Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

Repo-Man is a chromatin-associated PP1 targeting subunit that coordinates chromosome re-organisation and nuclear envelope reassembly during mitotic exit. At the onset of mitosis, Repo-Man association with the chromosomes is very dynamic; at anaphase, Repo-Man targets to the chromatin in a stable manner and recruits PP1 to de-phosphorylate histone H3 at Thr3, Ser10 and Ser28. Previous studies have suggested that CDK1 and AuroraB are the kinases responsible for the inactivation of the complex and for its dispersal at the onset of mitosis respectively. We have previously shown that the binding of Repo-Man to PP1 is decreased in mitosis and we have identified a region adjacent to the RVTF motif that contains multiple mitotic phosphosites (RepoSLIM). This region is conserved only in another PP1 targeting subunit: Ki-67. In order to understand the importance of this region for the complex formation and stability, we have conducted mutational analyses on several residues, and addressed their contribution towards Repo-Man chromosome targeting and PP1 binding in vivo. We have identified new sites in Repo-Man that, when phosphorylated, contribute to the weakening of the binding between Repo-Man and PP1. Interestingly, our results also indicate that several kinases are involved in the mitotic regulation of the complex. We have also identified Lamin A/C as a Repo-Man substrate and introduced a new model for Lamin A/C regulation at interphase. Furthermore, we identified Repo-Man as a marker of malignancy in tripe egative breast cancer, which controls cell movement and levels of important oncogenic markers Aurora A and C-Myc, and propose Repo-Man/PP1 complex as a therapeutic target for the treatment of triple negative breast cancer through the newly identified RepoSLIM.

Orientadores: Hiroshi Aoyama, Marcio Andre Miranda / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T01:06:42Z (GMT). No. of bitstreams: 1 Camargo_CamiladeAndrade_M.pdf: 1153945 bytes, checksum: d090e6ed4e436a5029c6e36de7786df1 (MD5) Previous issue date: 2007 / Resumo: Os flavonóides são fitocompostos polifenólicos, caracterizados quimicamente como heterosídeos flavônicos. Apresentam diversas atividades biológicas devido às suas propriedades antioxidantes e habilidades em modular a atividade de diversas enzimas ou receptores celulares, tornando-os responsáveis pelo efeito protetor contra doenças relacionadas ao sistema cardiovascular; certas formas de câncer e doenças de fotossensibilidade e envelhecimento. As fosfatases ácidas, enzimas que hidrolisam ésteres fosfatos em meio ácido, encontram-se amplamente distribuídas no organismo. Estas enzimas são importantes no catabolismo de diversas substâncias, acreditando-se que a alteração da atividade destas enzimas esteja relacionada com modificações induzidas por drogas e por várias doenças. As transaminases são enzimas hepáticas cujo nível aumenta quando há lesão das células hepáticas (hepatócitos) provocada por qualquer tipo de agressão, como vírus, consumo excessivo de álcool ou drogas. O acetaminofeno é uma droga frequentemente usada como analgésico e antipirético. O dano hepático causado pelo uso frequente ou exagerado do acetaminofeno é comum hoje em dia. A manutenção correta dos sistemas metabólicos hepáticos é de grande importância para a manutenção da saúde. Desta forma, o estudo do flavonóide quercetina como possível protetor dos efeitos hepatotóxicos provocados pelo acetaminofeno pode ser muito promissor. O objetivo do presente trabalho foi estudar os efeitos preventivos e terapêuticos, in vivo, do flavonóide quercetina sobre as atividades de fosfatases ácidas (total, tartarato-resistente e de baixa massa molecular relativa) e de transaminases glutâmica oxalacética (TGO) e glutâmica pirúvica (TGP) no fígado de camundongos, tratados ou não com acetaminofeno. Para o