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Polyamines are polycationic molecules that perform many functions essential to cell growth and proliferation. These compounds can be synthesized inside the cell or taken into the cell exogenously. Many tumor types have been shown to contain elevated polyamine levels and an activated polyamine transporter (PAT) for importing exogenous polyamines. Thus, the PAT represents a potential target for anti-cancer strategies. To date, the mechanism of polyamine uptake into cells of multicellular eukaryotes is poorly understood, primarily because the only PAT proteins that have been identified are in the unicellular organisms Escherichia coli, Saccharomyces cerevisiae, and the protozoan parasite Leishmania major. In order to identify a PAT, in a higher eukaryote, a novel Drosophila imaginal disc epithelial development assay was employed. In this assay, development of the imaginal disc epithelium is inhibited by polyamine-toxin conjugates with results (PAT selectivity and toxicity) similar to those shown in vertebrate Chinese Hamster Ovary (CHO) cells. Using the Gene Ontogeny database at Fly Base, 39 candidates for a Drosophila PAT were identified based on their putative function as cation transport proteins or polyamine transport proteins. Twenty of the candidate genes were selected for further analysis based on their expression in imaginal discs and availability of strains carrying mutations in these genes. Imaginal discs dissected from the candidate mutant strains were tested for defects in polyamine uptake following exposure to polyamine-toxin conjugates. Mutations in two genes, Orct2 and CG9413, conferred increased resistance to the polyamine-toxin in the epithelial assay, as would be expected for imaginal disc cells defective in polyamine transport. Therefore, these two genes represent candidates for the polyamine transporter in a multicellular eukaryote

This research has investigated the gene expression of protein disulfide isomerase (PDI; EC 5.3.4.1) and thioredoxin (Trx) in the marine macroalga Ulva fasciata Delile in response to hypersaline (90‰) for 1 h. 90‰ induced H2O2 accumulation, reflecting the occurrence of oxidative stress. The contents of free and protein-bound SH were increased by 90‰. Trx transcripts increased in response to 90‰. PDI transcripts and enzyme activities increased in response to 90‰. H2O2 accumulation under 90‰ condition was increased by putrescine (Put) but decreased by spermidine (Spd) and spermine (Spm). By treatment of spermidine and spermine, the contents of free SH was increased and the contents of protein-bound SH decreased, showing that spermidine and spermine can increase free SH against oxidative stress. The gene expression and activity of PDI were further increased by Spd and Spm. Overall, the gene expression of PDI and Trx were responded to 90‰ for 1 hour and were adjusted protein¡VSH in polyamines treatment.

The effects of polyamines, indole-3-acetic acid ( IAA ) and gibberellic acid ( GA3 ) on root elongation in radish ( Raphanus sativus L. cv. Luh Chin ) were studied. Incubation of radish seedlings in spermine or spermidine at 0.01 mM for 1hour, and then transferred to deionized water for 24 hours at 25¢J in the dark promoted root elongation as compared with concentration at 0.1 or 2 mM. When roots were treated with spermine or spermidine at 1 mM for 5 minutes, and then transferred to deionized water for 24 hours, root length increased significantly compared with controls. However, root length reduced gradually with increasing treatment times. Putrescine did not affect root elongation when treated in the same manner as spermine or spermidine. Exogenous spermidine synthesis inhibitor ( cyclohexylamine ) at 0.01, 0.1, 1 or 2 mM to the roots inhibited root elongation. The inhibition of root elongation was parallel to cyclohexylamine doses. Root length increased when spermine at 1 mM plus IAA at 10¡Â¹ ¡Ñ 6 nM was applied for 1 hour, and then transferred to deionized water for 24 hours compared with spermine at 1 mM plus IAA 10¡Â² or 1¡Ñ 6 nM. Root length was longer when treated with spermidine at 1 mM plus IAA at 10¡Â²¡Ñ 6 nM than at 10¡Â¹ or 1¡Ñ 6 nM. Root treated with spermine at 1 mM plus GA3 at 10¡Â²¡Ñ3 £gM Resulted in a longer root than treated with spermine at 1 mM plus GA3 at 10¡Â¹ or 1¡Ñ 3 £gM. Roots treated with spermidine at 1 mM plus GA3 at 10¡Â²¡Ñ3 £gM promoted root elongation. However, any treatments of spermine or spermidine in combination with IAA or GA3 significantly reduced the root length when compared with controls. Furthermore, either IAA or GA3 could not restore the inhibitory effects of root elongation caused by spermine or spermidine treatment at 1 mM for 1 hour and then transferred to deionized water for 24 hours. Endogenous spermidine and spermine contents after exogenous spermine plus GA3 treatment increased by increasing GA3 concentrations. But endogenous spermidine and spermine contents was the least in spermine 1 mM plus IAA 10¡Â¹ ¡Ñ 6 nM treatment. However, endogenous spermidine contents after exogenous spermidine plus IAA or GA3 application reduced significantly when compared with controls. But there is no significant difference of spermidine content between different exogenous IAA doses. In contrast, spermidine content maintained at a high level in spermidine at 1 mM plus GA3 at 10¡Â²¡Ñ3 £gM as compared with other spermidine plus GA3 combinations. However, endogenous spermine contents were not affected by exogenous spermidine plus IAA or GA3.

