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Orientador: Celso Eduardo Benedetti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T14:13:17Z (GMT). No. of bitstreams: 1 Lira_NayaraPatriciaVieirade_M.pdf: 3098417 bytes, checksum: 6441392e6b6c275daba6bd89e88cbaf8 (MD5) Previous issue date: 2014 / Resumo: Xylella fastidiosa e Agrobacterium tumefaciens são bactérias fitopatogênicas que infectam, respectivamente, o interior do xilema e de tecidos vasculares de raiz, ambientes cuja tensão de oxigênio é relativamente baixa. Uma vez que Xylella e Agrobacterium são bactérias estritamente aeróbicas, elas apresentam o operon bigR, responsável pela detoxificação do sulfeto de hidrogênio ou gás sulfídrico, um potente inibidor do citocromo c oxidase e respiração aeróbica. O operon bigR codifica cinco proteínas denominadas Blh (Beta-lactamase-like hydrolase), BigR (biofilm growth-associated repressor), um repressor transcricional e regulador do operon, e MP1-3, proteínas que compõem um transportador de membrana. Em trabalho anterior, foi demonstrado que mutantes de Agrobacterium deficientes na produção de Blh acumulavam gás sulfídrico, enquanto mutantes no repressor BigR secretavam mais sulfito, indicando que a proteína Blh convertia gás sulfídrico em sulfito e que este, que também é tóxico, seria exportado pelo complexo MP1-3. Além disso, dados de modelagem molecular indicaram que Blh poderia desempenhar funções de sulfotransferase e dioxigenase de enxofre, uma vez que apresenta os domínios DUF442 (rodanase) e ETHE1 (dioxigenase). A fim de testar tais hipóteses, este trabalho teve como principais objetivos a caracterização enzimática dos domínios DUF442 e ETHE1 da Blh de Xylella e Agrobacterium, como também confirmar interações proteína-proteína entre os componentes do operon bigR. Ensaios de atividade enzimática usando-se proteínas recombinantes purificadas confirmaram a função de dioxigenase de enxofre e de rodanase dos domínios ETHE1 e DUF442, respectivamente. Além disso, verificou-se que ambos os domínios produzem sulfito como produto final da reação, embora atuando em substratos diferentes. Ainda, ensaios de duplo híbrido de levedura mostraram haver inúmeras interações entre as proteínas do operon bigR, mas não entre os dois domínios DUF442 e ETHE1 de Blh que, de acordo com os ensaios enzimáticos, atuam de forma independente. / Abstract: Xylella fastidiosa and Agrobacterium tumefaciens are phytopathogenic bacteria that infect, respectively, the xylem vessels and root vascular tissues, where the oxygen tension is relatively lower. Since Xylella and Agrobacterium are strict aerobic organisms, they use the bigR operon for the detoxification of hydrogen sulfide, a potent inhibitor of cytochrome c oxidase and aerobic respiration. The bigR operon encodes five proteins designated Blh (Beta-lactamase-like hydrolase), BigR (biofilm growth-associated repressor), a transcriptional repressor that regulates the operon, and MP1-3, proteins that act as a membrane transporter. In a previous work, it was shown that Agrobacterium mutants deficient in Blh production accumulated hydrogen sulfide, whereas BigR-deficient mutants secreted sulfite at higher levels than the wild type bacteria, indicating that Blh converted hydrogen sulfide into sulfite, which would be exported by the MP1-3 complex. In addition, molecular modeling indicated that Blh could function as a sulfur transferase and sulfur dioxigenase, since it carries a DUF442 (rhodanese) and ETHE1 (dioxygenase) domains. To test such hypothesis, this work aimed to demonstrate the enzymatic activities of the DUF442 and ETHE1 domains of Blh from Xylella and Agrobacterium, as well as to confirm protein-protein interactions between components of the bigR operon. Enzyme activity assays using the purified proteins confirmed the sulfur dioxygenase and rhodanese activities of the ETHE1 and DUF442 domains, respectively. In addition, it was found that both domains produce sulfite as a final product, although having different substrates. Furthermore, yeast two-hybrid assays showed that many of the bigR operon proteins interact with each other, suggesting the formation of a protein complex. However, no physical interactions were detected between DUF442 and ETHE1 domains, which, according to the enzyme activity assays, act independently. / Mestrado / Microbiologia / Mestra em Genética e Biologia Molecular

A indoleamina 2,3-dioxigenase (IDO) desempenha um papel importante na tolerância materno-fetal devido á sua ação de catabolizar o triptofano e, consequentemente, impedir a proliferação de linfócitos T, que necessitam desse aminoácido para se manter. Hormônios da reprodução também participam do processo de sobrevivência do feto alogênico, como a progesterona que bloqueia o estímulo mitogênico da proliferação de células T, modula a produção de anticorpos, favorece a produção de IL-10, etc. e o estradiol, que pode modular o perfil imune Th1 ou Th2 na gestação, dependendo de sua concentração. Contudo, a existência ou não de correlação da ação da IDO com esses hormônios ou vice-versa ainda encontra-se pouco evidenciada. Desta forma este trabalho verificou a influência da expressão da IDO e às ações de hormônios da reprodução e citocinas no