desenvolvimento deste trabalho foi realizado um tratamento agudo (24 horas), de camundongos machos da linhagem Swiss, com quercetina e acetaminofeno, utilizando-se óleo de milho ou nujol como veículo para o flavonóide. A dosagem da TGO e da TGP confirmou que o acetaminofeno pode realmente ser considerado hepatotóxico, quando administrado ou ingerido em grandes concentrações no organismo. A quercetina, dissolvida em óleo mineral, reverteu a hepatotoxicidade induzida pelo acetaminofeno, nos tratamento terapêutico e, em conjunto com acetaminofeno. A quercetina dissolvida em óleo de milho, no tratamento preventivo, pode não ser a única responsável pela reversão da hepatotoxicidade causada pela administração do acetaminofeno, uma vez que quando se substituiu o veículo utilizado, óleo de milho pelo óleo mineral, este efeito não foi mais observado. Comparando-se os resultados obtidos entre fosfatases e transaminases pode-se observar que a atividade da FATR, no tratamento com nujol, demosntra uma semelhança muito grande com os resultados observados nos gráficos das atividades das transaminases e provavelmente pode se considerada uma marcadora de dano hepático. O alfa-tocoferol, presente no óleo de milho, pode ter exercido um efeito antioxidante, e mascarado o efeito protetor da quercetina e a hepatotoxicidade do acetaminofeno. Este estudo foi importante para mostrar que a atividade dos flavonóides no organismo vivo pode ser influenciada por diversos fatores, como: a natureza do veículo utilizado na sua administração, ordem de administração e o tempo de permanecia no organismo / Abstract: Flavonoids are polyphenolic phytocompounds chemically characterized as flavonic heterosides. These compounds present several biological activities due to their antioxidant properties and hability to modulate the activities of enzymes or cellular receptors, making them responsible for the protector effect against diseases related to the cardiovascular system, certain forms of cancer, photosensibility diseases and aging. Acid phosphatases, enzymes that hydrolyze phosphate esters at acid medium, are largely distributed in the organisms. These enzymes are important in the catabolism of several compounds and could be related to the modifications induced by drugs and diseases. Transaminases are hepatic enzymes which levels increase in consequence of hepatic cells (hepatocytes) lesions provoked by agressions such as virus and excessive alcohol and drug consumption. Acetaminophen is a drug frequently used as analgesic and antipyretic. It is common the hepatic damage caused by the frequent or exaggerated use of acetaminophen. The correct maintenance of the hepatic metabolic systems is of great importance for the health. In this way, the study of the flavonoid quercetin as a possible protector of the hepatotoxic effects provoked by acetaminophen seems to be promising. The objectif of the present work was to study the in vivo effects of the flavonoid quercetin on the activities of acid phosphatases (total, tartrate-resistant and relative low molecular weight) and of transaminases glutamic oxalacetic (GOT) and glutamic pyruvic (GPT) in mice livers treated with acetaminophen. To develop this work, it was performed an acute treatment (24 hours) of Swiss male mice, with quercetin and acetaminophen, using corn oil or nujol as a vehicle for the flavonoid. The determination of GOT and GPT activities confirmed that acetaminophen can be considered hepatotoxic, when administered or ingested in great amount. In the therapeutic treatments, when applied with acetaminophen, quercetin, solubilized in mineral oil, reverted the acetaminophen-induced hepatotoxicity. In the preventive treatment, quercetin, solubilized in corn oil, might not be the only responsible for the reversibility, since this effect was no more observed when the vegetal oil was replaced by the mineral oil. The results with FATR, in