Kwan Pui-Chun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 140-155). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Iron homeostasis --- p.5 / Chapter 1.3 --- Natural Resistance Associated Marcophage Protein (Nramp) Family --- p.15 / Chapter 1.4 --- Divalent Metal Transporter 1 (DMT1) --- p.18 / Chapter 1.5 --- Iron Responsive Element (IRE) and Iron Regulatory Protein (IRP) --- p.23 / Chapter 1.6 --- Expression and localization of DMT-1 in human --- p.27 / Chapter 1.7 --- Iron and the developing feus --- p.31 / Chapter 1.8 --- Objectives of the study --- p.36 / Chapter Chapter 2 --- Materials and Method / Chapter 2.1 --- Study population --- p.37 / Chapter 2.2 --- Procedure of surgical termination of pregnancy --- p.38 / Chapter 2.3 --- Tissues collection and preparation --- p.39 / Chapter 2.4 --- Semi-quantitative Reverse Transcription-Polymerase Chain Reaction --- p.44 / Chapter 2.5 --- Immunohistochemistry --- p.49 / Chapter 2.6 --- Statistical analysis --- p.55 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Description of subjects --- p.56 / Chapter 3.2 --- Existence of human DMT-1 isoforms at early pregnancy --- p.58 / Chapter 3.3 --- Relative expression of DMT-1 isoforms to β -actin mRNA expression at different week gestation --- p.67 / Chapter 3.4 --- Cellular localization of DMT-1 isoforms at early pregnancy --- p.91 / Chapter 3.5 --- Relative expression of DMT-1 proteins at early pregnancy --- p.101 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Existence of DMT-1 at early pregnancy --- p.116 / Chapter 4.2 --- Expression of DMT-1 isoforms at early pregnancy at gene level --- p.118 / Chapter 4.3 --- Expression of DMT-1 isoforms at early pregnancy at protein level --- p.120 / Chapter 4.4 --- "Comparison expression of DMT-1 between human fetus, human adult and animal studies" --- p.121 / Chapter 4.5 --- Functional importance of DMT-1 at developing fetus at early pregnancy --- p.130 / Chapter 4.6 --- Conclusion --- p.138 / Chapter 4.7 --- Further study --- p.139 / Chapter Chapter 5 --- Reference --- p.140 / Appendix I: Calculation of EM --- p.156

Direct methanol fuel cells (DMFCs) are considered a clean source of electrical power for future energy demand, creating a potential to reduce our dependency on fossil fuels. Despite their advantages, including high energy density, efficiency and easy handling and distribution of fuel, the commercialization of DMFCs has suffered from some drawbacks, including methanol crossover and contamination of the system. Metal cation contaminants (such as Ni, Co, etc) introduced through the degradation of fuel cell components (bipolar plate and electro-catalyst layer) can significantly affect the Nafion-membrane properties and overall fuel cell performance. In the current study, a systematic approach is taken to characterize and identify the mechanism of the effect of metal solution contaminants on the activities of electro-catalysts of DMFCs. Cyclic voltammetry and rotating disk electrode (RDE) techniques were utilized in order to characterize the effect of various concentrations (i.e., 2x10-x M (x=1-7)) of six metal solution contaminants (i.e., Co, Ni and Zn with sulfate and nitrate as counter-anions) on the voltammetric properties and electro-catalytic activity of polycrystalline Pt during methanol oxidation reaction (MOR) and oxygen reduction reaction (ORR). The results showed a decrease in the MOR and ORR activities of Pt as the concentration of metal solution increased. The effect of counter-anion on the Pt activity was further investigated. The results showed that a combined effect of counter-anions and metal cations may be responsible for the decrease in the electro-catalytic activity of Pt. The effect of metal solution contaminants on the Nafion-ionomer of anode electro-catalysts was investigated using Nafion-coated Pt electrode. Voltammetric properties and MOR activities of Nafion-coated and bare Pt electrodes in the presence of Ni solution contaminants were characterized using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The overall results showed a significant negative effect of Ni solution contaminants on the electro-catalytic activity of bare Pt electrode as compared to the Nafion-coated Pt electrode. Based on the results, it appears that Nafion-ionomer film may interact with metal cations (through its sulfonate groups) and repel them away from the Pt active sites, partially inhibiting the negative effect of metal cations on the Pt activity of Nafion-coated Pt electrode. The effect of metal solution contaminants on the carbon-supported platinum nanoparticle (Pt/C) with various Nafion-ionomer distributions and contents (i.e., Nafion-incorporated Pt/C and Nafion-coated Pt/C electrodes) was further investigated. Cyclic voltammetry and EIS techniques were employed to characterize the effect of Ni solution contaminants on the voltammetric properties and MOR activities of Nafion-incorporated and Nafion-coated Pt/C electrodes. The overall results showed a stronger negative effect of Ni solution contaminants on the electro-catalytic activity of Nafion-incorporated Pt/C electrodes as compared to the Nafion-coated Pt/C electrodes. This further confirms previous observations showing the sulfonate groups of Nafion-ionomer film may attract the Ni metal cations, localize them away from the Pt active sites, and subsequently suppress the negative effect of cations on the activity of Nafion-coated Pt/C electrodes.

