This dissertation thesis is concerned with the topic of socioeconomic demography in the Zdar nad Sazavou district. The first part is devoted to literary research. It provides readers not only with a definition of the term demography, a survey of demographics but also with division of demographical structure of the population from different perspectives: sex, age, education and marital status. Then, there are described demographical processes such as birthrate, miscarriage rate, death rate, marriage rate, migration and economical activity. The practical part focuses on the development of the number of inhabitants in the Zdar nad Sazavou district in the years 1991 2015 with the prediction for five forthcoming years. The Zdar nad Sazavou district is described in detail from the perspective of demographical structure according to sex, age and education. There are presented the most important indexes: economic activity in the district and the analysis of commuting people. At the end of the thesis, a cluster analysis is made according to demographical, economical and business level.

The subject of the diploma thesis is based on the specific intention of the town of Žďár nad Sázavou for the reconstruction of the original nursery school at Okružní street to the community center. The diploma thesis follows the analytical part of the previous semester, which examined the possibilities, risks and potential of the city. The main starting point is the work with the existing building - reconstruction and extension of the original kindergarten building. Compared to the conventional new "green meadow" construction, this assignment emphasizes the use of existing values, the concentration and strengthening of the existing city as opposed to the contemporary colonization of the free landscape. The design of a building modification of an existing building must also be based on the perception of the continuity of the development, in particular the constructional nature of the building, so that the newly designed "layer" of development does not become a denial of the previous layers.

Reconstruction and completion of the community center in Ždár nad Sázavou. The aim of the project is to create a multifunctional community center with operational and administrative facilities. On the basis of a specific intention of the contracting authority, the following building program for individual community center operations was set up.

The subject of the diploma thesis is based on the specific intention of the town of Žďár nad Sázavou for the reconstruction of the original nursery school at Okružní street to the community center. The diploma thesis follows the analytical part of the previous semester, which examined the possibilities, risks and potential of the city. The main starting point is the work with the existing building - reconstruction and extension of the original kindergarten building. Compared to the conventional new "green meadow" construction, this assignment emphasizes the use of existing values, the concentration and strengthening of the existing city as opposed to the contemporary colonization of the free landscape. The design of a building modification of an existing building must also be based on the perception of the continuity of the development, in particular the constructional nature of the building, so that the newly designed "layer" of development does not become a denial of the previous layers.

