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Since many estuarine zones are impacted by copper contamination, there is an on-going effort to develop Biotic Ligand Models (BLMs) predicting copper toxicity in transitional environments. In the first stage of this project, a critical analysis of the BLM framework identified some aspects of the model that required further investigation. In particular, a BLM for estuaries needed (a) a better characterization of the dissolved organic matter (DOC) and its effect on copper availability, and (b) the inclusion in the model’s equation of a salinity-correction factor modulating the relationship between copper accumulation on the biotic ligand and toxicity. The first issue was addressed by modelling the data produced using a Chelex resin method to determine the labile fraction of copper in samples of mixed riverine and estuarine waters. A refined and simplified BLM equation was then presented, accounting for both the DOC characteristics and the relevance of the osmotic gradient in modulating the relationship between copper accumulation and toxicity. A critical analysis of the literature on copper toxicity and salinity led to the hypothesis that copper-exposed fish are more sensitive to osmotic stresses, as copper interferes with their osmoregulatory pathways. In particular, the cytosolic isoform-2 of the enzyme carbonic anhydrase (CA2) was identified as an osmotic effector protein targeted by copper and involved in osmotic stress response pathways, hence representing a mechanistic link between the combined effects of copper exposure and osmotic stress. To test this hypothesis, two in vivo studies were performed, using the euryhaline fish sheepshead minnow (Cyprinodon variegatus) and applying different rates of salinity changes as a way of dosing osmotic stress. The results showed a disturbance in plasma ion homeostasis after the salinity transitions, but notably the magnitude of the disturbance was greater in the copper-exposed individuals, suggesting a sensitizing effect of copper on the responses of fish to osmotic stress. Gene expression data demonstrated that CA2 is targeted by copper and confirmed the role of the enzyme in osmoregulatory pathways, as further supported by a promoter analysis of the gene coding for zebrafish CA2, which revealed the presence of osmotic-stress related elements. Overall, these results suggest that CA2 is an osmotic effector protein whose response can be activated by a medium level of osmotic stress through a combination of transcriptional and post-translational control circuits.

The major aim of this work has been to gain further insights into the catalytic mechanism of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1). One approach has been to replace the essential Zn(II) ion by Cd(II) which has favourable spectroscopic properties. The Cd(II)-enzymes have appreciable 4-nitrophenyl acetate hydrolase activities. These activities increase with pH as if dependent on the basic form of a group with pKa near 10. The Cd(II)-carbonic anhydrases also have significant carbon dioxide hydration activities. Jhe Cd(II) derivatives are strongly inhibited by monovalent anions. The 113-Cd(II) derivatives have also been studied by 113-Cd NMR as a function of pH and bicarbonate or inhibitor concentration. Plots of chemical shift versus pH give sigmoidal titration curves in the studied pH range, 10.3. The p«a values vary from 9.2 to 9.7 correlating reasonably well with the activity profiles. When bicarbonate is added to the samples the 113-Cd resonances shift upfield to new characteristic positions. The inhibitors CN", SH", and SCN” bind directly to the metal ion with their C, S, and N atoms, respectively. The results are best explained by assuming a rapid exchange between three species in which the open coordination site of the metal ion is occupied by'hydroxide, water, or bicarbonate. Another approach has been to study kinetic properties of the active en­zyme. A number of monovalent anions were investigated as inhibitors of carbon dioxide hydration catalyzed by human carbonic anhydrase II. Predominantly uncompetitive inhibition patterns were observed at pH near 9 in all cases. The inhibition of human carbonic anhydrase II by the organic compounds tetrazole, 1,2,4-triazole, 2-nitrophenol, and chloral hydrate was also investigated. These inhibitors, together with phenol, can be classified in three groups depending upon the kinetic patterns of inhibition of carbon dioixde hydration at pH near 9. The first group, represented by tetrazole and 2-nitrophenol, yields predominantly uncompetitive inhibition under these conditions in analogy with simple, inorganic anions. The second group, represented by 1,2,4-triazole and chloral hydrate gives rise to essentially noncompetitive inhibition patterns whereas phenol, representing the third group, is a competitive inhibitor of carbon dioxide hydration. These results are analyzed in terms of two rivaling mechanism models, a kinetic scheme originally proposed by Steiner et al. (Eur. 3. Biochem. (1975) 59, 253-259) and a rapid-equilibrium kinetic scheme proposed by Pocker and Deits (3. Am. Chem. Soc. (1982) 104, 2424-2434). It is concluded that the observed steady-state inhibition patterns are compatible with both models, but hat discriminatory data, strongly favouring the model of Stêiner et al., are available in the literature. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1984, härtill 4 uppsatser</p> / digitalisering@umu