A mathematical model was developed to predict the exchange of one cation by another in a soil column undergoing one dimensional cation solution displacement under steady state flow conditions. The model allowed prediction of both the solution and exchanger phase concentration of the cation in question. The model consists of a material balance equation which is a parabolic type partial differential equation. The assumption was made that equilibrium was reached instantaneously between the cations in the solution phase and the exchanger phase. This assumption reduced the material balance equation to a form that allowed numerical solution providing the data concerning the cation exchange isotherm and the initial and boundary conditions are available. FORTRAN programs were written for the numerical computation of the problem involved. The computation was done on a digital computer. The model was verified by comparing the theoretically computed cation concentration profile with data from actual soil column experiments. The cation exchange of Mg→Ca was tested on Yolo fine sandy loam, Nibley clay loam and Haford Sandy loam columns. The exchange of Na→Ca was also tested on Yolo fine sandy loam. Satisfactory agreement between the column experiment values and the theoretically computed values was obtained.

Rule-based systems implemented as event-condition-action (ECA) rules utilize a powerful and flexible paradigm when it comes to specifying systems that need to react to complex situation in their environment. Rules can be specified to react to combinations of events occurring at any time in any order. However, the behavior of a rule based system is notoriously hard to analyze due to the rules ability to interact with each other. Formal methods are not utilized in their full potential for enhancing software quality in practice. We argue that seamless support in a high-level paradigm specific tool is a viable way to provide industrial system designers with powerful verification techniques. This thesis targets the issue of formally verifying that a set of specified rules behaves as indented. The prototype tool REX (Rule and Event eXplorer) is developed as a proof of concept of the results of this thesis. Rules and events are specified in REX which is acting as a rule-based front-end to the existing timed automata CASE tool UPPAAL. The rules, events and requirements of application design are specified in REX. To support formal verification, REX automatically transforms the specified rules to timed automata, queries the requirement properties in the model-checker provided by UPPAAL and returns results to the user of REX in terms of rules and events. The results of this thesis consist of guidelines for modeling and verifying rules in a timed automata model-checker and experiences from using and building a tool implementing the proposed guidelines. Moreover, the result of an industrial case study is presented, validating the ability to model and verify a system of industrial complexity using the proposed approach. / Avhandlingen presenterar en ny ansats för att formellt verifiera regel-baserade system. En verktygsprototyp, REX, är utvecklad inom ramen för detta projekt i syfte att stödja ansatsen genom realisering av de teoretiska resultaten. De regler som avses är Event-Condition-Action (ECA) regler, vilket betyder att en regel exekverar ett stycke kod (Action) om ett villkor (Condition) är sant när en specifik händelse (Event) inträffar. ECA-regler är användbara för att speci¯cera beteendet av system som måste reagera på komplexa situationer i sin interagerande miljö. En regel kan till exempel reagera på en kombination av händelser som kan inträffa när som helst och i vilken ordning som helst. Avhandlingen fokuserar på hur man med hjälp av formella metoder kan påvisa att en regelmängd beter sig som förväntat. Användandet av formella metoder för att öka kvalitén på mjukvara är inte så utbrett som det skulle kunna vara. Några av anledningarna kan vara att formella metoder anses svåra att använda och att de kräver extra tid och kunskap. Avhandlingen handlar om en approach där utvecklare kan uttrycka sitt system i ett för dem enkelt språk och där detaljer rörande det formella verktyget döljs av ett verktyg som sköter interaktionen med det formella verktyget. Regler och händelser specificeras som indata till verktyget REX som agerar som en regelbaserad front-end till det formella verktyget UPPAAL. Regler, händelser och egenskaper som modellen ska uppfylla specificeras i REX. Formell verifiering stöds genom att REX automatiskt överför regler och egenskaper till en tidsautomat som kan verifieras av Uppaal. REX startar model-checkern i UPPAAL och returnerar resultatet från analysen till användaren. Resultatet från avhandlingen består av riktlinjer för hur man kan modellera och verifiera regler i en tidsautomat samt erfarenheter från att bygga och använda ett verktyg som implementerar dessa riktlinjer. Därutöver presenteras resultat från experiment och en fallstudie som genomförts för att validera den framtagna ansatsen.