The effluent quality from mining & processing operations is monitored to ensure that maximum allowable limits are not exceeded. Recently, copper concentration levels in the effluent discharge flows of a copper and nickel mining company in Ontario have indicated increasing trends. A chemical particular to the problem is use of diethylenetriamine (DETA) in the process. Adsorption tests were conducted to investigate the ability of various adsorbents to remove and retain copper complexed with DETA and triethylenetetramine (TETA) in solutions. The tests were divided into two sections: gangue adsorbents (silica and pyrrhotite) and high capacity adsorbents (natural bentonite, peat, zeolite Y and zeolite ZSM-5). Pyrrhotite as a sulphide gangue had a greater adsorption capacity than silica for the concentration range studied. At 1 ppm initial concentration, over 80% of copper chelate was removed by minus 400 mesh pyrrhotite compared to 72% of the same size silica. Freundlich and Langmuir isotherm models of adsorption are applicable. However, the Langmuir adsorption isotherm was found to more closely represent the experimental data with a maximum adsorption capacity of 129.9 μg/g for copper complexed with DETA on pyrrhotite. For the high capacity adsorbents, natural bentonite, zeolite Y and peat each worked well at removing the copper chelates. Zeolite Y had the highest capacity for copper chelates and a maximum adsorption capacity of 55.9 mg/g. Freundlich and Langmuir adsorption isotherm models were studied with the Langmuir isotherm model more closely representing the experimental data. iii Studies were also conducted on the effect of temperature. This led to a thermodynamic analysis of adsorption and estimation of activation energies. The standard free energies estimated for adsorption of copper chelated on adsorbents studied were nearly always negative, typically varying from around -2 kJ/mol to -7 kJ/mol with increasing temperature. The activation energy was found to be highest for the natural bentonite system suggesting a strong adsorption (e.g. 40.5 kJ/mol for CuTETA). Desorption experiments on the peat indicated very poor reversal for the process, confirming that the adsorption of copper chelates on high capacity adsorption was indeed very strong. Settling experiments indicated copper chelates were highly effective as coagulants on bentonite. / Thesis (Master, Mining Engineering) -- Queen's University, 2013-12-25 15:00:39.553

Arabidopsis thaliana contains two genes annotated as methylthioadenosine nucleosidases (MTN): MTN1, At4g38800 and MTN2, At4g34840. This enzyme activity hydrolyzes the methylthioadenosine (MTA) produced by nicotianamine (NA), polyamine (PA), and ethylene biosynthesis to methylthioribose (MTR) within the Yang cycle. Comprehensive analysis of the mtn1-1mtn2-1 mutant line with 14 % residual MTN activity revealed a complex phenotype that includes male and female infertility and abnormal vascular development. Based on metabolite profiling, mtn1-1mtn2-1 has a reduced NA content, altered PA profiles with higher putrescine (Put) and lower spermidine (Spd) and spermine (Spm) levels, disrupted metal ion profiles, and abnormal auxin distribution. The modeling of Arabidopsis PA synthases developed by comparison with the crystal structures of human Spd and spermine synthases complexed with MTA suggests that Arabidopsis PA synthases are product inhibited by MTA. Thus, these pleiotropic mutant phenotypes possibly are the result of one metabolite directly inhibiting numerous pathways. By creating and analyzing a series of mutants and transgenic lines with moderate levels of MTN activity the complex phenotype of mtn1-1mtn2-1 was dissected in order to determine the fundamental trait associated with MTN deficiency. Two double mutants were identified by crossing single T-DNA mutants, and an artificial micro RNA (amiRNA) line was generated by transforming mtn1-1 with amiRNA specific to MTN2. The T-DNA double mutants, mtn1 4mtn2-1, and mtn1-1mtn2-5 had 98 % and 28 % MTN activity, respectively, whereas the amiRNA line has 16 % MTN activity. The growth, development, and metabolite analysis of these mutants revealed that their delayed bolting, correlated with an increased number of leaves, was the common trait observed across all lines. Xylem proliferation defects and increased number of vascular bundles