In an effort to produce the tetrahedrally coordinated, catalytically active zinc center, three families of tris(2-pyridyl)methanol derivatives were synthesized and characterized. Zinc binding studies revealed that the binding behaviors of the ligands depended on the steric and electronic properties of the substituents on the pyridyl rings, as well as the functional group on the tertiary alcohol. A novel tris-pyridyl macrocyclic receptor was synthesized. The receptor possesses both hydrogen bond donors and acceptors. NMR titration experiments revealed that the receptor simultaneously bound both ammonium cation and the counter anion. The counter anion significantly influences the association between the receptor and the ammonium cation. Chiral ditopic macrocycles, which enantioselectively bind chiral ammonium cations, have also been synthesized. Their enantioselective binding properties, as well as the ditopic recognition properties were investigated

A obesidade e a cárie dentária são problemas de saúde pública, que atingem a população infantil. O objetivo deste estudo foi identificar a prevalência de cárie dentária e relacioná-la com a concentração da enzima anidrase carbônica VI, do íon cálcio, fluxo salivar e quantidade de biofilme dentário em crianças com sobrepeso/obesidade. Foram avaliadas 112 crianças de 4 a 6 anos de idade, de ambos os gêneros. A análise antropométrica foi realizada (percentil do IMC) e através dessa análise as crianças foram divididas em dois grupos: G1 sobrepesos/obesos (n=41) e G2 normais (n=71). Os exames bucais realizados para a cárie dentária foram os índices ceo-s e ICDAS II, quantidade de biofilme dentário pelo Índice de Placa de Turesky e volume de fluxo salivar estimulado. A concentração do íon Cálcio na saliva foi analisada pelo kit colorimétrico e da enzima Anidrase Carbônica VI pelo kit ELISA. Na sequência, as crianças de cada grupo foram divididas em 3 subgrupos: LC (livres de cárie), LI (com lesões iniciais) e C (com cárie). Os testes Wilcoxon, Mann-Whitney, teste t e correlação de Spearman foram aplicados (p<0,05). Não houve diferença significativa no ceo-s entre os grupos. Houve maior concentração média de cálcio salivar no G1 (G1=2847,96mM; G2=1230,90mM;p=0,001) e maior concentração da Anidrase Carbônica VI no G2 (G1=3455,18 pg/mL; G2=442428,9pg/mL;p=0,000). No G1 houve correlação negativa entre o ceo-s e íon Cálcio (r=-0,444;p=0,010). Já no G2, houve correlação negativa entre placa e a Anidrase Carbônica VI (r=-0,551;p=0,014). Pode-se concluir que o íon cálcio é fator protetor para cárie dentária em crianças. Já a anidrase carbônica VI parece não ser biomarcador para a cárie dentária. / Obesity and dental caries are public health problems that affect the child population. The aim of this study was to identify the prevalence of dental caries and relate it to the concentration of the enzyme carbonic anhydrase VI, calcium ion, salivary flow, and dental plaque in overweight/obesity children. The study was conducted on 112 children aged 4-6, of both genders. Anthropometric analysis was performed (BMI percentile) and by this analysis the children were divided into two groups: G1 - overweight/obese (n=41) and G2 - normal (n=71). The oral examinations performed for dental caries were the dmfs and ICDAS II indexes, measurement of the amount of dental plaque by the Turesky Board Index and volume of stimulated salivary flow. The concentration of calcium ion in saliva was measured by a colorimetric kit and the enzyme carbonic anhydrase VI by an ELISA kit. Then, children from each group were divided into three subgroups: CF (caries-free), IL (initial lesions) and D (decayed teeth). The Wilcoxon test, Mann-Whitney, t test and Spearman correlation (p<0.05) were applied. There was no significant difference in the dmfs between groups. There was higher concentration of salivary calcium in G1 (G1=2847.96mM; G2=1230.90mM; p=0.001), and higher concentration of carbonic anhydrase VI in G2 (G1 = 3455.18 pg/ml; G2 = 442428.9pg/ml; p = 0.000). In G1, there was negative correlation between dmfs and salivary calcium (r = -0.444; p = 0.010). In G2, there was negative correlation between dental plaque and carbonic anhydrase VI (r=-0.551; p=0.014). It can be concluded that the calcium ion is a protective factor for dental caries in children. The carbonic anhydrase VI does not seem to be a biomaker of dental caries.

