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Regulation of gene expression is known to be a critical factor involved in proper development, responses to environmental cues, metabolism, energy conservation, and disease. Gene expression is regulated at several levels including transcription, mRNA splicing, post translational modification, and the rate of protein degradation. The developmental control of <i>alkaline phosphatase</i> (alp) in <i>Dicytostelium</i> has provided a focal point for the study of gene regulation at the level of <i>de novo</i> synthesis. The localization of <i>alkaline phosphatase</i> (alp) expression during development was characterized by fusing the 5' flanking sequence to the <i>lacZ</i> reporter and using an <i>in situ</i> β-galactosidase staining method. The localization of </i>lacZ</i> expression corresponds with that of the endogenous ALP enzyme suggesting that <i>alp</i> is regulated at the level of transcription. In order to identify temporal regulatory elements within the <i>alp</i> promoter a series of 5' and internal promoter deletions were generated and fused to the <i>lacZ</i> reporter. The data from these promoter deletion constructs indicated a regulatory element within the -683 to -468 bp sequence that is required for normal expression of <i>alp</i> during development. A series of small internal and 5' promoter deletions were designed within the -683 to -468 bp regulatory sequence. The results from these promoter deletion-reporter gene fusions suggested a DNA regulatory element is located within a 26-bp sequence beginning at the -620 bp site. The function of <i>cis</i>-acting regulatory elements were evaluated using the electromobility shift assay (EMSA) to identify sequence specific DNA-protein interactions on the <i>alp promoter</i>. We report the characterization of three DNA-binding activities with the 20% ammonium sulfate (AS) slug nuclear fraction. These DNA-binding activities appear to be related as they all require magnesium or calcium for effective binding to the <i>alp</i> promoter. Interestingly, the DNA-binding proteins appeared to interact with a GT-rich sequence that contained a G-box binding factor (GBF) consensus element. Additionally, a DNA-binding activity observed in the 80% AS slug nuclear extract was characterized and sequentially purified using conventional and affinity chromatography techniques. The DNA-binding protein was identified as TFII, a protein that was previously identified during the investigation of <i>glycogen phosphorylase-2 (gp2)</i> regulation. A comparison of the <i>alp</i> and <i>gp2</i> probes used to identify TFII suggests a DNA-binding site, ACAATGN₈₋₁₂CACTA. The ability of TFII to bind specifically with the promoter of two functionally different genes suggests that it may regulate the temporal and/or spatial expression of several <i>Dictyostelium</i> genes. / Ph. D.

Li Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 182-202). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.v / List of Abbreviations --- p.vi / CONTENTS --- p.viii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Alkaline phosphatase --- p.1 / Chapter 1.1.1 --- Distribution --- p.1 / Chapter 1.1.2 --- Molecular characteristics of alkaline phosphatase --- p.4 / Chapter 1.1.3 --- Properties of alkaline phosphatase --- p.8 / Chapter 1.1.4 --- Role of alkaline phosphatase --- p.10 / Chapter 1.2 --- Mouse embryonic brain development --- p.18 / Chapter 1.2.1 --- General developing process --- p.18 / Chapter 1.2.2 --- The crainal nerve nuclei in the embryonic mouse brainstem --- p.20 / Chapter 1.2.3 --- The process of neurogenesis in central nerve system --- p.22 / Chapter 1.3 --- Alkaline phosphatase expressed in developing neural tube --- p.26 / Chapter 1.4 --- Summary --- p.30 / Chapter 1.5 --- Objectives of study --- p.31 / Chapter Chapter 2 --- AP expression pattern in embryonic mouse brainstem --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.1.1 --- AP expressed in developing neural tube --- p.33 / Chapter 2.1.2 --- Methods for alkaline phosphatase detection --- p.35 / Chapter 2.2 --- Materials and methods --- p.39 / Chapter 2.2.1 --- Animal and procedure --- p.39 / Chapter 2.2.2 --- Preparation of tissue sections and histochemistry --- p.39 / Chapter 2.2.3 --- Electron microscopy study of AP location --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Histochemical demonstration of AP --- p.42 / Chapter 2.3.2 --- Stage-specificity and tissue-specificity of AP activity in the neural tube --- p.43 / Chapter 2.3.3 --- Cytochemical localization of AP activity --- p.46 / Chapter 2.3.4 --- Sencitivity to pH of the histochemical staining for AP --- p.46 / Chapter 2.3.5 --- Inactivation of AP activity --- p.47 / Chapter Chapter 3 --- Quantitative studies of AP activity in embryonic mouse brainstem --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- Basic knowledge about enzyme kinetic study --- p.48 / Chapter 3.1.2 --- Enzyme assay for alkaline phosphatase --- p.50 / Chapter 3.2 --- Materials and methods --- p.52 / Chapter 3.2.1 --- Animals and sample preparation --- p.52 / Chapter 3.2.2 --- Measurement of AP activities --- p.53 / Chapter 3.2.3 --- Data analysis --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- "Determination of reaction duration, initial velocity and Km of AP activity" --- p.54 / Chapter 3.3.2 --- Comparision of AP activity in the brainstem and cortex and at different stages --- p.55 / Chapter 3.3.3 --- Effects of physical and chemical factors on AP activity --- p.55 / Chapter Chapter 4 --- Electrophoresis study of AP activity --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and methods --- p.60 / Chapter 4.2.1 --- AP extraction --- p.60 / Chapter 4.2.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.61 / Chapter 4.2.3 --- Detection of AP activity --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Demonstration of AP activity on the gels --- p.62 / Chapter 4.3.2 --- Comparison of AP from the brain at different stages --- p.62 / Chapter 4.3.3 --- "Comparison of AP in the embryonic brainstem with those in the adult mouse placenta, kidney, liver and intestine" --- p.63 / Chapter 4.3.4 --- Effect of heating and chemical factors on AP activity in the embryonic brainstem --- p.63 / Chapter Chapter 5 --- Study of the cell types expressing AP activity --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and methods --- p.67 / Chapter 5.2.1 --- Materials --- p.67 / Chapter 5.2.2 --- Immunostaining of AP in the embryonic brainstem --- p.68 / Chapter 5.2.3 --- Double staining for AP and cells markers --- p.70 / Chapter 5.3 --- Results --- p.70 / Chapter 5.3.1 --- Effectiveness of anti-TNAP antibody on the embryonic mouse brain --- p.70 / Chapter 5.3.2 --- Expression pattern of different neural cell markers at E13.5 --- p.71 / Chapter 5.3.3 --- Co-localization of AP and specific cell markers in E13.5 brain --- p.72 / Chapter Chapter 6 --- Discussion --- p.74 / Chapter 6.1 --- Stage-dependence and tissue-specificity of AP expression in the developing mouse brainstem --- p.75 / Chapter 6.2 --- Possible molecular nature of AP expressed in the developing mouse brainstem --- p.80 / Chapter 6.3 --- The possible cell types that express the enzyme activity --- p.83 / "Figures, Tables, Graphs and Legends" --- p.87 / Appendices --- p.165 / Appendix A: Sources of materials --- p.165 / Appendix B: The process of sample for staining --- p.167 / Appendix C: Protocol of histochemical staining for AP --- p.170 / Appendix D: Protocol of electron microscopy study for AP activity --- p.172 / Appendix E: Protocol of enzyme assay for AP activity --- p.174 / Appendix F: Protocol of immunostaining (ABC method) --- p.175 / Appendix G: Protocol of double staining with fluorescent detection --- p.177 / Appendix H: Protocol of electrophoresis analysis for AP --- p.179 / References --- p.182

Diploma thesis studies historical bonding agents on the base of geopolymers. The aim of the thesis is to find a proper material composition, especially made of natural clay materials (kaolinite, bentinite) and their modifications (metakaoline). These bonding agents have to have a suitable type of an activator that guarantees good bonding properties. Therefore testing of various kinds of alkaline activating ingredients on the same mineral composition was done. Except of usual hydroxides and a water glass, ancient natrons (mixtures of alkaline carbonate with addition of appropriate chlorides) and a lime mash were used as the activators. From the above mentioned mixtures, series of samples (columns 20 x 20 x 100 mm) stored at the laboratory temperature were prepared. In the prepared mashes, their workableness and moulding were investigated. In the developed samples, their surface appearance was observed together with a number of efflorescence and its types. An indivisible part of the research was formed by determination of mechanical properties of the experimental columns such as a compressive strength and a tensile strength in bending. Furthermore, phase composition of the samples and its changes with a temperature increase were investigated. For these tests, XRD and TG – DTA methods were used. A multi-seat isoperbolic calorimeter was used to study hydratation that was also the important part of the general evaluation of designed mixtures.