per unit area were shared in all lines except mtn1 4mtn2-1. Based on these results, auxin distribution is proposed as the key target of the accumulated MTA that results from MTN deficiency. The infertility related to MTN-deficiency was restored by supplying 100 ??M of Spd to the mtn1-1mtn2-1 seedlings over 14 days. The data presented in this thesis reveals two potential links that work synergistically to recover fertility in this mtn1-1mtn2-1 line. Based on a detailed analysis of the female gynoecia morphology, transcript, hormone and metabolite profiles, it is proposed that the Spd partially reverses the mutant phenotypes through the recovery of auxin distribution and /or vascular development. Interestingly, the Spd effect seems to be transgenerational: they give rise to plants that are genotypically mtn1-1mtn2-1 but phenotypically WT over generations. Taken together, all of the results suggest that MTN-deficient mutants provide the potential for unraveling the molecular mechanism associated with nicotianamine, polyamines, auxin, and vascular development with respect to enhancing the efficiency of nutrient use and yields in plants.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-04-10T17:17:19Z No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-04-10T17:17:27Z (GMT) No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) / Made available in DSpace on 2015-04-10T17:17:27Z (GMT). No. of bitstreams: 1 Tayane Gonçalves Fernandes Efeito....pdf: 3094368 bytes, checksum: 87dc6aa455511d7e1892b6398cc286a4 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A giardíase é uma doença causada pelo protozoário flagelado Giardia lamblia, e sua sintomatologia é caracterizada pela eliminação de fezes esteatorréicas, dores abdominais e náuseas. Segundo o CDC estima-se que há cerca 1,2 milhões de casos por ano de giardíase, acometendo principalmente crianças em idade escolar. Atualmente, o tratamento da giardíase é realizado principalmente pelo uso do fármaco da família dos 5-nitromidazóis, metronidazol (Flagyl®), secnidazol e tinidazol em particular. Estes são confrontados em casos de resistência clínica causada pelo frequente uso inadequado do medicamento e/ou abandono do tratamento. Além disso, o metronidazol pode apresentar efeito carcinogênico em longo prazo em humanos. Desta forma, novos estudos com análogos e/ou inibidores de poliaminas podem levar à elucidação dos mecanismos de ação envolvidos, favorecendo o estabelecimento de novos regimes terapêuticos mais seguros e eficazes. Em nosso trabalho, foram testadas as substâncias ciclohexilamina (CHA) e o metronidazol que são produtos sintéticos, com o objetivo de avaliar os seus efeitos na proliferação celular, caracterização dos moduladores do metabolismo de poliaminas, avaliação nas mudanças no potencial redox e elucidação de seus possíveis mecanismos de ação nos trofozoítos de Giardia lamblia. Foi realizada uma avaliação da proliferação celular na presença de CHA para trofozoítos de Giardia lamblia, onde observamos que a substância demonstrou ter ação siginficativa apresentando um efeito dosedependente. Observamos que os trofozoítos de G. lamblia apresentam uma inibição significativa do crescimento em presença de concentrações milimolares do CHA, cujo IC50 em 72 horas foi de 1,646 mM. Ao avaliar a produção de lipoperóxidos nos trofozoítos foi observado o possível papel do CHA como promotor de estresse oxidativo neste parasito. Ao realizar microscopia eletrônica de varredura (MEV) os trofozoítos apresentaram morfologias completamente irregulares em diferentes concentrações da CHA, com internalização do disco adesivo, sendo corroborado com os resultados da microscopia eletrônica de transmissão (MET) que mostram o processo de encistamento seguido de necrose celular. Esses resultados indicam que a CHA é possível candidata para o uso terapêutico contra a giardíase. / Giardiasis is a disease caused by the flagellate protozoan Giardia lamblia, and its symptomatology is characterized by steatorrhea, abdominal pain and nausea. According to the CDC, an estimate number of 1.2 million cases of giardiasis happen every year, affecting especially schoolchildren.Nowadays, giardiasis treatment is based on drugs from the 5-nitroimidazole family, particularly metronidazole (Flagyl), secnidazole and tinidazole. Those drugs are indiscriminately used by the population, and