Branchial carbonic anhydrase was purified from silver seabream (Sparus sarba) and antibody against the enzyme was obtained by immunization in rabbits. An assay for quantifying the activity of carbonic anhydrase was developed. Using enzymatic and immunological techniques, the activity, expression and distribution of branchial carbonic anhydrase of silver seabream acclimated to different salinities were studied. Fish gill is one of the most important organs involved in various homeostatic processes. The ability of euryhaline fish to maintain constant internal ionic balance is crucial for the survival of the fish upon change in salinity. The presence of carbonic anhydrase in the chloride cells was suggested to be an important enzyme involved in ion regulation of fish. / In the present study, branchial carbonic anhydrase and erythrocyte carbonic anhydrase were purified from the gill cells of silver seabream with p-aminomethylbenzenesulfonamide-agarose affinity column. They were predominantly cytosolic with a molecular size of 26.6 k Da for branchial carbonic anhydrase and 28.6 k Da for erythrocyte carbonic anhydrase. Investigation of kinetic properties towards the inhibitor acetazolamide has helped determine the inhibition constants (Ki of branchial carbonic anhydrase: 0.54 x 10-9; Ki of erythrocyte carbonic anhydrase: 0.22 x 10-9). The difference in molecular size and inhibition constant towards acetazolamide supported the view that branchial carbonic anhydrase and erythrocyte carbonic anhydrase were two different isozymes. Polyclonal antibody specific to seabream branchial carbonic anhydrase was obtained by immunization in rabbit. The distribution of branchial carbonic anhydrase in the gill of seabream acclimated to different salinities was studied with immunohistochemical method. The enzyme was mainly located at the interlamellar region. The effect of salinity (0, 6, 12, 33, 50 and 70 &permil;) acclimation on the expression and activities of branchial carbonic anhydrase has shown a U-shape pattern from freshwater to double-strength seawater on the quantity of seabream branchial carbonic anhydrase. Higher amount of branchial carbonic anhydrase in freshwater was consistent with the current view that the enzyme was actively involved in the ion uptake process through the hydration of carbon dioxide to produce bicarbonate ion and proton for the exchange of chloride and sodium ions, respectively. An interesting finding was obtained with elevated amount of branchial carbonic anhydrase in seabream acclimated to double-strength seawater and the possible role of the enzyme in such extreme environment was discussed. / This study has provided useful information on the properties, localizations and activities of branchial carbonic anhydrase in silver seabream for the understanding of the involvement of the enzyme in salinity adaptation of silver seabream. / Ma, Wing Chi Joyce. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3250. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 127-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Biomineralization refers to the production of mineralized tissues by organisms. The fine control which organisms can exert over this process produces crystals with morphologies and properties contrasting to that of non-biogenic crystals and specifically altered to suit the required functional need. A key model system of biomineralization are a unicellular marine algae, coccolithophores, which produce calcium carbonate scales known as coccoliths. These coccoliths are comprised of arrangements of single crystals of calcite interlocked to form a plate-shaped structure. Coccoliths are developed intracellularly in a specialised compartment called the coccolith vesicle, before being extruded to the cell surface. In this work, two vital components of the coccolith biomineralization process are investigated - a soluble polysaccharide thought to act as a habit modifier and an insoluble organic scaffold known as a baseplate that provides the surface for nucleation and growth of the crystals. Whilst both these elements are thought to play a key part in the biomineralization process, the role of each is not fully understood. To investigate the effect of coccolith-associated polysaccharides (CAPs) on nucleation and polymorph selection, two systems that promote different polymorphs of calcium carbonate were utilised. In both systems, the intracrystalline polysaccharide fraction extracted from one species, Gephyrocapsa oceanica, was able to promote calcite nucleation in vitro, even under conditions favouring the kinetically-privileged polymorphs of calcium carbonate: vaterite and aragonite. As this property is not observed with CAPs extracted from its 'sister species', Emiliania huxleyi, the in vivo function of CAPs may differ between the two species. Both cryo-transmission electron microscopy (cryoTEM) and scanning electron microscopy (SEM) were used to determine the mechanism of calcite growth in the presence of G. oceanica CAPs, showing its impact on the forming amorphous calcium carbonate (ACC), decreasing the size of the particles and producing irregular, angular particles. Using cryo-electron tomography (cryoET), it was possible to create a 3D representation of the structure of the baseplate from the coccolithophore Pleurochrysis carterae, revealing its two-sided organisation. Examination of several stages of the coccolith growth process demonstrated the interlocking nature of the calcite crystals that make up the coccolith and the progression of the crystal morphologies over time, and the interaction of these crystals with the baseplate rim. Additionally, the effect of inhibiting carbonic anhydrase (CA), an enzyme involved in the regulation of carbonate species, revealed that inhibition of CA can affect coccolithogenesis as well as cell proliferation.