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Previous studies suggest that dietary patterns that promote acidosis may have a negative effect on bone density, whereas a more alkaline‐based profile would be associated with better bone health. Thus, the aim of this study was to assess, in omnivores, vegetarians, and vegans bone mineral density using Dual‐energy X‐ray absorptiometry (DEXA) and compare it to their acid‐base status as indicated by urinary pH, Potential Renal Acid Load (PRAL) and serum anion gap. Our hypothesis was that plant‐based diets would be associated with a more alkaline acid‐base profile than omnivorous diets, and thus have a higher bone mineral density. Methods: We conducted a cross‐sectional study where we compared plant based vs. omnivorous diets. Eighty‐two subjects were enrolled in the study (27 omnivores, 27 vegetarians, and 28 vegans). Subjects were asked to fill out a medical history form and a 24‐ hour diet recall, and to complete a 24‐hour urine collection. After a few weeks, subjects returned to the test site to complete a DEXA scan. Acid base‐balance and bone health were determined using PRAL, urine pH, and anion gap as biomarkers for pH, and DEXA as an indicator of bone density. Our results showed that bone mineral density did not differ significantly between groups, although lacto‐ovo and vegan diets were more alkaline compared to meat based diets (6.5  0.4, 6.7  0.4, and 6.2  0.4 pH respectively, p = 0.003). Protein intake was found to be reduced by ~30% in individuals adhering to a lacto‐ovarian or vegan diet; yet protein was only associated with bone mineral density in those following vegan diets. Conversely, urinary pH was associated with bone mineral density only in those following a meat‐based diet. The significance of this study is that it provides knowledge in the area of osteoporosis prevention and perhaps specific recommendations based on diet groups: increased fruit and vegetable intake for those with high meat consumption, to improve the acid‐base homeostasis, and increased plant protein intake for individuals who follow a plant-based diet.

The Twin arginine translocase (Tat) system catalyzes movement of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. This transport process requires energy in the form of the transmembrane proton motive force (PMF). The Tat system can be studied in vitro using inner membrane vesicles (IMVs) from E. coli overproducing the Tat components, TatA, TatB and TatC. However, the transport efficiencies of current in vitro Tat transport assays are low. In this work, current in vitro Tat transport assays were compared and parameters that affect transport efficiencies were identified. Mild French press treatment of IMVs resulted in larger IMVs with higher transport efficiencies. Chloride ions were shown to inhibit Tat transport in vitro. Generation of a PMF by the activity of ATP synthase gave higher transport efficiencies than generating a PMF by NADH respiration. This understanding was applied to develop an optimized in vitro Tat transport assay that showed a higher transport efficiency than currently published methods. Fluorescently labelled Tat substrates were developed to allow quantitative analysis of Tat transport. The transport of the purified native Tat substrate, CueO into IMVs was characterized using the optimized in vitro Tat transport assay. It was shown that the proton concentration (ΔpH) component of the PMF was sufficient to support Tat transport in vitro. It was observed that transport of CueO ceased in a time-dependent manner in the in vitro Tat transport assays. This loss of transport efficiency could be due, at least in part, to the presence of a PMF since transport efficiency was reduced when IMVs were pre-energized. Substrates for future in vitro single molecule fluorescence microscopy studies of the Tat transport were developed in this work. One of the substrates is fluorescently labelled CueO. The second substrate is the native Tat substrate alkaline phosphatase PhoX from Vibrio fischeri which was able to cleave the fluorogenic compound AttoPhos® and can thus be used as an enzymatic reporter of Tat transport. The structure of a native Tat substrate from Pseudomonas fluorescens, PhoX, was solved by X-ray crystallography at a resolution of 1.4Å. PhoX is a monomeric six blade β propeller with two α-helical bundle subdomains. PhoX was shown to have optimum activity at pH8.0. PhoX has a novel catalytic site which requires two Fe³⁺ (including a Cys-coordinated Fe³⁺) and three Ca²⁺ as cofactors. Mutagenesis studies showed that all the metal ions are required for the integrity of the active site. Co-crystallization of PhoX with vanadate, an inhibitor of PhoX which mimics the transition state, showed that hydrolysis of phosphomonoesters does not involve formation of a covalent phosphoenzyme intermediate. Instead, dephosphorylation of substrates is proposed to occur via a SN2 reaction with OH- as the attacking nucleophile.