the treatment with nujol, showed great similarity with the results obtained with transaminases, suggesting that this class of phosphatases could be considered as markers of hepatic damage. The alphatocopherol, present in the corn oil, could be exerting an antioxidant effect, masking the protector effect of quercetin and the acetaminophen hepatotoxicity. The importance of the present study was to stress that the in vivo flavonoids activities can be influenced by several factors, such as, the nature of the vehicle used in the administration, the order of administration and the retention time in the organism / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientadores: Hiroshi Aoyama, Claudio Martín Jonsson / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:07:04Z (GMT). No. of bitstreams: 1 Dantzger_Miriam_M.pdf: 2456182 bytes, checksum: ce7e4445d55ff9a2572a6ad4f3dea90d (MD5) Previous issue date: 2010 / Resumo: Daphnia similis é um microcrustáceo da família dos cladóceros que se encontra amplamente distribuído em ambientes de água doce. Devido a sua natureza cosmopolita e sensibilidade, o seu uso em estudos de ecotoxicidade, como biomarcador, é recomendado em protocolos nacionais e internacionais. Agentes poluentes como os agrotóxicos, efluentes industriais e metais são diariamente despejados nos corpos d'água, influenciando sistemas locais e podendo causar efeitos toxicológicos aos organismos aquáticos. Tendo em vista a necessidade da normalização de biomarcadores enzimáticos, nossa proposta foi analisar a atividade enzimática de fosfatases de D. similis como uma alternativa.Especificamente, nossos objetivos foram: a) analisar o efeito de poluentes agrícolas e metais nas atividades in vitro de fosfatases ácidas e alcalinas de D. similis, com o intuito de selecionar a enzima mais sensível à ação dos poluentes; b) realizar estudos mais detalhados in vitro com a fosfatase mais sensível na presença de poluentes préselecionados; c) avaliar a atividade in vivo desta enzima perante concentrações subletais de poluentes provenientes de ensaios de toxicidade aguda. Dessa forma, foram analisados os efeitos in vitro de vários agentes poluentes (metais e pesticidas) nas atividades da fosfatase ácida total (FAT) e fosfatase alcalina. A FAT apresentou maior alteração em relação à fosfatase alcalina quando exposta aos poluentes in vitro, sendo que, dentre estes, os metais apresentaram efeitos mais significativos. Dos metais testados, Al3+, Se3+ e Mo6+, exibiram maior efeito inibitório, enquanto permetrina e Cd2+ apresentaram efeito ativador. A energia de ativação da reação catalisada pela FAT, usando-se p-nitrofenilfosfato (pNPP) como substrato, foi reduzida de 13 para 10 Kcal.mol-1 na presença de 0,5mM de Cd2+. Na reação catalisada pela FAT, os valores de IC50 (concentração do metal que promove 50% de inibição da atividade enzimática) obtidos para Al3+, Se3+ e Mo6+, foram, respectivamente: 1,23 mM, 0,54 mM e 0,9 µM. A inibição da FAT mostrou-se do tipo não competitiva para oAl3+ e do tipo competitiva para Se3+ e Mo6+, com os seguintes valores de constante de inibição (Ki): 0,9 mM para o Al3+; 0,62 mM para o Se3+ e 1,32 µM para o Mo6+. Nos ensaios in vivo de toxicidade aguda 48h com D. similis, a permetrina apresentou maior toxicidade comparada aos demais poluentes, sendo que a ordem de toxicidade, em mM, foi: Permetrina (0,000056) > Cd2+ (0,00139) > Se4+ (0,004) > Mo6+ (0,024) > Al3+(0,072). A ordem de inibição da atividade da FAT de D. similis exposta ao EC50 (48h) assemelhouse à obtida nos ensaios de toxicidade aguda (Cd2+ > permetrina > Al3+ > Mo6+), sugerindo uma relação entre alteração de atividade enzimática com a toxicidade dos poluentes ao modelo biológico em estudo. Nos ensaios de toxicidade aguda também foram estudados os efeitos da mistura binária dos metais Al3+, Cd2+, Mo6+ e Se4+. Observou-se que somente Al3+ + Cd2+ (S = 0,588) e