Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22496号 / 農博第2400号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5276(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

X-linked hypophosphataemia (XLH) is the most common form of inherited rickets that leads to deformities in the lower limbs, poor tooth and skeletal mineralization, and disproportionate short stature in children. In adults, it is often complicated by enthesopathy, an abnormal mineralization of the tendon-bone attachment. Enthesopathy causes pain and stiffness in affected joints, particularly in the knee, hip, and ankle joints. Enthesopathy is reported as one of the most debilitating symptoms in XLH patients. Previous studies showed that entheses from mice with XLH (Hyp) are characterized by enhanced Bone morphogenic protein (BMP) and Indian hedgehog (IHH) signaling. This study aims to investigate the role of GDF5 in the development of enthesopathy in the Hyp mice. The study has two specific aims: (1) to determine if deleting GDF5 in enthesis (scleraxis-expressing (Scx+)) cells affects BMP/IHH signaling in entheses and (2) to determine if deleting GDF5 in Scx+ cells of Hyp mice attenuates XLH enthesopathy. The study hopes to gain a better understanding of role of GDF5 in enthesis maturation and XLH enthesopathy development. This study finds that deleting GDF5 in wild-type mice does not change normal enthesis maturation. However, deleting GDF5 in Hyp mice attenuates enthesopathy as indicated by decreased BMP/IHH signaling in Hyp entheses. / 2026-03-19T00:00:00Z