Inorganic cations play decisive roles in many cellular and physiological processes and are essential components of plant nutrition. Therefore, the uptake of cations and their redistribution must be precisely controlled. Vacuolar antiporters are important elements in mediating the intracellular sequestration of these cations. CAXs (for CAtion eXchanger) are members of a multigene family and appear to predominately reside on vacuoles. Defining CAX regulation and substrate specificity have been aided by utilizing yeast as an experimental tool. Studies in plants suggest CAXs regulate apoplastic Ca2+ levels in order to optimize cell wall expansion, photosynthesis, transpiration and plant productivity. CAX studies provide the basis for making designer transporters that have been used to develop nutrient enhanced crops and plants for remediating toxic soils. In my second study, I have characterized and defined autoinhibitory domain of Arabidopsis CAX3. Several CAX transporters, including CAX1, appear to contain an approximately 40 amino acid N-terminal regulatory regions (NRR) that modulates transport through N-terminal autoinhibition. Deletion of the NRR from several CAXs (sCAX) enhances function in plant and yeast expression assays; however, to date, there are no functional assays for CAX3. In this report, we create a series of truncations in the CAX3 NRR and demonstrate activation of CAX3 in both yeast and plants by truncating a large portion of the NRR. Experiments on endomembrane-enriched vesicles isolated from yeast expressing activated CAX3 demonstrate that the gene encodes Ca2+/H+ exchange with properties distinct from CAX1. These studies demonstrate shared and unique aspects of CAX1 and CAX3 transport and regulation. My third study is to express and purify CAX proteins for X-ray crystallographic analysis. In this study, I initiated crystallization of vacuolar membrane localized CAX protein from eukaryotes. Membrane proteins continue to be challenging targets for structural biology because of their hydrophobic nature. We have demonstrated here that eukaryotic Ca2+/H+ exchanger can be successfully expressed in E. coli based expression system. Collectively, our findings suggest that CAX protein can be successfully expressed, detergent solublized and purified from E. coli with a yield sufficient for functional and structural studies.

La présence de cations de métaux de transition en grandes concentrations dans les eaux présente des risques pour la santé humaine et pour l'environnement. Une des méthodes les plus utilisées pour les éliminer est la coagulation-floculation. Il s'agit d'une étape essentielle dans la chaîne de traitement des eaux. Cependant, l'utilisation de cette méthode pour traiter une eau de faible turbidité est difficile. Le recours à l'ajout de montmorillonite est d'un grand intérêt dans ce cas, car cela permet d'intensifier le processus de coagulation-floculation. Afin d'améliorer ce processus, l'ajout d'un floculant, non polluant, non toxique et biodégradable, s'avère nécessaire, d'où l'utilisation du chitosane qui joue deux rôles essentiels: il favorise à la fois la floculation et la complexation des cations des métaux de transition. Le présent travail a pour objet d'étudier l'élimination de trois cations de métaux de transition, à savoir le cobalt, le nickel et le cuivre, par coagulation-floculation du système montmorillonite-chitosane. L'élimination des cations métalliques et de la turbidité de l'eau dépend de plusieurs facteurs, dont le pH et le rapport massique (chitosane/montmorillonite). De même, les concentrations de cations, de chitosane et de montmorillonite peuvent, elles aussi, avoir un effet sur l'élimination de ces cations. Étant donné la multitude des facteurs pouvant influencer l'élimination de ces cations par la montmorillonite et par le chitosane, ainsi que la coagulation-floculation du système montmorillonite-chitosane, l'utilisation d'un plan factoriel s'est avérée nécessaire pour surmonter cette difficulté et déterminer les conditions favorables à une élimination optimale des trois cations étudiés. Les résultats obtenus prouvent qu'il peut exister une synergie entre le chitosane et la montmorillonite dans l'élimination des cations métalliques. La montmorillonite, dont l'efficacité a augmenté après modification chimique, et le chitosane ont montré une affinité différente pour chacun des trois cations métalliques. Cette affinité augmente dans l'ordre suivant: CO²⁺ < Ni²⁺ < Cu²⁺ . Une meilleure coagulation-floculation ne donne pas forcément une meilleure élimination des cations métalliques. Ainsi, l'élimination des cations dans une solution contenant 1 à 3 (g/L) de montmorillonite augmente avec l'augmentation du pH entre 3 et 7, tandis que la turbidité minimale est obtenue à un pH = 5,4 et pour un rapport massique (chitosane/montmorillonite) =0,6%. Ces conditions jugées optimales dans le cadre du présent travail peuvent faire l'objet d'une étude ultérieure plus poussée, visant la mise en oeuvre d'une technologie de démétallisation des eaux. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Chitosane, Montmorillonite, Coagulation, Floculation, Cations métalliques, Synergie.