Polyheterocycles such as polythiophene and its derivatives comprise an important class of conducting polymers used for electronic applications. They have been of great interest for use in electronic materials due to their increased environmental stability as well as novel electronic properties in their polymer states. We have been interested in exploring the electronic properties of organometallic analogues of the low-band-gap polymer poly(benzo[3,4-c]thiophene) (polyisothianaphthene) that incorporates η5-cyclopenta[c]thienyl monomers such as ferroceno[c]thiophene. First chapter of this dissertation involved synthetic attempts to ferroceno[c]thiophene. Exploring a shorter synthetic route to starting material, 1,2-di(hydroxymethyl)ferrocene was the first task. This was followed by attempts to synthesize an important precursor, 1,3-dihydroferroceno[c]thiophene to our target molecule, ferroceno[c]thiophene. In order to achieve our target precursor molecule, 1,3-dihydroferroceno[c]thiophene, we reacted 1,2-di(hydroxymethyl)ferrocene with H2S/H2SO4 and Na2S/HBF4 respectively. Reaction of 1,2-di(hydroxymethyl)ferrocene with either H2S/H2SO4 or Na2S/HBF4 results in 2,16-dithia[3.3](1,2)ferrocenophane instead of monomeric 1,3-dihydroferroceno[c]thiophene. Dehydration of 1,2-di(hydroxymethyl)ferrocene with dilute H2SO4 resulted in 2,16-dioxa[3.3](1,2)ferrocenophane. Formation of the five-membered tetrahydrothiophene or tetrahydrofuran rings is probably disfavored compared to formation of the ten-membered ferrocenophane rings because of greater strain in the five-membered rings. Thus, in order to achieve our target molecule ferroceno[c]thiophene, we took an alternate route. We decided to pursue the route with 1,4-dihydro-2,3-ferrocenodithiin being the precursor to our final target molecule. This was successfully accomplished. 1,2-Di(hydroxymethyl)ferrocene reacts with thiourea in the presence of catalytic trifluoroacetic acid to give a water-soluble thiouronium salt, which reacts with aqueous potassium hydroxide in air to give 1,4-dihydro-2,3-ferrocenodithiin, via oxidation of the intermediate 1,2 di(mercaptomethyl)ferrocene. 1,4-dihydro-2,3-ferrocenodithiin, an important precursor to our desired heterocyclic chemistry was synthesized. The increased emission of CO2, a greenhouse gas, to the atmosphere is a matter of serious worldwide concern. Every year a few gigatons of CO2 are added to the atmosphere by various anthropogenic activities like burning of fuel for electricity, running industry and transportation. Thus, developing ways to reduce the emission of CO2 to the atmosphere is of major importance. Although the amine-based absorption method is considered the most reliable, it is an expensive alternative. The catalyzed enhancement of CO2 absorption is a critical component to reduce the capital cost of CO2 capture. Specifically, an effective catalyst will increase the CO2 hydration rate, thereby decreasing the size of the absorber tower needed. In biological systems, CO2 hydration is catalyzed by the enzyme carbonic anhydrase, which contains ZnII in its active site. Carbonic anhydrase typically is not stable enough to be used in an industrial process, therefore, there is a need to synthesize robust, inexpensive CO2 hydration catalysts. Majority work of this dissertation focuses on designing catalysts that show high CO2 hydration rate similar to carbonic anhydrase while showing superiority towards temperature, pH and inhibitors. We focused our efforts on complexes of Zn, Cu and Co with ligands such as 1,4,7,10-tetraazacyclododecane (cyclen), 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (teta and tetb), tris(benzimidazolylmethyl)amine (BIMA) and anionic tris(pyrazolylborate)s that mimic the enzyme, carbonic anhydrase. Several of these complexes have been reported for their interesting CO2 capture properties but they contain hazardous perchlorate ion. We desired to replace them with benign, non-coordinating counterions like PF6-, BF4-, Cl-, CH3COO-, NO3-, CF3SO3-, SiF62- that avoid the potentially explosive perchlorate salts. In order to test the activity of synthesized catalysts under industrial capture conditions, we designed a quick experimental screening pH drop method. [[Zn(cyclen)(H2O)][SiF6]•2H2O as well as a number of other catalysts have been synthesized and tested for their post-combustion CO2 capture enhancement capabilities in aqueous solvent mixtures under both pH-drop screening and stopped-flow conditions. [Zn(cyclen)(H2O)][SiF6]•2H2O, which has an unreactive counteranion, is found to catalyze CO2 hydration in aqueous solvent mixtures under both pH-drop screening and stopped-flow conditions. However, under pH-drop which has conditions similar to industrial post combustion capture, activity of Zn(cyclen)(H2O)][SiF6]•2H2O drops as compared to observed in