ciclo celular de células oriundas de fetos e placenta de gestação a termo de fêmeas de ratas e camundongos em cultivo. Este estudo foi realizado em complementação a um trabalho anterior, no qual foi realizada uma avaliação da expressão da IDO por citometria de fluxo em células uterinas, de placentas e de embriões de ratas e camundongos fêmeas prenhes e não prenhes que foram mantidas em cultivo e suplementadas com estradiol, progesterona, interferon γ, triptofano e 1-metil-DL- triptofano. A avaliação das fases do ciclo celular foi realizada pela citometria de fluxo. De acordo com os resultados, em relação ao efeito dos tratamentos no comportamento das células no ciclo celular, podemos observar que, em ratas prenhes e não prenhes, ao adicionar estradiol, houve maior predominância das células em fase G1 nos períodos de 4 e 24 horas, bem como no grupo de camundongos fêmeas prenhes. Algumas células uterinas de ratas não prenhes tratadas com estradiol, assim como as tratadas com progesterona e interferon γ progrediram no ciclo para fase de síntese nos tempos de 24 e 48 horas. Com a adição de triptofano podemos notar nos grupos de ratas não prenhes, camundongos fêmeas prenhes e não prenhes, um aumento da quantidade de células na fase G1 nos três períodos analisados. No grupo de ratas prenhes foi observado uma predominância celular em fase G1 nos tempos de 4 e 24 horas, sendo que em 48 horas, as células sofreram fragmentação de DNA. As células que foram suplementadas com 1- metil DL - triptofano + triptofano mantiveram o mesmo comportamento no ciclo celular , se mantendo em fase G1 nos tempos de 4, 24 e 48 horas grupos prenhes e não prenhes de ratas e nos tempos de 4 e 24 horas nos grupos de camundongos fêmeas prenhes e não prenhes, estando com seu DNA fragmentado e em fase de síntese, respectivamente, no tempo de 48 horas. A suplementação dos diversos fatores aos cultivos celulares permitiu observar alterações na dinâmica do ciclo celular, representada principalmente por um aumento de células em fase G1 nos grupos de células placentárias e embrionárias que receberam progesterona, estradiol, interferon γ e triptofano e apresentaram um significativo aumento da expressão de IDO, e que permite inferir que provavelmente a síntese de RNAm esteja correlacionada à produção da IDO. / The indoleamine 2,3-dioxygenase (IDO) plays an important role in maternal-fetal tolerance due to its capacity to catabolize tryptophan and thereby preventing the proliferation of T lymphocytes, necessary for their maintenance. Reproductive hormones are also involved in the process of survival of semi-allogeneic fetus, as progesterone that blocks mitogenic stimulation of T cell proliferation, modulates antibodies production, promotes production of IL-10, etc. and estradiol, that can modulate the Th1 or Th2 immune profile during pregnancy, depending on its concentration. However, there is very few evidences regarding a possible correlation between IDO expression and these hormones; thus this study examined the influence of the expression of IDO and the actions of reproductive hormones and cytokines in the cell cycle of cells derived from fetuses and placenta at term gestation of female rats and mice in culture. This study was conducted as a complement to an earlier work, in which an evaluation was made of the IDO expression by flow cytometry in uterine cells, placentas and embryos of pregnant rats and mice and non-pregnant females that were maintained in culture and supplemented with estradiol, progesterone, interferon γ, tryptophan and 1-methyl-DLtryptophan. The cell cycle evaluation was conducted by flow cytometry. According to the results, regarding the effect of treatments on the behavior of the cells in the cell cycle, it was observed that in pregnant and non-pregnant rats after the addition of estradiol, there is a greater predominance of cells in G1 phase at periods of 4 and 24 hours, that also was noted in the group of pregnant female mice. Uterine cells from non-pregnant female rats treated with estradiol and progesterone as well as treated with interferon γ progressed to the phase of synthesis on the cycle, at 24 and 48 hours. With the addition of tryptophanin the groups of non-pregnant rats, pregnant and non-pregnant mice , an increased amount of cells in the G1 phase were observed in the three periods analyzed. In the group of pregnant rats a predominance of cells in the G1 phase was observed in periods of 4 and 24 hours, and 48 hours, where the cells undergone DNA fragmentation. In pregnant and non-pregnant rats the cells supplemented with 1 - methyl - DL - tryptophan + tryptophan remained at G1 phase in periods of 4, 24 and 48 hours and 4 and 24 hours for cells from pregnant and non-pregnant mice , that show ragmented DNA followed by synthesis at 48 hours period. Supplementation of the various factors to cell cultures allowed to observe dynamic changes in the cell cycle, represented primarily by an increase of cells in G1 phase in groups of embryonic and placental cells that received progesterone, estradiol, interferon γ and tryptophan and showed a significant increase in the expression of IDO, that allows us to infer that the mRNA synthesis is probably correlated to the production of IDO.