Al3+ + Mo6+ (S = 0,951) apresentaram efeito sinérgico sobre D. similis, constatado pelo valor de S<1; nas mesmas condições, a mistura Al3+ + Se4+ não mostrou nenhuma alteração significativa. A ordem de inibição da atividade da FAT para a concentração de EC50 e seus respectivos percentuais foram: Al3+ + Cd2+ (90%) > Al3+ + Mo6+ (78%) > Se4+ + Cd2+ (72%) > Mo6+ + Cd2+ (32%). Contraditoriamente, a mistura Se4+ + Mo6+ promoveu uma ativação de 50% na atividade enzimática nos mesmos níveis de toxicidade analisados para as demais misturas. Esses resultados evidenciam a necessidade de complementar os testes de toxicidade aguda com mistura de poluentes a partir da utilização de biomarcadores enzimáticos, tais como a FAT. Concluiu-se que a FAT, através da alteração de sua atividade in vivo, pode ser utilizada como biomarcadora de toxicidade em nível letal (EC50), para os poluentes permetrina e Al3+ e, em níveis subletais, para Cd2+ e Se4+, podendo-se predizer o impacto ambiental / Abstract: Daphnia similis, a microcrustacean of the cladoceran's family, is widely found in freshwater environment. Because of its cosmopolitan nature and sensitivity, this organism is recommended by national and international protocols as a biomarker in ecotoxicity analyses. Agrochemicals, industrial effluents and metals are daily released in the rivers, impairing local living systems and causing toxicological effects on aquatic organisms. In order to contribute with enzymatic biomarkers normalization, our purpose was to evaluate D. similis phosphatases activities in the presence of several pollutants. Specifically, our aim was: a) to analyze in vitro effects of metals and agrochemicals on D. similis alkaline and acid phosphatases activities, in order to select the more pollutants-sensitive enzyme; b) to perform more detailed in vitro studies with those more sensitive enzyme and selected pollutants; c) to analyze the in vivo activity of this enzyme at sub-lethal levels of pollutants, derived from acute toxicity assays. In this way, in vitro effects of several pollutants (metals and pesticides) were evaluated on total acid phosphatase (TAP) and alkaline phosphatase activities. Our results showed that TAP presented higher alterations in relation to alkaline phosphatase when exposed to in vitro pollutants, from which the metals showed more remarkable effects. The metals Al3+, Se3+ and Mo6+ exhibited higher inhibitory effects, whereas permethrin and Cd2+ presented activator effects. The activation energy of the reaction catalyzed by TAP, by using p-nitrophenylphosphate as substrate, was decreased from 13 to 10 kcal.mol-1, in the presence of 0,5mM Cd2+. IC50 (concentration of metal that promotes 50% of enzyme inhibition) values for Al3+, Se3+ and Mo6+ were determined to be: 1.23 mM, 0.54 mM e 0.9 µM, respectively, in the TAP catalyzed reaction. The TAP inhibition was of non-competitive type for Al3+, and competitive for Se3+ and Mo6+, with inhibition constant (Ki) values ranging 0.9 mM, 0.62 mM and 1.32 µM, respectively. In the 48 hours acute toxicity assays with D. similis, permethrin presented higher effect in relation to the other pollutants, with the following toxicity order (mM): Permethrin (0.000056) > Cd2+ (0.00139) > Se4+ (0.004) > Mo6+ (0.024) > Al3+ (0.072). The inhibition order of D. similis TAP activity exposed to 48 hours EC50 was similar to the acute toxicity values (Cd2+ > permethrin > Al3+ > Mo6+), suggesting a relationship between enzymatic activity alteration and the pollutants toxicity. Further, we decided to evaluate the effects of binary metals mixtures of Al3+, Cd2+, Mo6+ and Se4+ on the acute toxicity assays, it was observed that only Al3+ + Cd2+ (S = 0.588) and Al3+ + Mo6+ (S = 0.951) exhibited synergic effects on D. similis, evidenced by the S<1 values; at the same conditions the mixture Al3+ + Se4+ did not show significant alterations. The TAP activity