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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

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

by Paul, Liu Po Lung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 86-94). / ACKNOWLEDGMENT --- p.i / TABLE OF CONTENT --- p.ii / "LIST OF TABLE, FIGURE & PHOTO" --- p.viii / ABSTRACT --- p.x / Chapter CHAPTER ONE : --- INTRODUCTION --- p.1 / Chapter 1.1 --- ALKALINE PHOSPHATASE / Chapter 1.1.1 --- Alkaline Phosphatase Isoenzyme --- p.2 / Chapter 1.1.2 --- The Properties of Alkaline Phosphatases --- p.4 / Chapter 1.1.3 --- Serum Alkaline Phosphatases --- p.6 / Chapter 1.1.3.1 --- Placental Alkaline Phosphatase --- p.7 / Chapter 1.1.3.2 --- Intestinal Alkaline Phosphatase --- p.7 / Chapter 1.1.3.4 --- Skeletal Alkalne Phosphatase --- p.8 / Chapter 1.1.3.5 --- Hepatic Alkaline Phosphatase --- p.8 / Chapter 1.1.3.3 --- Renal Alkaline Phosphatase --- p.9 / Chapter 1.1.3.6 --- Miscellaneous Alkaline Phosphatase --- p.9 / Chapter 1.1.4 --- Problems in Discriminating the Skeletal and Hepatic Alkaline Phosphatase in Serum --- p.11 / Chapter 1.1.5 --- Quantitative measure of the Bone-Specific Alkaline Phosphatase --- p.12 / Chapter 1.1.6 --- Qualitative Detection of ALP isoenzymes --- p.14 / Chapter 1.2 --- OSTEOSARCOMA --- p.17 / Chapter 1.2.1 --- Definition --- p.17 / Chapter 1.2.2 --- Epidemiology and Statistics --- p.17 / Chapter 1.2.3 --- Clinical Presentation --- p.18 / Chapter 1.2.4 --- Radiographic finding --- p.19 / Chapter 1.2.5 --- Staging of Musculoskeletal Neoplasms --- p.20 / Chapter 1.2.6 --- Treatment of osteosarcoma --- p.21 / Chapter 1.2.6.1. --- Chemotherapy in Prince of Wales Hospital --- p.21 / Chapter 1.3 --- PLASMA AND TISSUE ALKALINE PHOSPHATASE IN NORMAL AND NEOPLASTIC CONDITION --- p.23 / Chapter 1.3.1 --- Normal values of plasma alkaline phosphatase --- p.23 / Chapter 1.3.2 --- Clinical use of elevated plasma & tissue alkaline phosphatase level in neoplastic conditions --- p.25 / Chapter 1.3.2.1 --- Helping the Diagnosis of the Osteosarcoma --- p.25 / Chapter 1.3.2.2 --- Monitoring the effect of chemotherapy --- p.26 / Chapter 1.3.2.3 --- Predicting the clinical course --- p.26 / Chapter 1.3.3 --- Qualitative measurement of ALP in plasma and tissue extract of osteosarcoma patient --- p.29 / Chapter 1.4 --- AIM AND SCOPE OF THE PRESENT DISSERATION --- p.30 / Chapter CHAPTER TWO : --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- DIFERENT GROUPS OF PATIENTS --- p.33 / Chapter 2.1.1 --- Monitering the plasma bone specific ALP --- p.33 / Chapter 2.1.1.1 --- Osteosarcoma group --- p.33 / Chapter 2.1.1.2 --- Benign bone tumour group --- p.34 / Chapter 2.1.1.3 --- Metastasis group --- p.34 / Chapter 2.1.2 --- Collection of plasma samples preserve of tumor tissue --- p.34 / Chapter 2.2 --- QUANTITATIVE ANALYSIS OF THE PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.36 / Chapter 2.2.1 --- Extraction of tissue ALP --- p.36 / Chapter 2.2.1.1. --- Reagent --- p.36 / Chapter 2.2.1.2. --- Homogenization of the bone tissue --- p.36 / Chapter 2.2.1.3. --- Extraction of ALP --- p.37 / Chapter 2.2.2 --- Assay for Bone-specific ALP --- p.38 / Chapter 2.2.2.1. --- Reagents --- p.38 / Chapter 2.2.2.2. --- Procedures --- p.38 / Chapter 2.3 --- QUALITATIVE MEASUREMENT OF ALP ISOENZYME --- p.40 / Chapter 2.3.1 --- Equipment required --- p.40 / Chapter 2.2.2 --- Practical procedure --- p.40 / Chapter 2.3.3.1 --- Gel casting --- p.40 / Chapter 2.3.3.2 --- Sample