Abstract Lysyl hydroxylases (E.C. 1.14.11.4, LHs) have three isoenzymes that are found in humans and mice, and they hydroxylate lysine residues in collagens and other proteins containing collagenous sequences. The hydroxylysines formed are crucial for the intermolecular collagen crosslinks that stabilise collagen fibres, thereby providing the stiffness and stability required by various tissues. In addition, hydroxylysines serve as attachment sites for carbohydrates, whose functions on collagen molecules are not completely understood yet. In humans, lack of LH1 causes Ehlers-Danlos syndrome (EDS) VIA, which is characterised, for example, by severe progressive kyphoscoliosis and muscular hypotonia with joint laxity. Mutations in the LH2 gene are associated with Bruck syndrome, which is characterised by fragile bones with congenital joint contractures. In the present work recombinant human lysyl hydroxylases were produced in insect cells and purified to homogeneity. Limited proteolysis revealed that LHs consist of at least three structural domains. The N-terminal domain plays no role in the lysyl hydroxylase activity, but instead, is responsible for the recently reported glucosyltransferase activity of LH3, and the galactosyltransferase activity reported here for the first time. The LH polypeptide lacking the N-terminal domain is a fully active LH with Km values identical to those of full-length enzyme. In addition, direct evidence is shown that LH2, but not LH1 or LH3, hydroxylates the telopeptide lysine residues of fibrillar collagens. All three recombinant LHs were able to hydroxylate the synthetic peptides representing the helical hydroxylation sites in types I and IV collagens, with some differences in the Vmax and Km values. In addition, all three LHs hydroxylated the collagenous domain of coexpressed type I procollagen chain to similar extend. In this study mouse lines lacking LH3 or LH1 were created and analysed. Unexpectedly, the LH3 null mice died during the embryonal period due to fragmentation of basement membranes. Type IV collagen, one of the major components in basement membranes, aggregates on its way to extracellular space and is absent from the basement membranes making them fragile. This is most probably caused by abnormal processing of type IV collagen due to decreased glucosyltransferase activity of the LH3 null embryos. The first mouse model for human EDS VIA is presented here. The LH1 null mice did not have kyphoscoliosis characteristic of EDS VIA, but showed gait abnormalities due to muscular hypotonia and possible joint laxity, as also seen in EDS VIA patients. In addition, the null mice died occasionally from aortic ruptures. Ultra structural analysis revealed degradation of smooth muscle cells and abnormal collagen fibres even in non-ruptured aortas of LH1 null mice. The hydroxylation of lysine residues and crosslinking in LH1 null mice were also abnormal, as in human EDS VIA patients. The LH1 null mouse line provides an excellent tool for analysing several aspects of human EDS VIA, including muscular hypotonia, abnormalities in collagen fibres and their crosslinking.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine, 2-oxyglutarate, 5-dioxygenase, Plod) catalyzes the hydroxylation of certain lysine residues in collagens and in other proteins with collagenous domains. Three lysyl hydroxylase isoforms have been cloned from human and rat. The importance of lysyl hydroxylase 1 in collagen biosynthesis is demonstrated by the heritable disorder, Ehlers-Danlos syndrome type VI, which is characterized by joint laxity, progressive scoliosis, muscle hypotonia, scleral fragility and rupture of the ocular globe. An alternatively spliced form of lysyl hydroxylase 2 seems to function as a telopeptide lysyl hydroxylase. Lysyl hydroxylase 3 has three enzyme activities, lysyl hydroxylase, hydroxylysyl galactosyltransferase (EC 2.4.1.50), and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activities that have been demonstrated earlier with in vitro experiments. In this thesis study, the cDNAs of mouse lysyl hydroxylase isoforms 1, 2 and 3 were cloned and characterized and the gene structures of lysyl hydroxylase 2, Plod2, and lysyl hydroxylase 3, Plod3, were determined. Mouse