Orientador: Luis Henrique Souza Guimarães / Banca: Ariela Veloso de Paula / Banca: Hamilton Cabral / Resumo: A investigação biotecnológica, acompanhada da aplicação das enzimas, tem sido realizada em microrganismos para a produção de enzimas para fins industriais. Entre estas enzimas, as fosfatases, responsáveis por hidrolisar ésteres e anidridos de ácido fosfórico, e as fitases microbianas, que catalisam a hidrólise do fitato (mio-inositol hexaquisfosfato) em mio-inositol e fosfato inorgânico, têm sido amplamente utilizadas em diferentes setores como, por exemplo, em experimentos de biologia molecular e na alimentação animal. De acordo com o pH ótimo de reação, as fosfatases são divididas em alcalinas (EC 3.1.3.1) e ácidas (EC 3.1.3.2). As fitases são enzimas que também pertencem à classe das fosfatases, hidrolisando, no entanto, de forma específica, o ácido fítico. Em recentes trabalhos, o fungo filamentoso Rhizopus microsporus var. microsporus apresentou potencialidade na produção de fosfatases e fitases. Diante disto, este estudo visou a produção, a purificação e caracterização da fosfatase e da fitase alcalina produzidas por R. microsporus var. microsporus. No processo de otimização em Fermentação Submersa (FSbm), a maior produção enzimática foi em meio Khanna com 0,4 mM de KH2PO3 e adicionado de 0,5% de farinha de centeio por 76 h, 32ºC, pH 6,3, a 100 rpm. Em colunas cromatográficas, a fosfatase alcalina foi purificada 10 vezes e com recuperação de 13%, e a fitase alcalina foi purificada 86 vezes com recuperação de 167%. A massa molecular nativa da fosfatase e da fitase alcali... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Biotechnological research, accompanied by the application of enzymes, has been carried out in microorganisms for production of enzymes for industrial purposes. Among these enzymes, microbial phosphatases, responsible for hydrolyzing phosphoric acid esters and anhydrides, and phytases, which catalyzes the hydrolysis of phytate (myo-inositol hexaquisphosphate) in myo-inositol and inorganic phosphate, have been widely used in different sectors as, for example, in molecular biology experiments and in animal feed. According to the optimum reaction pH, phosphatases are divided into alkaline (EC 3.1.3.1) and acidic (EC 3.1.3.2). Phytases are enzymes that also belong to the class of phosphatases, however, hydrolyzing phytic acid. In recent works, the filamentous fungus Rhizopus microsporus var. microsporus presented potential for production of phosphatases and phytases. In view of this, this study aimed at the production, purification and characterization of phosphatase and alkaline phytase produced by R. microsporus var. microsporus. In the optimization of Submerged Fermentation (FSbm), the highest enzymatic production was in Khanna medium with 0.4 mM KH2PO3 and added with 0.5% rye flour for 76 h, 32ºC, pH 6.3, at 100 rpm. In chromatographic columns, alkaline phosphatase was purified 10 folds and recovered at 13%, and alkaline phytase was purified 86 folds with recovery of 167%. The native molecular mass of alkaline phosphatase and phytase produced by R. microsporus var. microsporus... (Complete abstract click electronic access below) / Mestre

