pelo qual eles controlam a estruturação do anel Z in vivo. O objetivo do presente trabalho foi estudar a interação entre FtsZ e um modulador de divisão, a proteína ZapA, da bactéria gram-positiva Bacillus subtilis. Para isso construímos uma biblioteca de mutantes de ftsZ por \"Error Prone PCR\", com aproximadamente 1 substituição por cópia de ftsZ e contendo um total de 1x105 clones. A partir dessa biblioteca, utilizamos duas triagens genéticas para identificar mutantes incapazes de interagir com ZapA. Na primeira estratégia, selecionamos 12 mutantes de FtsZ resistentes à superexpressão de uma forma tóxica de ZapA, que bloqueia a divisão, causando filamentação e morte das células. Surpreendentemente, apesar destes mutantes serem insensíveis ao efeito de ZapA, ensaios citológicos mostraram que nenhum deles perdeu a interação com ZapA. Como as mutações foram mapeadas nas vizinhanças do sítio catalítico e de polimerização de FtsZ, e como a maioria delas confere resistência cruzada aos efeitos de outros moduladores de FtsZ, suspeitamos que elas afetassem a estabilidade do polímero de FtsZ e, consequentemente, o comportamento do anel Z. Essas suspeitas foram confirmadas em ensaios de FRAP e cálculos de proporção de FtsZ no anel Z, indicando que os mutantes formam um anel Z mais estável que o normal. Como não obtivemos mutantes que perderam a interação com ZapA na primeira triagem, aplicamos a biblioteca em uma segunda estratégia de triagem genética, procurando um mutante de FtsZ que voltasse a interagir com um mutante de ZapA que não se liga mais a FtsZ (ZapAN62A). Esta estratégia de ganho de função identificou um candidato, FtsZE91V , que, tanto por critérios genéticos como citológicos, voltou a interagir com ZapAN62A. Apesar do mutante FtsZE91V mostrar-se capaz de restaurar a interação com ZapAN62A, ele não afetou a interação com ZapA selvagem, segundo nossos ensaios de microscopia de fluorescência e viabilidade. O mutante FtsZE91V, mapeia na hélice H3 de FtsZ. Esta hélice está exposta na superfície de FtsZ (compõe um dos lados da molécula de FtsZ) de uma maneira compatível com a idéia de que ela seria importante para interações laterais entre polímeros de FtsZ. Nossos resultados apontam, portanto, que a hélice H3 deve ser o sítio de interação para ZapA em FtsZ. / The bacterial cytokinesis is ruled by a number of proteins that constitute the divisome complex. FtsZ, a homologue of eukaryotic tubulin, is the main component of the divisome and self-associates in a structure named Z ring. The Z ring works as a scaffold and recruits the other components of divisome, establishing itself where the septum will be synthesized in the cell. Some of these proteins interact directly with FtsZ and control self-association, promoting polymerization or preventing it. Although there have been discovered many of FtsZ modulators, little is known about the mechanisms that control the formation of the Z ring in vivo. The aim of this work was study de interaction between FtsZ e one of its division modulators, ZapA protein, on Bacillus subtilis grampositive bacteria. We created a mutagenized ftsZ plasmid library by error prone PCR, which contained 1,0x105 transformants and exhibited a mutation rate of one substitution per ftsZ copy. The library was transformed into a modified Bacillus subtilis strain and we performed two genetic screenings to select cells with FtsZ mutants incapable of interacting with ZapA. In first strategy, we selected 12 resistant ftsZ mutants for a toxic ZapA overexpression, that blocked division and caused filamentation and cell death. Surprisingly, although these mutants were insensitive to ZapA effect, cytological assays showed that none of them lost interaction with ZapA. As the substitutions were mapped around the catalytic and interaction site of FtsZ structure and showed resistance to other modulators, we suspected that the mutations were affecting the polymer stability of FtsZ and, consequently, the behavior of Z ring. This hypothesis was confirmed by FRAP experiments and by calculations of FtsZ proportions in Z ring, pointing out that the mutants form more stable Z rings. As we didnt\' find mutants that lost their ZapA´s interaction, we applied our library in a second genetic screen, looking for mutants that return to interact with a ZapA mutant (ZapAN62A) that doesn´t bind to FtsZ anymore. This gain of function strategy identified one candidate, FtsZE91V, which returns to interact with ZapAN62A in our genetic and cytological assays. Although the mutant FtsZE91V showed itself capable to interact with ZapAN62A, that didn´t affect the interaction with wild type ZapA by our fluorescent microscopy and viability assays. The substitution E91V was mapped on H3 helix of FtsZ structure. This helix is exposed on FtsZ surfaces (on FtsZ´s lateral side), being compatible with the idea that lateral interaction is important in FtsZ polymers. So, we concluded that helix H3 is the binding site of ZapA in FtsZ.