it's not uncommon to find them causing clinical resistance due to inappropriate utilization and/or tratment abandon. Besides that, metronidazole can present longterm carcinogenic effect in humans. Thus, new studies with analogs and/or polyamines inhibitors can lead to the clarification of the drugs action mechanis, favouring the establishment of new, safer and more efficient therapeutic regimens.Our work tested cyclohexylamine (CHA) and metronidazole, wich are synthetic products, in order to evaluate their effects on cell proliferation and on changes in redox potential, characterize polyamines metabolism modulator and describe their possible action mechanisms on Giardia lamblia trophozoites. We evaluated Giardia lamblia trophozoites cell proliferation in the presence of CHA; it was observe that the substance shows significant action, presenting dose-dependent effect. We also observed that G. lamblia trophozoites presented significant growth inhibition when exposed to millimolar concentrations of CHA - its IC50 in 72 hours was 1,646mM. When assessed the lipoperoxides production in trophozoites, we observed a possible role of CHA as an oxidative stress promoter in the parasite.Under Scanning Electron Microscopy, trophozoites showed completely irregular morphologies in different CHA concentrations, with internalization of the adhesive disc; this results are corroborated by the Transmission Electron Microscopy results, wich showed the process of encystment followed by cell necrosis. This makes CHA a possible candidate for therapeutic use against giardiasis.

Malaria, a disease transmitted by female mosquitoes, has plagued the world for many centuries. The disease is associated with high mortality rates, severe poverty, and economic burden. These are factors which hamper effective eradication of the disease. Drug resistant forms of the parasite have caused increasing concerns and questioned the longevity of current effective antimalarials. Efforts are therefore aimed at the identification and exploitation of essential parasite proteins as potential drug targets. The polyamine pathway of Plasmodium falciparum is an exploitable pathway which contains two distinct, chemically validated drug targets; a bifunctional PfAdoMetDC-ODC protein and PfSpdSyn. These enzymes ensure intricate regulation of polyamine production and the pathway contains various distinctive features which could be selectively targetable from the mammalian counterpart pathways. However, inhibition of polyamine production through the use of specific enzyme inhibitors has revealed various compensatory responses that negate the efficacy of these inhibitors. An account is given of the metabolomic fluctuations in the parasite during inhibition of polyamine biosynthesis. From co-inhibited P. falciparum extracts, it could be demonstrated that the characteristic growth-arrest coincided with the depletion in spermidine, the metabolic product of PfSpdSyn. The co-inhibition strategy therefore emphasised the importance of spermidine biosynthesis by PfSpdSyn. Moreover, adenosyl-related metabolite levels were not disrupted during polyamine depletion, supporting the notion that these metabolites are intricately recycled within the parasites. The identified metabolic compensatory mechanisms have further potential for exploitation, and can strategically be combined with polyamine biosynthesis inhibition to ensure parasitic attenuation. In addition, several novel inhibitors were previously computationally identified, based on a dynamic receptor-based pharmacophore model of PfSpdSyn. The in vitro inhibiting activity of these compounds was determined against PfSpdSyn. Results from the in vitro experiments supported the in silico predictions, and emphasized the supportive role of pharmacophore modelling has for the identification of novel inhibitors. The research contributed in understanding parasitic polyamine metabolite regulation, and will aid in the future optimization of therapeutic strategies, aimed at exploitation of the polyamine pathway as a potential antimalarial drug target. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Biochemistry / unrestricted

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13344号 / 論農博第2887号 / 新制||農||1079(附属図書館) / 学位論文||R2||N5251(農学部図書室) / (主査)教授 小川 順, 教授 木岡 紀幸, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.