L'agriculture sur brûlis le long d'un front de colonisation du Bas Tapajos (Para, Brésil) favorise l'émergence d'une double situation d'exposition au risque pour les communautés. D'une part, le brûlis occasionne le transfert du mercure inorganique du sol vers le milieu aquatique utilisé par les populations humaines; de l'autre, les grands palmiers arborescents du genre Attalea dont la fréquence est stimulée par l'agriculture sur brûlis sont des indicateurs importants de l'aléa de la Trypanosomiase américaine, car ses vecteurs, les triatomes (Hemiptera : Triatominae), nichent principalement dans la matière organique qui s'accumule dans leurs couronnes. Notre étude a comme objectif de déterminer comment la distribution des espèces du genre Attalea (Palmae) est liée aux propriétés édaphiques d'un front de colonisation de la région du Bas Tapajos, dans le bassin amazonien. Nous avons identifié 3 espèces d'Attalea et un quatrième palmier considéré comme pouvant être une variété acaule d'Attalea speciosa (A. sp. nain). Nos résultats montrent qu'A. phalerata préfère des sols aux teneurs en cations disponibles plus élevées et moins acides. A. maripa est inversement distribué à A. phalerata et se limite aux sols les plus pauvres en cations disponibles et plus acides. Malgré que la distribution de A. speciosa n'a pas varié fortement avec les propriétés édaphiques mesurées, nos données indiquent que A. sp. nain serait associée à des sols aux teneurs plus élevées en chlore ainsi qu'à des caractéristiques minéralogiques spécifiques des Alfisols. L'effet important des cations majeurs échangeables sur la présence et l'absence des espèces d'Attalea nous a amené à étudier comment ces nutriments sont associés aux propriétés physicochimiques du sol. Nos résultats montrent que le Ca2+ échangeable est corrélé aux oxyhydroxydes de fer associés aux limons fins (2-10 f-lm). Le K+ échangeable est principalement associé à des réserves non échangeables de Ktf. Le Mg2+ échangeable, de taille intermédiaire, est d'une part fortement lié aux facteurs du sol qui contrôlent le Ca2+ échangeable, mais est aussi significativement associé au Mgtf non échangeable. Nos résultats suggèrent de privilégier le rôle individuel de chacun des cations échangeables ainsi qu'une catégorisation granulométrique détaillée pour la recherche en écologie végétale. De plus, la présence de quantités appréciables de Mgtr et de Ktf indique que l'altération des minéraux joue un rôle significatif dans le cycle des éléments nutritifs de ces forêts de terre ferme amazonienne. Enfin, ces découvertes complètent les connaissances actuelles sur le cycle du mercure de la région considérant le rôle capital des cations échangeables dans son relargage du sol. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : palmier, Attalea, cations, sol, texture, minéralogie, Trypanosomiase américaine

Stockholm Water is a water and sewage company with Henriksdal as one of two wastewater treatment plants (WWTPs). At Henriksdal wastewater sludge generated in the wastewater treatment process is digested which generate biogas; a mixture of mainly methane and carbon dioxide. If purified to methane content of 96 - 98 % this gas is called biomethane. Biogasmax is a project aiming to reduce the use of fossile fuels in Europe by providing that biogas is a good technical, economical and environmental alternative as vehicle fuel. The specific aim for Stockholm Water is to increase the biogas production at the existing plant in Henriksdal. Enzymatic treatment of wastewater sludge is an innovative technique earlier proofed to increase the biogas production from wastewater sludge with up to 60 %. The enzyme activity is in turn proven to significantly increase in the presence of a cation binding agent. One aim with this thesis was to investigate if the sludge from Henriksdal wastewater treatment process at all is affected of enzymatic treatment in presence of a cation binding agent since this has shown to have some significance. The chemical oxygen demand (COD) was measured in the liquid phase of sludge after treatment and used as a measurement of treatment effect. Another aim of this thesis was to look into the possibility to increase the methane production from sludge at Henriksdal WWTP. This was investigated through batch laboratory digestion tests. The sludge from Henriksdal WWTP was shown to be a good substrate for the enzymes added. COD in the liquid phase was increased with 17 – 32 % depending on the dose of enzymes and sodium citrate added. Digestion of sludge with a total addition of 18.6 mg enzymes per 1 g total solids (TS) and a concentration of 5 mM sodium citrate increased the methane production with almost 18 % compared to untreated sludge. This equals an increase of 18.3 % when converted to represent a totally blended and continuous digestion chamber at Henriksdal WWTP. The increased methane production also results in a sludge reduction out from the digestion chambers. The increased methane production and sludge reduction though does not fulfil the increased costs for the enzymes and sodium citrate applied. These doses must be decreased and the costs for both enzymes and sodium citrate must be reduced for this technique to be economically feasible in a full scale operation.