preparation and application --- p.42 / Chapter 2.3.3.3 --- Electrofocusing --- p.42 / Chapter 2.3.3.4 --- Western blotting of the protein --- p.43 / Chapter 2.3.3.5 --- Detection methods --- p.45 / Chapter 2.4 --- METHOD OF STATISTICAL ANALYSIS --- p.48 / Chapter CHAPTER THREE : --- RESULTS --- p.49 / Chapter 3.1 --- QUANTITATIVE MEASUREMENT OF PLASMA AND TISSUE BONE SPECIFIC ALKALINE PHOSPHATASE --- p.50 / Chapter 3.1.1 --- General Information of the patients monitoring --- p.50 / Chapter 3.1.2 --- Pretreatment evaluation --- p.52 / Chapter 3.1.3 --- Correlation between the pretreatment plasma ALP levels and prognosis in the osteosarcoma patient group --- p.57 / Chapter 3.1.4 --- "Correlation between the pre-operational, post- operational plasma ALP levels and the prognosis of osteosarcoma" --- p.59 / Chapter 3.1.5 --- Analysis of plasma ALP levels at the time of relapse in osteosarcoma patients --- p.61 / Chapter 3.1.6 --- Usefulness of the plasma ALP levels for monitoring the effectiveness of chemotherapy --- p.62 / Chapter 3.1.7 --- Correlation between the ALP levels in the tumor extract and the prognosis of the osteosarcoma --- p.64 / Chapter 3.2 --- QUALITATIVE ANALYSIS OF THE PLASMA AND TISSUE ALKALINE PHOSPHATASE LEVEL --- p.67 / Chapter 3.2.1 --- Comparison of the result of Isoelectric focusing of the plasma ALP of the osteosarcoma patients and the normal subjects --- p.67 / Chapter 3.2.1 --- Result of Isoelectric focusing of the ALP isoenzymes in the tissue extract of the osteosarcoma and normal bone --- p.70 / Chapter CHAPTER FOUR : --- DISCUSSION --- p.72 / Chapter 4.1 --- USE OF QUANTITATIVE MONITORING OF PLASMA ALP AND MEASURING TISSUE ALP IN OSTEOSARCOMA PATIENTS --- p.73 / Chapter 4.2 --- ISOELECTRIC FORCUSING AS A TECNIQUE FOR QUALITATIVE MEASUREMENT OF PLASMA AND TISSUE ALKALINE PHOSPHATASE --- p.80 / Chapter CHAPTER FIVE : --- CONCLUSION --- p.83 / Chapter CHAPTER SIX : --- BIBILOGRAPHY --- p.85

by Au Sze Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves xii-xix). / Acknowledgement --- p.i / Table of Content --- p.ii / List of Abbreviation --- p.vi / Abstract --- p.viii / Chapter Chapter One : --- Introduction / Chapter 1.1. --- Osteosarcoma --- p.1 / Chapter 1.1.1. --- Definition --- p.1 / Chapter 1.1.2 --- "Incidence, geographic patterns of distribution and epidemiological consideration" --- p.1 / Chapter 1.1.3 --- "Age, sex and sites" --- p.3 / Chapter 1.1.4 --- Type and grade --- p.5 / Chapter 1.1.4.1. --- Grade --- p.5 / Chapter 1.1.4.2. --- Site --- p.5 / Chapter 1.1.4.3. --- Metastasis --- p.5 / Chapter 1.1.5 --- Histological features --- p.9 / Chapter 1.1.6 --- Clinical features --- p.9 / Chapter 1.1.7 --- Radiological features --- p.11 / Chapter 1.1.8. --- Molecuar genetics --- p.13 / Chapter 1.1.9 --- Treatment --- p.13 / Chapter 1.2 --- Biochemical Markers of Osteosarcoma --- p.14 / Chapter 1.2.1 --- Tumor marker --- p.14 / Chapter 1.2.2 --- Biochemical markers of bone turnover --- p.15 / Chapter 1.2.3 --- Change of biochemical marker in osteosarcoma --- p.17 / Chapter 1.3 --- Alkaline Phosphatase (ALP) --- p.17 / Chapter 1.3.1 --- ALPs Family --- p.20 / Chapter 1.3.2 --- Membrane binding --- p.22 / Chapter 1.3.3 --- Biochemical function and physiological role of ALP --- p.24 / Chapter 1.4 --- Normal values of serum ALP --- p.28 / Chapter 1.5 --- Clinical applications of ALP --- p.28 / Chapter 1.6 --- "Separation, identification and quantification of ALP isozymes" --- p.31 / Chapter 1.6.1 --- Themostability --- p.31 / Chapter 1.6.2 --- Inhibition studies --- p.31 / Chapter 1.6.3 --- Electrophoresis --- p.33 / Chapter 1.6.4 --- Isoelectric focusing --- p.34 / Chapter 1.6.5 --- Affinity precipitation --- p.34 / Chapter 1.6.6 --- Immunological studies --- p.35 / Chapter 1.7 --- Plasma BALP level as biochemical marker of osteosarcoma --- p.35 / Chapter 1.8 --- ALP in malignancies --- p.37 / Aim of study --- p.x / Chapter Chapter Two : --- Methods and Materials / Chapter 2.1 --- Plasma BALP measurement as a biochemical markerin osteosarcoma --- p.40 / Chapter 2.1.1 --- Patient groups --- p.40 / Chapter a) --- Normal subjects --- p.40 / Chapter b) --- Osteosarcoma patients --- p.40 / Chapter 2.1.2 --- Collection and preparation of patient bloods samples of patients --- p.40 / Chapter 2.1.3 --- Plasma total ALP measurement --- p.41 / Chapter a) --- Reagent --- p.41 / Chapter b) --- Procedure --- p.43 / Chapter 2.1.4 --- Plasma BALP measurements --- p.43 / Chapter a) --- Wheat germ lectin precipitation of BALP --- p.44 / Chapter i) --- Reagent / Chapter ii) --- Procedure / Chapter b) --- ABBOTT methods for plasma BALP activity measurement --- p.45 / Chapter c) --- COBAS MIRA methods for BALP measurement --- p.45 / Chapter d) --- ALKPHASE-B method of BALP measurement --- p.46 / Chapter 2.1.5 --- Inter-conversion of plasma BALP activity measurement in different methods --- p.47 / Chapter 2.1.6 --- Statistical analysis --- p.48 / Chapter 2.2 --- Alkaline phosphatase isozymes expression in human osteosarcoma --- p.48 / Chapter 2.2.1 --- In Vitro cultures of human SaOS-2 and U-2 OS osteosarcoma cell line --- p.48 / Chapter a) --- Reagent --- p.49 / Chapter b) --- Procedure --- p.50 / Chapter i) --- Storage of U-2 OS and SaOS-2 / Chapter ii) --- Subculture of confluent monolayer / Chapter 2.2.2 --- Protein assay --- p.51 / Chapter a) --- Standard Assay --- p.51 / Chapter i) --- Reagent / Chapter ii) --- Procedure / Chapter b) --- Mircoassay --- p.51 / Chapter i) --- Reagent / Chapter ii) --- Procedure / Chapter 2.2.3 --- Extraction of ALP from the cultured osteosarcoma cells --- p.52 / Chapter a) --- Reagent --- p.52 / Chapter b) --- Procedure --- p.52 / Chapter 2.2.4 --- "ALP extraction from human liver, placenta and osteosarcoma tissue" --- p.53 / Chapter a) --- Reagent --- p.53 / Chapter b) --- Procedure --- p.54 / Chapter 2.2.5 --- Isoelectric focusing of ALP --- p.55 / Chapter a) --- Preparation of the agarose IEF gel --- p.55 / Chapter b) --- Samples preparation --- p.56 / Chapter c) --- Isoelectric focusing --- p.57 / Chapter d) --- Protein detection --- p.59 / Chapter i) --- Reagent / Chapter ii) --- Procedure / Chapter e) --- Visualization of ALP isozyme --- p.60 / Chapter 2.2.6 --- Biochemical differentiation of ALP expressed in human osteosarcoma --- p.61 / Chapter a) --- Thermodenaturation of ALP --- p.61 / Chapter b) --- Ammino acid inhibition of ALP --- p.61 / Chapter 2.2.7 --- Immunohistostaining of placental ALP in human Osteosarcoma --- p.62 / Chapter a) --- Reagent --- p.62 / Chapter b) --- Preparation of human osteosarcoma cell line --- p.63 / Chapter c) --- Preparation of human osteosarcoma tissue --- p.63 / Chapter d) --- Immunohistostaining --- p.64 / Chapter Chapter Three : --- Results / Chapter 3.1 --- General information of the patients --- p.65 / Chapter 3.1.1 --- Age and sex distribution --- p.65 / Chapter 3.1.2 --- Sites --- p.65 / Chapter 3.1.3 --- Treatment and survival rate --- p.66 / Chapter 3.2 --- Clinical significance of