Abstract Hypericum perforatum L. (St. John’s wort) is a medicinal plant widely utilized for the treatment of depression. The antidepressant activity is mainly attributed to the phenolic compounds hypericins and hyperforins, which also have a wide range of other pharmacologically interesting properties. The biosynthetic routes leading to hypericins and hyperforins are poorly understood, although a polyketide pathway including type III polyketide synthases (PKSs) has been suggested to be involved. Furthermore, a gene called hyp-1 is assumed to attend to the final stages of the hypericin biosynthesis. In the present work, the biosynthesis of hypericins and hyperforins in H. perforatum was further studied by focusing on the elucidation of the precursors and genes involved. The incorporation of isotopically labelled branched-chain amino acids into hyperforins was investigated as well as the possibilities to enhance the production of hyperforins in H. perforatum in vitro cultures by feeding them with amino acid precursors. Furthermore, two novel cDNAs encoding for type III PKSs were isolated from H. perforatum. The functions of these new genes, designated HpPKS1 and HpPKS2, as well as the role of hyp-1 were elucidated by comparing their expression with the levels of hypericins and hyperforins in H. perforatum tissues. The enzymatic activity of the recombinant HpPKS2 protein was also analyzed. To study Hyp-1 at a protein level, a protein extraction method was optimized for tissues of Hypericum species. The results show the incorporation of valine and isoleucine into the acyl side chain of hyperforin and adhyperforin, respectively. Through the biotransformation of the amino acid precursors, it is possible to enhance the levels of adhyperforin, but not hyperforin, in H. perforatum shoot cultures, which demonstrates the tight regulation of the hyperforin biosynthesis. A correlation between HpPKS1 expression and hyperforins was detected in H. perforatum tissues. The localization of HpPKS2 mRNA in dark glands in which hypericins accumulate as well as the octaketide synthase activity of the recombinant HpPKS2 suggest that HpPKS2 is associated with possible co-operating tailoring enzymes in the biosynthesis of hypericins. The presence of both hyp-1 mRNA and Hyp-1 protein in distinct places compared with hypericins in H. perforatum tissues does not support the idea that Hyp-1 would be involved in the biosynthesis of hypericins in dark glands, although mobility of the Hyp-1 protein was shown to be possible. The present thesis extends knowledge about the biosynthesis of hypericins and hyperforins in H. perforatum by providing new candidate genes for their biosynthesis and by identifying precursors for hyperforins. Moreover, new information was obtained about the role of hyp-1 in H. perforatum.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-09-29T18:54:06Z No. of bitstreams: 1 2015 - Peter Sana.pdf: 2426905 bytes, checksum: 4886ee552c348aa742f8706df208997b (MD5) / Made available in DSpace on 2017-09-29T18:54:06Z (GMT). No. of bitstreams: 1 2015 - Peter Sana.pdf: 2426905 bytes, checksum: 4886ee552c348aa742f8706df208997b (MD5) Previous issue date: 2015-04-27 / The work titled as "The educational dimension of the thought and the action D. Adriano Hyp?lito in the social struggles of the Baixada Fluminense during the military dictatorship (1964-1985)", investigated since the formation of the thought of the Catholic Church in Brazil, it's share the colonial, imperial and republican education to date that corresponds to the decision-making by senior ecclesiastical hierarchy, depending on the situation which was driven to coexist in opting for the poorest in society. During the first half of the twentieth century Church, with its conservative nature, seeks the most influential means to ally and so take him a role in active participation of society, whether in the case of formal education means such as schools and seminars, even in the informal means such as the press, radio and television media. The political education now has an importance for the Church in as observing situation in Latin America, it is noticed that the less affluent classes of society suffered from injustice and social inequality. It was in this way that there were some sectors of the Catholic Church, strengthened by the Vatican Council and the Medellin and Puebla conferences, sought more active forms of political activity in the mobilization and politicization of the masses, uniting faith to fight for social justice. In this context that the figure of the Bishop Adriano Mandarino Hyp?lito in the Baixada Fluminense, when in 1966, already observing the political structure set up after the 1964 coup, took over the Nova Igua?u Diocese and joined the movement called Liberation Theology and spent to take a leadership attitude toward the union, social movements, and lay and religious who sought social equality and improvements in the most important sectors of society such as education, health, sanitation, land reform and policy, etc. The participation of Nova Igua?u bishop made the conservative sectors of the Church reacted against his bishopric, as well as the current military government and the economic elite, seeking to end the wave of political awareness of the people, exploited by organizations such as the Christian Communities Base and Neighborhood Friends Movement, these who had the guidance and funding for the Church proposed by Don Adriano de bishopric in line with the situation of struggle and resistance against the military regime and social inequalities and injustices caused by the current system. / No trabalho intitulado como "A dimens?o educativa do pensamento e da a??o de D. Adriano Hyp?lito nas lutas sociais da Baixada Fluminense no per?odo da ditadura militar (1964-1985)", investigamos desde a forma??o do pensamento da Igreja Cat?lica no Brasil, sua participa??o na educa??o colonial, imperial e republicana at? o momento que corresponde ? tomada de decis?o por parte da alta hierarquia eclesi?stica, em fun??o da conjuntura a qual foi impulsionada a coexistir, em optar pelos mais pobres da sociedade. Durante a primeira metade do s?culo XX a Igreja com seu car?ter conservador busca os meios mais influentes para aliar-se e assim tomar para si uma import?ncia na participa??o ativa da sociedade, quer seja no ?mbito dos meios formais de educa??o como escolas e semin?rios, at? nos meios informais como a m?dia impressa, radiof?nica e televisiva. A forma??o pol?tica passou a ter uma import?ncia para a Igreja na medida que observando conjuntura da Am?rica Latina, percebe-se que as classes menos abastadas da sociedade sofriam com a injusti?a e desigualdade social. Foi desta maneira que surgiram alguns setores da Igreja Cat?lica que, fortalecidos pelo Conc?lio Vaticano e as Confer?ncias de Medell?n e Puebla, buscaram formas mais ativas de atua??o pol?tica na mobiliza??o e politiza??o das massas, unindo f? ? luta por justi?a social. Neste contexto que surgiu a figura do Bispo Dom Adriano Mandarino Hyp?lito na Baixada Fluminense, quando no ano de 1966, j? observando a estrutura pol?tica montada ap?s o golpe de 1964, assumiu a Diocese de Nova Igua?u e aderiu o movimento denominado Teologia da Liberta??o e passou a ter uma postura de lideran?a diante dos movimentos sindicais, sociais, e dos leigos e religiosos que buscavam igualdade social e melhorias nos setores mais importantes da sociedade como a educa??o, sa?de, saneamento b?sico, reforma agr?ria e pol?tica, etc. A participa??o do bispo de Nova Igua?u fez com que os setores conservadores da pr?pria Igreja reagissem contra seu bispado, assim como o governo militar vigente e a elite econ?mica, buscando terminar com a onda de conscientiza??o pol?tica do povo, instrumentalizada por organiza??es como as Comunidades Eclesiais de Base e Movimento Amigos de Bairro, estes que tiveram a orienta??o e financiamento pela Igreja proposta pelo bispado de Dom Adriano em conson?ncia com a conjuntura de luta e resist?ncia contra o regime militar e as desigualdades e injusti?as sociais provocadas pelo sistema vigente.

In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases. The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW). A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 722

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<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>