Metal chalcogenide nanomaterials are interesting due to their size dependent properties and potential use in numerous types of devices or applications. The synthetic methods of binary phase metal chalcogenide nanoparticles are well established, but finding simple ways to make even more complex nanostructures is important. To this end, two techniques were studied: the cation exchange of metal chalcogenide nanocrystals, CdE → MxEy (E = S, Se, Te; M = Pd, Pt) and the solution phase synthesis of ternary chalcogenide nanoparticles. The effects of cation solvation and the volume change (Delta V) of reaction on the equilibrium and the morphology change in the cation-exchange reactions of CdE → MxEy were investigated. A two-phase solvent environment was particularly efficient in increasing the thermodynamic driving force. The effect of Delta V of reaction on the morphology of the product nanocrystals was also investigated. Depending on the stress developed in the lattice during the reaction, product nanocrystals underwent varying degrees of morphological changes, such as void formation and fragmentation, in addition to the preservation of the original morphology of the reactant nanocrystals. The knowledge of the effect of ion solvation and Delta V of reaction on the equilibrium and product morphology provides a new strategy and useful guide to the application of cation-exchange reactions for the synthesis of a broader range of inorganic nanocrystals. Using a solution phase method, the morphology of CuInSe2 nanoparticles could be tuned from small 10 nm spheres to micron length nanowires by varying the relative amount of strong and weak surfactants passivating the surface. Oleylamine and trioctylphosphine oxide were chosen as the strong and weak surfactants, respectively. Small isotropic structures were formed when the oleylamine was the only surfactant with the size of the nanospheres increasing as the amount of oleylamine decreased. For the CuInSe2 nanowires, weakly-binding dioctylphosphine oxide (DOPO), an impurity in the TOPO, was found to be the key surfactant that enables the anisotropic one-dimensional growth. Detailed analysis of the structure of the nanowires indicated that they grow perpendicular to (112) planes, with twinning around the growth axis by ~60 degree rotation. The nanowires exhibit a saw-tooth surface morphology resembling a stack of truncated tetrahedral.

Clinoptilolite is a natural zeolite which can be used favorably in heavy metal removal. The main mechanisms for metal removal via clinoptilolite are adsorption and ion exchange. Several sources propose to keep the normality constant to obtain equilibrium isotherms for ion exchange systems, while many studies use constant sorbent mass with varying normalities of the sorbate. The objective of this study is to investigate the prevailing mechanisms of clinoptilolite during cadmium removal and the impact of the methodology for obtaining equilibrium isotherms. Batch Cd2+ removal experiments were conducted by using the two different methodologies (i.e. keeping the sorbent mass constant vs. keeping the normality constant) with clinoptilolite in as-received (AsC) and conditioned form (CnC), an ion exchange resin and granulated activated carbon. Exchangeable and framework cations, conductivity and pH were monitored during experiments. The equilibrium results were then fitted to isotherm models. The prevailing mechanisms for Cd2+ removal with clinoptilolite are discussed considering all monitored parameters and isotherm model fits. Use of the methodology was seen to have an effect on the overall Cd2+-clinoptilolite interaction. For example, differences between methodologies are observed regarding maximum sorbed Cd2+ and the distribution of exchangeable cations. Conductivity profiles provided a good indication of presence of ion exchange and demonstrated that it is more dominating for CnC than for AsC. The Cd2+ removal capacities observed in this study (0.65 meq/g for AsC and 1.46 meq/g for CnC) are the highest recorded for a clinoptilolite sample, as can be found in the literature.