plasma bone-specific alkaline phosphatase (BALP) activity measurementin osteosarcoma patients --- p.71 / Chapter 3.2.1 --- Plasma BALP activity measurement --- p.71 / Chapter 3.2.2 --- Normal reference of plasma BALP determination --- p.72 / Chapter 3.2.3 --- Diagnostic value of plasma BALP measurement in osteosarcoma --- p.75 / Chapter a) --- Plasma BALP level at admission --- p.75 / Chapter b) --- Plasma Total ALP level at admission --- p.78 / Chapter 3.2.4 --- Prognosis value of plasma BALP measurement in osteosarcoma Patients --- p.78 / Chapter a) --- Correlation of plasma BALP-Adm with the local relapse of the disease --- p.78 / Chapter b) --- Correlation of plasma BALP-Adm with survival rate of the patients --- p.90 / Chapter i) --- One year survival Rate / Chapter ii) --- two-year survival Rate / Chapter iii) --- Three-year survival rate / Chapter c) --- Correlation of the plasma BALP-Adm with the tumor volume --- p.90 / Chapter 3.2.5 --- Using plasma BALP measurement for monitoring of the disease --- p.91 / Chapter a) --- Effectiveness of pre-operative chemotherapy --- p.91 / Chapter b) --- Change of plasma BALP level during the treatment --- p.92 / Chapter i) --- Monitoring of pre-operative chemotherapy / Chapter ii) --- Detection of local recurrence and secondary metastasis / Chapter 3.3 --- Alkaline phosphatase isozyme expressionin osteosarcoma --- p.103 / Chapter 3.3.1 --- Isoelectric point (pI) gradient in isoelectric focusing (IEF)gel --- p.10? / Chapter 3.3.2 --- ALP isozyme standard --- p.103 / Chapter 3.3.3 --- Ectopic expression of ALP in human osteosarcoma cell line: U-2 OS and SaOS-2 --- p.104 / Chapter a) --- Isoelectric focusing separation --- p.104 / Chapter b) --- Biochemical differentiation of ALP extracts --- p.110 / Chapter 3.3.4 --- Alkaline phosphatase expression in osteosarcoma patient plasma sample --- p.110 / Chapter 3.3.5 --- Alkaline phosphatase isozyme expression in human osteosarcoma biopsy tissue --- p.111 / Chapter 3.3.6 --- Ectopic expression of placental ALP in human osteosarcoma by immunohistochemistry --- p.111 / Chapter a) --- Ectopic expression of placental ALP in human osteosarcoma cell line U-2 OS --- p.111 / Chapter b) --- Ectopic expression of placental ALP in human osteosarcoma tissue sections --- p.112 / Chapter Chapter Four : --- Discussion --- p.128 / Chapter Chapter Five : --- Conclusion --- p.142 / Bibliography --- p.xii / Appendix --- p.xx

Alkaline phosphatase (ALP) is an enzyme present in intestinal mucosa, bile, bone and renal tubule cells. Bile acids have been shown to decrease ALP activity from bone and kidney but not those from intestinal origin. This action can be mimicked in serum and peritoneal fluid samples by the use of an L-phenylalanine buffer which specifically measures intestinal ALP activity only; while the standard buffer measures total ALP activity. We sought to assess the diagnostic and prognostic relationship of intestinal and total ALP activity between serum and peritoneal fluid in 126 horses with acute colic. Blood and peritoneal fluid samples were analyzed for ALP activity using both the standard and L-phenylalanine based buffers. Neither total nor intestinal serum ALP activity was useful in classifying type or severity of intestinal damage. Total and intestinal peritoneal fluid ALP activity were lowest in horses suffering simple medical colic and non-strangulated surgical lesions, and highest in surgical cases with suspected ulceration, strangulation, peritonitis