O maciço alcalino da Serra da Baixa Verde abrange uma área de exposição de aproximadamente 4O0 km² e localiza-se na divisa entre os Estados de Paraíba e Pernambuco, no Nordeste brasileiro. Do ponto de vista geológico encontra-se incluído na chamada \"zona transversal\" de EBERT (1958), constituída por rochas predominantemente pré-cambrianas, delimitada ao Sul pelo lineamento de Pernambuco e ao Norte pelo de Patos ou Paraíba. Petrográficamente, trata-se de uma intrusiva ígnea classificada como quartzo augita sienito, podendo ser considerada como uma diferenciação menos ácida de um magma granítico encontrado na região e localmente situado na parte leste da área estudada. Suas relações de contato são geralmente concordantes e parcialmente discordantes com as estruturas encaixantes e, tal fato, ligado a outras evidências, levou-nos a supor uma origem tarditectônica para o maciço. Do ponto de vista estratigráfico, as rochas encaixantes são metamórficas pertencentes aos Grupos Uauá e Cachoeirinha (BARBOSA et al, I970), de idades pré--cambriano inferior e superior, respectivamente. Estes dois Grupos são constituídos localmente por micaxistos gnaissificados e fenitizados nas bordas da intrusão e dobrados aproximadamente na direção EW-NE. O maciço apresenta-se cortado por falhas de natureza transcorrente, chegando algumas a medir mais de 25 km de comprimento. Estas feições disruptivas estão associadas na sua maioria aos lineamentos de Patos e Pernambuco. A idade do sienito supõe-se que seja de aproximadamente 500 milhões de anos, em analogia com datações K-Ar efetuadas em corpos similares. O autor acredita que esta ígnea constitui parte de um conjunto de corpos sienito-graníticos introduzidos tarditectonicamente durante o Eo Cambriano. / Não existente na dissertação.

Long relegated to the background by the pre-eminence of magnesium-based, stoichiometric Grignard reagents, a distinct chemistry of the heavier alkaline earth metals, calcium, strontium and barium, is only now starting to emerge. As similarities have been drawn between the large, electropositive, redox-inert and d0 alkaline earth Ae2+ dications and the Ln3+ cations of the lanthanide series, a growing group 2-mediated catalytic chemistry has developed over the last decade, including polymerisation reactions, heterofunctionalisation reactions of multiple bonds and some rare examples of dehydrocoupling reactions. Among these catalytic reactions the magnesium- and calcium-catalysed intramolecular hydroamination of aminoalkenes has attracted particular interest. Mechanistic studies have demonstrated many parallels with the lanthanide-mediated catalytic cycle based upon successive σ-bond metathesis and insertion steps. In the first part of this thesis, further investigations into the hydroamination/cyclisation reaction have demonstrated the prominent role of the charge density of the catalytic group 2 cation (M = Mg, Ca, Sr, Ba), the beneficial influence of stabilising spectator ligands, and the importance of the choice of the reactive co-ligand for efficient catalyst initiation. Kinetic analyses of reactions monitored by NMR spectroscopy have given new insight into activation energies, entropic effects, substrate and product inhibition, and kinetic isotope effects, leading to a review of the previously suggested lanthanide-mimetic mechanism. In a second part, this study seeks to address two of the main challenges posed by the intramolecular hydroamination reaction in particular, and heavier alkaline earth-catalysed reactions in general: (i) The need to design new monoanionic spectator ligands capable of stabilising heteroleptic heavier alkaline earth complexes and preventing deleterious Schlenk-type ligand redistribution processes in solution; (ii) The stabilisation of highly reactive heteroleptic group 2 alkyl functionalities for fast, irreversible catalyst initiation and novel reactivity.

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).