Abiotic stresses are constantly rising and pose a very high risk to global agricultural productivity and food security. Some plants have evolved several innate pathways for defense against these stresses. Hence, understanding stress signaling pathways can help develop crop plants with higher stress tolerance. The salicylic acid-mediated signaling pathway is important in plants experiencing biotic and abiotic stresses. In previous studies, SABP2-Interacting Protein (SIP-428) has been shown to be a negative regular of plant growth under abiotic stress. This study aimed to investigate the roles of SIP-428 in the ROS signaling of tobacco plants. We investigated transgenic RNAi-silenced lines of SIP-428 and wild-type tobacco plants for the activities of guaiacol peroxidase and catalase enzymes in Mannitol and NaCl-stressed plants for 7 and 14 days. Our results showed that SIP-428 plays a significant role in ROS signaling in Mannitol and NaCl-stressed plants via the activities of guaiacol peroxidase.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24475号 / 医博第4917号 / 新制||医||1062(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Enzymatic pathways have evolved over billions of years to carry out essential cellular processes and ensure the survival of their host species. These reaction pathways rely on the interconnectedness of multiple enzymes and substrate, encouraging cross-talk and, at times, competition. In many cases, enzymes require the assistance of a diffusible secondary biomolecule, known as a cofactor, to participate in catalytic reactions. This network of reactions is unfavorable when trying to optimize the production of a specific product. In order to circumvent surrounding reactions, researchers have been engineering orthogonal enzymatic pathways that operate independently from endogenous reactions within a cell. Orthogonal pathways can be created by utilizing biomimetics molecules; most enzymes have not naturally evolved affinity and activity with these are non-canonical cofactors. Nicotinamide adenine dinucleotide (NAD(H) and nicotinamide adenine dinucleotide 2’-phosphate (NADP(H)) are vital cofactors that participate in redox reactions within cells. NAD(P)(H) have been the target of enzymatic research for several decades due to their extensive involvement in reactions across species and their utility in the biotechnology industry. Creation of orthogonal pathways dependent on NAD(P)(H) analogs has massive potential in various industries, such as biofuels and biopharmaceuticals. Nicotinamide mononucleotide (NMN(H)) is a precursor molecule in the biosynthesis of NAD(H); it currently exists within cells but, in general, does not participate as a cofactor. Nicotinamide adenine dinucleotide 3’-phosphate (3’-NADP(H)) is another analog that closely resembles NAD(P)(H) for which most enzymes have not evolved natural affinity and activity. Computation and structural inspection techniques were used in an attempt to engineer formate dehydrogenase from Candida boidinii (CbFDH) for activity with the non-canonical cofactors NMN(H) and 3’-NADP(H). Amino acid positions proximal to the NAD(H) binding site were input into a PyRosetta algorithm, which then outputted a list of recommended mutations ranked by their Rosetta energy scores. Structural alignment and visual inspection were also used to design mutations. The mutations were recombinantly expressed, and the purified enzymes were assays with NAD+, NADP+, NMN+ and 3’-NADP+. None of the designed single mutations led to CbFDH activity gain with NMN+ to any meaningful degree; however, various mutations led to the removal of NAD+ activity. A strength of PyRosetta was identifying key mutations that would lead to activity removal. The single mutants D195A and D195G attained the largest specific initial rates with 3’-NADP+ under the screening assay conditions. Kinetics parameters of a simplified ordered bi bi model were calculated for these mutants. Double mutants were created in an attempt to further enhance activity. The double mutations resulted in decreased activity but enhanced the specificity for 3’-NADP+ over NAD+. To complete the 3’-NADP(H) enzymatic cycle, a non-specific cofactor oxidizer, xenobiotic reductase A (XenA), was expressed and assayed with 3’-NADPH. It was found that XenA is able to oxidize 3’-NADPH back to 3’-NADP+. The avirulence factor AvrRxo1 from a rice plant pathogen was explored since it specifically catalyzes NAD+ phosphorylation at the 3’ position. The AvrRxo1 gene was expressed in LysY/IQ competent E. coli cells and it was found that the presence of AvrRxo1 caused a longer lag phase, but the bacteria were later able to recover. Co-expressing AvrRxo1, XenA, and D195A CbFDH has the potential to create an orthogonal pathway depending on biosynthesized 3’-NADP(H) in vivo. Another in vivo non-canonical cofactor source is NAD(H)-capped RNA, which have recently captured researchers’ attention. NAD+-RNA was synthesized using the polymerase chain reaction, and it was shown that D195A and D195G CbFDH were able to reduce the NAD+ cap.

The NAD glycohydrolase (NADase) from Bungarus fasciatus venom was purified over 1000-fold to electrophoretic homogeneity through a 3-step procedure which included affinity chromatography on Cibacron Blue agarose. The enzyme exhibited a broad pH profile with the optimum range between 7-8. Studies on the substrate specificity of B. fasciatus venom NADase demonstrated that alterations in the purine ring were less pronounced then alterations in the pyridinium moiety of NAD. Product inhibition studies indicated nicotinamide to be a noncompetitive inhibitor with a K_i = 1.4 mM and ADP-ribose to be a competitive inhibitor with a K_i =0.4 mM. The purified enzyme was inactivated by both 2,4-pentane-dione and Woodward's Reagent K suggesting the involvement of a lysine and carboxyl group in the catalytic process. In contrast to other known NADases, the snake venom enzyme did not self-inactivate. The purified B. fasciatus venom NADase catalyzed a transglycosidation reaction (ADP-ribose transfer) with a number of acceptor molecules. The functioning of a variety of substituted pyridine bases as acceptor molecules was demonstrated through the formation of the corresponding NAD analogs. The enzyme also catalyzed the transfer of ADP-ribose to aliphatic alcohols (methanol to hexanol, inclusive) and a positive chainlength effect was observed in the functioning of these acceptors. Kinetic studies of transglycosidation reactions were consistent with the partitioning of an enzyme-ADP-ribose intermediate between water and nucleophilic acceptors as has been proposed in earlier studies of mammalian NADases. The partitioning of this intermediate between water and pyridine bases can be correlated with the basicity of the ring nitrogen of the pyridine derivative. The K_i of pyridine bases in the hydrolytic reaction did not equate to the K_m of these bases in the pyridine base exchange reaction suggesting two forms of the NADase with varying affinity for the pyridine bases. This implys the pyridine base exchange reaction to be more complicated than originally proposed. / Ph. D.

No / Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer’s glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.