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Estudo genético da interação entre FtsZ e o modulador de divisão ZapA em Bacillus subtilis / Genetic Study of the interaction between FtsZ and the division modulator ZapA in Bacillus subtilisAlexandre Wilson Bisson Filho 01 April 2009 (has links)
A citocinese bacteriana é controlada por diversas proteínas que se agrupam em um complexo chamado divisomo. O cerne do divisomo é constituído por FtsZ, uma proteína homóloga à tubulina eucariótica, que se auto-associa formando uma estrutura chamada anel Z. O anel Z serve como arcabouço e recruta diversas outras proteínas componentes do divisomo para o sítio onde o septo será sintetizado na célula. A formação do anel Z é modulada por proteínas que se ligam diretamente a FtsZ e regulam a sua auto-associação, tanto induzindo como inibindo a sua polimerização. Apesar de muitos destes moduladores de FtsZ já serem conhecidos, muito pouco se sabe sobre o mecanismo pelo qual eles controlam a estruturação do anel Z in vivo. O objetivo do presente trabalho foi estudar a interação entre FtsZ e um modulador de divisão, a proteína ZapA, da bactéria gram-positiva Bacillus subtilis. Para isso construímos uma biblioteca de mutantes de ftsZ por \"Error Prone PCR\", com aproximadamente 1 substituição por cópia de ftsZ e contendo um total de 1x105 clones. A partir dessa biblioteca, utilizamos duas triagens genéticas para identificar mutantes incapazes de interagir com ZapA. Na primeira estratégia, selecionamos 12 mutantes de FtsZ resistentes à superexpressão de uma forma tóxica de ZapA, que bloqueia a divisão, causando filamentação e morte das células. Surpreendentemente, apesar destes mutantes serem insensíveis ao efeito de ZapA, ensaios citológicos mostraram que nenhum deles perdeu a interação com ZapA. Como as mutações foram mapeadas nas vizinhanças do sítio catalítico e de polimerização de FtsZ, e como a maioria delas confere resistência cruzada aos efeitos de outros moduladores de FtsZ, suspeitamos que elas afetassem a estabilidade do polímero de FtsZ e, consequentemente, o comportamento do anel Z. Essas suspeitas foram confirmadas em ensaios de FRAP e cálculos de proporção de FtsZ no anel Z, indicando que os mutantes formam um anel Z mais estável que o normal. Como não obtivemos mutantes que perderam a interação com ZapA na primeira triagem, aplicamos a biblioteca em uma segunda estratégia de triagem genética, procurando um mutante de FtsZ que voltasse a interagir com um mutante de ZapA que não se liga mais a FtsZ (ZapAN62A). Esta estratégia de ganho de função identificou um candidato, FtsZE91V , que, tanto por critérios genéticos como citológicos, voltou a interagir com ZapAN62A. Apesar do mutante FtsZE91V mostrar-se capaz de restaurar a interação com ZapAN62A, ele não afetou a interação com ZapA selvagem, segundo nossos ensaios de microscopia de fluorescência e viabilidade. O mutante FtsZE91V, mapeia na hélice H3 de FtsZ. Esta hélice está exposta na superfície de FtsZ (compõe um dos lados da molécula de FtsZ) de uma maneira compatível com a idéia de que ela seria importante para interações laterais entre polímeros de FtsZ. Nossos resultados apontam, portanto, que a hélice H3 deve ser o sítio de interação para ZapA em FtsZ. / The bacterial cytokinesis is ruled by a number of proteins that constitute the divisome complex. FtsZ, a homologue of eukaryotic tubulin, is the main component of the divisome and self-associates in a structure named Z ring. The Z ring works as a scaffold and recruits the other components of divisome, establishing itself where the septum will be synthesized in the cell. Some of these proteins interact directly with FtsZ and control self-association, promoting polymerization or preventing it. Although there have been discovered many of FtsZ modulators, little is known about the mechanisms that control the formation of the Z ring in vivo. The aim of this work was study de interaction between FtsZ e one of its division modulators, ZapA protein, on Bacillus subtilis grampositive bacteria. We created a mutagenized ftsZ plasmid library by error prone PCR, which contained 1,0x105 transformants and exhibited a mutation rate of one substitution per ftsZ copy. The library was transformed into a modified Bacillus subtilis strain and we performed two genetic screenings to select cells with FtsZ mutants incapable of interacting with ZapA. In first strategy, we selected 12 resistant ftsZ mutants for a toxic ZapA overexpression, that blocked division and caused filamentation and cell death. Surprisingly, although these mutants were insensitive to ZapA effect, cytological assays showed that none of them lost interaction with ZapA. As the substitutions were mapped around the catalytic and interaction site of FtsZ structure and showed resistance to other modulators, we suspected that the mutations were affecting the polymer stability of FtsZ and, consequently, the behavior of Z ring. This hypothesis was confirmed by FRAP experiments and by calculations of FtsZ proportions in Z ring, pointing out that the mutants form more stable Z rings. As we didnt\' find mutants that lost their ZapA´s interaction, we applied our library in a second genetic screen, looking for mutants that return to interact with a ZapA mutant (ZapAN62A) that doesn´t bind to FtsZ anymore. This gain of function strategy identified one candidate, FtsZE91V, which returns to interact with ZapAN62A in our genetic and cytological assays. Although the mutant FtsZE91V showed itself capable to interact with ZapAN62A, that didn´t affect the interaction with wild type ZapA by our fluorescent microscopy and viability assays. The substitution E91V was mapped on H3 helix of FtsZ structure. This helix is exposed on FtsZ surfaces (on FtsZ´s lateral side), being compatible with the idea that lateral interaction is important in FtsZ polymers. So, we concluded that helix H3 is the binding site of ZapA in FtsZ.
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Design and Syntheses of Potential Drugs Based on GABA(A) Receptor PharmacophoresClement, Ella Chow 11 August 2005 (has links)
Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Ã ). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4-benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2-ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (ï ¡-methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the ï ¡-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the ï ¢-carbons. / Ph. D.
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