lysyl hydroxylase isoforms were found to be highly homologous to the corresponding human isoforms and they were approximately 60% identical with each other. The mouse Plod3 gene has 19 exons as do the human PLOD1 and PLOD3 genes, and mouse Plod2, like the human PLOD2, has 20 exons including one alternatively spliced extra exon. The mouse isoforms were also found to have distinct tissue distributions. Phylogenetic analysis revealed that the lysyl hydroxylase genes have evolved from an ancestral gene through two gene duplication events. Lysyl hydroxylase 3 was demonstrated to be the oldest isoform, which is further supported by the association of glycosyltransferase activities with lysyl hydroxylase 3 and with the only lysyl hydroxylase of Caenorhabditis elegans. The roles of the different enzyme activities of lysyl hydroxylase 3 were determined in vivo by generating three genetically modified lysyl hydroxylase 3 mouse lines. The analysis of these mouse lines demonstrated that lysyl hydroxylase 3 possesses at least lysyl hydroxylase and glucosyltransferase activities in vivo and it functions as the main, if not the only glucosyltransferase during embryogenesis. The absence of lysyl hydroxylase 3 and, especially, its glucosyltransferase activity results in the abnormal glycosylation of type IV collagen, and thus causes a severe basement membrane defect leading to death during early development. By contrast, lysyl hydroxylase activity had no effect on embryonic development, but caused changes in the structure of the epidermal basement membrane and changes in collagen fibril organization and probably in their interactions.

Abstract HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH) are novel members of the 2-oxoglutarate dioxygenase family that play key roles in the regulation of the hypoxia-inducible transcription factor (HIF). They hydroxylate specific proline and asparagine residues in HIF-α, leading to its proteasomal degradation and inhibition of its transcriptional activity, respectively. These enzymes are inhibited in hypoxia, and as a consequence HIF-α becomes stabilized, forms a dimer with HIF-β, attains its maximal transcriptional activity and induces expression of many genes that are important for cell survival under hypoxic conditions. The three HIF-P4Hs and FIH were expressed here as recombinant proteins in insect cells and purified to near homogeneity. All these enzymes were found to require long peptide substrates. The three HIF-P4Hs and FIH acted differently on the various potential hydroxylation sites in the HIF-α isoforms. The HIF-P4Hs acted well on sequences with cores distinctly different from the core motif -Leu-X-X-Leu-Ala-Pro-, suggested based on sequence analysis studies, the alanine being the only relatively strict requirement in addition to the proline itself. Acidic residues around the hydroxylation site also played a distinct role. These results together with those of others provide evidence that there is no conserved core motif for the hydroxylation by HIF-P4Hs. The Km values of the HIF-P4Hs for O2 were slightly above its atmospheric concentration, while the Km of FIH was about one-third of these values but still 2.5 times that of the type I collagen P4H. The HIF-P4Hs are thus effective oxygen sensors, as even small decreases in the amount of O2 affect their activities, while a more severe decrease is required to inhibit FIH activity. Small molecule inhibitors of the collagen P4Hs also inhibited the HIF-P4Hs and FIH but with distinctly different Ki values, indicating that it should be possible to develop specific inhibitors for the HIF-P4Hs and FIH. The HIF-P4Hs were found to bind the iron cosubstrate more tightly than FIH and the collagen P4Hs, and the chelator desferrioxamine was an ineffective inhibitor of the HIF-P4Hs in vitro. Several metals were effective competitive inhibitors of FIH but they were ineffective inhibitors of the HIF-P4Hs. The well-known stabilization of HIF-1α by cobalt and nickel is thus not due to a simple competitive inhibition of the HIF-P4Hs, and is probably at least in part due to HIF-P4H-independent mechanisms. The effective inhibition of FIH by these metals nevertheless indicates that the stabilized HIF-1α is transcriptionally fully active.