stopped-flow conditions probably because of bicarbonate coordination to Zn active site in these systems. The Zn center is highly electron deficient and therefore easily coordinates anions, inhibiting the ability to reform hydroxyl species on the metal. Thus, we decided to test the catalysis of benchmark enzyme carbonic anhydrase under similar conditions to determine the threshold value. Carbonic anhydrases catalyze the hydration of carbondioxide at ambient temperatures and physiological pH with the highest known rate constant= 106 M–1 s–1, but in our system (CAER pH drop screening) came out to be 438797 M–1 s–1. The lower catalytic rate constant for carbonic anhydrase in 0.1000 M K2CO3, similar to Zn-cyclen, strengthens the conjecture that at high bicarbonate concentrations, HCO3– binding to the Zn(II) active site slows catalysis by inhibiting bicarbonate displacement with water to regenerate the active species. The complexes containing anionic ligands that donate electron density into the metal center may serve to remove anionic bicarbonates/carbamates from the secondary coordination sphere and away from the metal center, thereby facilitating bicarbonate/anion dissociation and increasing CO2 hydration rates. We studied catalysis of trispyrazolylborate molecule in 30% MEA and found the molecule to be catalytically active. We also developed an NMR-based method to see if the coordination of solvents to CO2 capture solvents can be studied.

Les benzoxaboroles (qui sont les dérivés cycliques des acides boroniques) s’imposent comme une nouvelle classe de molécules intéressantes, pour la formulation de nouveaux agents thérapeutiques (notamment avec la commercialisation de l’AN2690, dérivé fluoré du benzoxaborole, en tant qu’antifongique), tout comme la formation de matériaux fonctionnels. A ce jour, il n’y a pas eu de mise au point de synthèse systématique de petites molécules benzoxaboroles multivalentes, limitant ainsi leur utilisation. Dans le cadre de cette thèse, différentes approches de synthèse ont été développées et ont permis l’obtention de 12 nouveaux composés bi- ou tri-valents. La principale réactivité de ces composés est leur capacité à complexer les cis-diols. Étant donné que la force de l’interaction est dépendante de la nature du substrat, il est important de pouvoir l’analyser en détail. La complexation entre les benzoxaboroles avec les diols est généralement réalisée par la méthode spectrofluorimétrique qui est controversée, particulièrement dans le cas des molécules organoborées multivalentes. Pour analyser en détail la stœchiométrie de ces complexations, une méthode RMN multinucléaire en solution a ici été développée. Les composés bi- ou tri-valents ont ensuite été testés en tant qu’agent thérapeutique. En effet, récemment, l’activité d’inhibition contre l’anhydrase carbonique de motifs benzoxaboroles monovalents a été discutée dans la littérature. La multivalence nos molécules pouvant être un avantage, leur constante d’inhibition contre l’anhydrase carbonique ont été mesurées. Enfin, dans un tout autre domaine, la réactivité avec les diols et la multivalence de nos composés ont été mises en avant par la formation de nouveaux complexes moléculaires. / Benzoxaboroles (which are cyclic derivates of boronic acids) are emerging as an interesting class of molecules for the design of news therapeutic agents (in particular with the commercialization of AN2690, which is a fluorinated derivative of benzoxaborole, as an antifungal agent), as well as for the formation of functional materials. However, to date, only few syntheses of small molecules of multivalent benzoxaborole have been described in the literature. In this thesis, different approaches were explored to isolate 12 new bi- or tri-valent benzoxaboroles. The main reactivity of these compounds is their ability to bind to cis-diols. Given that the strength of interaction between an organoboron molecule and a diol depends on the nature of the interacting counterparts, it is important to be able to measure it in detail. The complexation between benzoxaboroles and cis-diols is usually studied by a spectrofluorimetric method, which is controversial, especially in the case of multivalent benzoxaboroles. To analyse in detail the stoichiometry of these complexation, a multinuclear NMR method in solution was developed in this thesis. The di- or tri-valent benzoxaboroles were then tested as therapeutic agents. Indeed, recently, the activity of monovalent benzoxaborole against carbonic anhydrases was discussed in the literature. Since the multivalence of our molecules could be a benefit, the measurement of their inhibition constants against carbonic anhydrases was performed. Finally, in a completely different type of application, their reactivity toward cis-diols and their multivalence was put forward to create new complexes.