inhibition order for EC50 concentration and its respective percentuals were: Al3+ + Cd2+ (90%) > Al3+ + Mo6+ (78%) > Se4+ + Cd2+ (72%) > Mo6+ + Cd2+ (32%). On the other hand, the mixture Se4+ + Mo6+ presented 50% enzyme activation at the same toxicity levels analyzed for the other mixtures. These results emphasize the need to complement acute toxicity tests with pollutants mixtures, using enzymatic biomarkers, such as TAP. In conclusion, TAP, through the alteration of its in vivo activity, could be used as a lethal level toxicity biomarker for the permethrin and Al3+, and at sub-lethal levels for Cd2+ and Se4+, predicting possible environmental impact / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Fernanda Ramos Gadelha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-11T13:57:30Z (GMT). No. of bitstreams: 1 MoralesNeto_Raphael_M.pdf: 499454 bytes, checksum: de4a1750fcb9184b1cfcef4f5e0cc3ee (MD5) Previous issue date: 2008 / Resumo: Trypanosoma cruzi, o agente etiológico da doença de Chagas, exibe um notável grau de heterogeneidade estrutural e funcional, o qual pode modular sua patogenicidade, sobrevivência e adaptação. Em eucariotos superiores, mecanismos de fosforilação e desfosforilação são fundamentais para a regulação de uma ampla variedade de eventos celulares. Por essa razão, um melhor entendimento da atividade de fosfatases em T. cruzi pode ser útil para a identificação de potenciais alvos para o desenvolvimento de terapias mais específicas. Neste trabalho, analisou-se o perfil das fosfatases, em homogenatos de duas cepas de T. cruzi, Tulahuen 2 e Y. A atividade de fosfatases foi determinada espectrofotometricamente utilizando-se o p-nitrofenilfosfato (p-NPP) como substrato. Na cepa Tulahuen 2, que apresenta maior resistência ao estresse oxidativo gerado pelo peróxido de hidrogênio (H2O2), foi observada uma atividade de fosfatase sete vezes maior quando comparada a cepa Y. Em relação ao pH ótimo a Tulahuen 2 mostrou uma expressiva atividade em pH 4,0, a qual foi aumentada na presença de Mg2+ exógeno em uma ampla faixa de pH (5,0 - 8,0). A cepa Y, embora em menores níveis, exibiu o mesmo perfil sem a adição de Mg2+, apresentando na presença desse íon um pico de atividade em pH 7,0. A adição de Ca2+ ao meio reacional afetou significativamente a cepa Tulahuen 2 em pH 8,0 e inibiu a atividade das fosfatases na cepa Y nos pHs 7,0 e 8,0. Inibidores clássicos de fosfatase ácida, bem como inibidores de proteína tirosina fosfatase e fosfatase alcalina, mostraram padrões distintos de inibição em ambas as cepas, reforçando a heterogeneidade entre os isolados de T. cruzi / Abstract: Trypanosoma cruzi, the etiological agent of Chagas'disease, displays a remarkably high degree of both, structural and functional intraspecific heterogeneity, which could modulate its pathogenicity, survival and adaptability. In higher eukaryotes, phosphorylation and dephosphorylation are fundamental pathways that regulate a wide variety of cellular events. Therefore, a better understanding of phosphatases activities in T. cruzi could be useful to identify potential targets for the development of a more specific therapy. In this work, we analysed the phosphatase activity profile in T. cruzi homogenates of two strains (Tulahuen 2 and Y). The phosphatase activities was determined spectrophometricaly using p-nitrophenylphosphate (p-NPP) as substrate. In the strain more resistant to the oxidative stress generated by H2O2, Tulahuen 2, it was observed a seven-fold higher phosphatase activity when compared to the other strain. In relation to optimum pH and substrate specificity, Tulahuen 2 had an expressive acid phosphatase activity (optimum pH at 4.0), which was even higher in the presence of Mg2+ and had a broad range (pH 5-8). Y strain, although at lower levels, showed the same activity profile in the presence or absence of Mg2+. The presence of calcium showed significant effect on the Tulahuen 2 strain only at pH 8.0 and had an inhibitory effect on phosphatase activity in the Y strain at pHs 7-8. Classical inhibitors of acid phosphatases, as well as inhibitors of phosphotyrosine phosphatase, showed distinct patterns of effects in phosphatases from both strains / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