and intestinal rupture. High total and intestinal peritoneal fluid ALP activity was associated with greater intestinal damage, increased probability of surgical intervention and a worse prognosis while low total and intestinal peritoneal fluid ALP activity was unable to accurately differentiate between simple medical colics and surgical colics. The use of L-phenylalanine buffer in both serum and peritoneal fluid did not improve the sensitivity of the test. Based on these results, determination of total ALP activity in peritoneal fluid may be helpful in identifying ischemic or inflammatory bowel lesions in horses with acute colic. A portable clinical analyzer (PCA) was used for the determination of venous blood and peritoneal fluid pH value, glucose, lactate and electrolyte concentrations in a hospital setting. Blood and peritoneal fluid glucose, lactate, sodium, chloride and potassium concentrations, and pH value were determined using both a portable clinical analyzer with test cartridges and an in-house analyzer in 56 horses with acute abdominal disease. Results were compared by the Bland-Altman method of comparison and linear regression. The PCA yielded higher blood and peritoneal pH values, with greater variability in the alkaline range and lower pH values in the acidic range. The PCA glucose concentrations (<150 mg/dL) were significantly lower, and were higher in the high range (>150 mg/dL). Venous lactate concentration (<5 mmol/dL) arid peritoneal fluid lactate concentration (<2 mmol/dL) had the smallest variability. On average, the PCA underestimated peritoneal lactate and glucose concentration. Peritoneal fluid sodium and chloride concentration had higher bias and variability than venous sodium and chloride concentration. Venous and peritoneal fluid potassium concentration was closely clustered around the mean with a low bias and variability. Correlation coefficients were >0.80 for all values except venous and peritoneal sodium concentration; venous chloride concentration and venous pH value. The PCA may be suitable for point-of-care biochemical analysis of blood and peritoneal fluid for horses suffering colic and may provide further diagnostic and prognostic information. The PCA may be of help in diagnosing metabolic acidosis, uroperitoneum, septic and non-septic peritonitis and intestinal ischemia. This may be of benefit to ambulatory equine clinicians. / Graduation date: 2004

Organic phosphorus (P$ sb{ rm o}$) in farm animal wastes must be mineralized to inorganic P for subsequent plant use. This study was conducted to determine if feed additives affect P$ sb{ rm o}$ mineralization, manure decomposition, and plant growth. Feed additives in aqueous systems affected the P mineralization of inositol hexaphosphate by phytase and of adenosine monophosphate by alkaline phosphatase. Pronounced effects were produced by bacitracin and both enzymes and by neomycin on phytase. Feed additives in dairy cattle (Bos taurus L.) manure produced effects on microbial activity as measured by gas production that differed from those produced on fecal phosphatase activity. Additives applied directly or with manure to Ste. Rosalie clay, Greensboro loam, or silica sand had no effect on barley (Hordeum vulgare L.) yield but did produce additive, rate, growth medium, and manure dependent effects on plant P concentration and soil phosphatase activity. Therefore, each feed additive must be independently evaluated to determine its effect on biological systems.