Chronic kidney disease (CKD), a reduction in kidney function, has reached pandemic proportions and imposes a major healthcare burden worldwide. A hallmark of CKD is the accumulation of several chemical compounds, called uremic toxins, which inflict systemic and renal-specific damage. Of the known uremic toxins, kynurenine (Kyn) is known to be particularly vasculotoxic and is implicated in several complications of CKD. Indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes the first step in the metabolism of Tryptophan (Trp), regulates immune response to inflammatory cytokines in tissues. IDO plays a role in apoptosis and damage during acute kidney injury (AKI), a transient decrease in kidney function. During metabolism of Trp, IDO generates Kyn, a uremic solute, and therefore IDO may play a role in the brain and kidney damage due to accumulation of Kyn. The objective of the current study was to investigate the role and regulation of IDO in CKD pathology. Studies were performed to determine whether IDO is protective or pathologic and to find how IDO is regulated in the kidney during CKD. IDO in renopathology was examined using murine models of CKD. CKD was induced via a 0.2% adenine-supplemented diet (AD) model for 21 days. IDO regulation was examined using an Indoxyl Sulfate (IS)-specific solute model. Renal function in the IDO+/+ and IDO-/- AD mice was assessed through weekly measurement of blood urea nitrogen (BUN). H&E and Masson’s trichrome stains were used to assess percentages of glomerulosclerosis (GS) and immune infiltration (II), and combined interstitial fibrosis and tubular atrophy (IFTA) score in IDO+/+ and IDO-/- mice with and without CKD. IDO protein concentration in the kidneys of all mice with and without CKD and IDO+/+ IS mice was determined via immunoblotting. Patients with kidney disease suffer from neuropsychological disorders and neurocognitive decline. The effects of uremic solutes on the CNS was examined using immortalized human umbilical endothelial vein cells (HUVEC-TERT), in vitro. Cell proliferation and viability, in the presence of IS, were measured by BrdU and Alamar blue assays, respectively. In both IDO+/+ and IDO-/-, 21 days of AD results in significant deterioration of renal function. The average IFTA score and percentage of II in IDO-/- mice increased with AD compared to ND (p<0.05, p<0.001). IDO expression was seen sporadically in the glomeruli and walls of major vessels in the kidneys of 4d AD IDO+/+ mice, and in the tubules and vessel walls in the kidneys of 14d AD IDO+/+ mice. In IDO+/+ ND mice, endogenous IDO protein expression was undetectable at a signal intensity of 119.86 ± 268.01, whereas IDO+/+ AD mice showed a 370-fold higher level of IDO protein expression compared to IDO+/+ ND (p<0.001). IDO-/- AD IDO protein expression was 9.5-fold higher than in IDO-/- AD (p<0.05). IDO expression was found to be 58-fold higher in IDO+/+ mice with IS treatment (p<0.05). In the IS mice, non-significant trends toward decrease in cellular proliferation and viability with time were also observed (p=ns). IDO is upregulated at the protein level both in a CKD model and directly by the uremic solute, IS. IDO appears to be protective in the kidney during CKD, given the trend toward increased percentage of GS and II in IDO-/- compared to IDO+/+ mice with CKD, though there is little difference seen in total kidney IFTA. IDO upregulation is linked to increased apoptosis. Blocking uremic solute production would therefore prevent IDO protein upregulation and reduce apoptosis, alleviating renal damage during CKD.

Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole source of carbon and energy without formation of any secondary waste product. Catechol-1,2- dioxygenase was isolated from cytosolic fraction of this yeast by the procedure consisting of chromatography on DEAE-Sepharose and gel permeation chromatography on Sephadex G- 100. The catechol-1,2-dioxygenase was purified to homogeneity. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation). The activity profiles at different temperatures showed temperature optimum of 30řC. Kinetic characterizations were studying in different values of pH. The values of Km and Vmax of 0,52 mM and 17,2 nM/min for consumption of catechol, respectively, and 0,34 mM and 12,6 nM/min for formation of cis,cis-muconic acid, respectively, were found at optimum pH of the reaction, pH 7,6.

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第17862号 / 薬博第793号 / 新制||薬||236(附属図書館) / 30682 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 髙倉 喜信, 教授 橋田 充, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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