RESUMO As fosfatases alcalinas sao enzimas que participam da atividade catalitica na hidrolise de ésteres fosfóricos. Nesta Dissertação, investigou-se a sua adsorção em monocamadas de Langmuir e também em filmes do tipo Langmuir-Blodgett (LB) de fosfolipídios em diferentes temperaturas. Esses sistemas são considerados como modelos para membranas biológicas. O efeito de um dissacarídeo (trealose), bem como as impurezas contidas na amostra do açúcar, foi testado na subfase que suporta a monocamada,. Constatou-se que a trealose nao purificada altera a isoterma superficial de monocamadas do sal de sódio do acido dimiristoilglicerofosfatidico, DMPA, e atua como um bom agente de ligação entre camadas sucessivas do fosfolipídio para a obtenção de filmes LB. Já para trealose purificada nao se observaram esses efeitos. A caracterização das impurezas foi realizada por espectroscopia na região do infravermelho e por absorção atômica, detectando-se a presença de moléculas anfifílicas e de íons metálicos (Na+, K+, Ca2+, Mg2+), respectivamente. Dois tipos de fosfatases alcalinas foram usadas: uma purificada de placa óssea de ratos por extração com tensoativo nâo-iônico, chamada de DSAP e outra purificada de conídia de Neurospora crassa, denominada CNCAP. As interações com o fosfolipídio foram estudas por meio das mudanças nas curvas -A e a adsorção em filmes LB de DMPA/Zn2+ e DMPA/trealose, por meio da técnica de microbalança a cristal de quartzo (QCM). A presença das enzimas nos filmes foi também comprovada pela detecção de suas atividades catalíticas na hidrolise do PNPP (p-nitrofenilfosfato). Para a DSAP, a melhor atividade foi obtida quando a imobilização da proteína ocorreu a baixa temperatura (10º C). / ABSTRACT Alkaline phosphatases (AP) are unspecific enzymes that hydrolyze phosphate esthers. In this Dissertation we have studied the adsorption of two forms of AP in phospholipid Langmuir monolayers and Langmuir-Blodgett films, considered as biomembrane models, at different temperatures. Further, the effect of the presence of a disaccharide, trehalose, as well as some impurities present in its commercial form, in the subphase of the monolayers was tested. We concluded that the use of trehalose without further purification alters the surface pressure-area (Pi-A) curves of the monolayers. In addition, it acts as a good linking agent between successive layers in the Langmuir-Blodgett films of the sodium salt of 1,2-dimirystoyl-sn-glicero-3-phosphate acid, DMPA. In the presence of purified trehalose instead, these effects were not observed. No difference was noticed in the Pi-A curves of DMPA for trehalose concentrations between 10-3 mmol L-1 to 0.1 mmol L-1. The characterization of the impurities by atomic absorption and infrared spectroscopy reveal the presence of amphiphilic compounds as well as electrolytes such as: sodium, magnesium, calcium and potassium Two forms of AP were used: one purified from rat osseous plates using detergent solubilization, named DSAP, and the other from Neurospora crassa conidia, CNCAP. Enzyme interaction with phospholipids was followed by changes in Pi-A curves, and their adsorption on DMPA/Zn2+ or DMPA/trehalose LB films using the quartz crystal microbalance technique, QCM. Their presence within the films was also monitored by catalytic activity assays, using PNPP (p-nitrophenylphosphate) as hydrolysis substrate. For DSAP, the highest enzymatic activity was observed when the immobilization was carried out at low temperature (10o C).

No description available.

PP1-Arch was verified as a protein phosphatase by both acid molybdate extraction and thin layer electrophoresis. Soluble fraction was prepared from <i>Sulfolobus solfataricus</i>, from which PP1-Arch was purified over 1OOO-fold by DE-52 ion-exchange, hydroxyapatite, gel filtration (G- 100), and Mono Q FPLC chromatography. PP1-Arch was identified from the final purified sample by renaturation on an SDS-polyacrylamide gel. The molecular size of PP1-Arch was determined by both gel filtration chromatography and SDS-PAGE as 28 kDa and 33 kDa, respectively, which suggests that PP1-Arch is a monomer. PP1-Arch was found stable at temperatures as high as 90°C. Activation constants for the divalent metal ions Mn²⁺ and Ni²⁺, and the K_m for phosphocasein were determined. Myosin light chain was found to be a substrate for PP1-Arch <i>in vitro</i>. EDTA, Cu²⁺, Zn²⁺, P_i' and PP_i were shown to be inhibitors of PP1-Arch, while many compounds known to affect eukaryotic protein phosphatase activities were found to be without noticeable effect. N-terminal and an internal peptide sequence of the enzyme were obtained. The gene for PP1-Arch was cloned by a combination of "touchdown" PCR and conventional cloning techniques. The PP1-Arch gene was sequenced on both strands, and the sequence was compared with ones from eukaryotes and bacteriophage λ. The sequence homology between PP1-Arch and PP1/PP2A/PP2B suggests that they belongs to the same genetic family. A recombinant plasmid which was derived from pT7-7 was constructed for expression of PP1-Arch. The PP1-Arch gene was expressed in <i>E. coli</i> and the activity of the expressed enzyme was tested and shown to be divalent metal ion-dependent. Formation of inclusion bodies of expressed PP1-Arch was demonstrated. / Ph. D.

The acid phosphomonoesterase activity associated with two major rooting strategies in arctic tundra plants was examined: that of Eriophorum vagina tum, a dominant plant in tussock tundra ecosystems, with its predominantly non-mycorrhizal root system; and that of ectomycorrhizal roots. Eriophorum has phosphatase activity which is evenly distributed along its root surface, has a pH optimum at soil pH (3.5-4.0), and continues at substantial rates at 1 °C. Inorganic phosphorus inhibits activity only 7 to 19%. In addition, Eriophorum has phosphatase activity associated with all the "below-ground" components of its tussock growth form: dead roots, leaf sheaths, and soil. Plants with higher tissue phosphorus growing in soils with higher available phosphate in general had higher live and dead root, leaf sheath, and soil phosphatase activity in both natural and manipulated sites of higher plant productivity. Yearly and seasonal variation sometimes exceeded differences among treatments, suggesting that enzyme activity would not provide a reliable measure of plant or soil phosphorus levels. Experiments with radiolabeled inositol hexaphosphate showed that Eriophorum is able to hydrolyze and absorb inorganic phosphate from an organic phosphate source. A comparison of enzyme hydrolysis rates with inorganic phosphate assimilation rates indicates that organic phosphate hydrolysis may occur as rapidly as inorganic phosphate absorption. Inorganic phosphate released by root surface phosphatase activity could satisfy approximately 65% of the annual phosphate demand of Eriophorum. Phosphatases of two ectomycorrhizal fungi (Cenococcum geophilum and Entoloma sericeum) responded similarly to growth in axenic culture at 2 or 50 micromolar KH₂PO₄ or sodium inositol hexaphosphate: surface Vmax estimates were significantly greater for 2 micromolar- than for 50 micromolar-grown isolates. The presence of constitutive extracellular soluble phosphatase activity resulted in the appearance of inorganic phosphate in media initially supplied only with organic phosphate. The surface acid phosphatase activity of field-collected ectomycorrhizal roots of arctic Salix and Betula, however, did not respond in a consistent way to differences in soil characteristics. Activity differed more among "color types" or fungal types than among sites of different soil characteristics. / Ph. D.

Montréal Trigonix inc. 2018