This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

This thesis is focussed on structural and functional studies of a novel glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. GpdQ is highly promiscuous and is the first known phosphatase which is capable of degrading all three classes of phosphate esters (mono-, di- and triesters). Remarkably, GpdQ is also able to hydrolyse stable aliphatic phosphate esters and has been shown to degrade the hydrolysis product of the nerve agent VX. For these reasons, GpdQ has been realised to have potential as a powerful bioremediator for the removal of organophosphate pesticides and nerve agents. GpdQ is a binuclear metallohydrolase in which one of the metal ions is very weakly bound. Chapter 1 introduces the catalytic mechanisms of binuclear metallohydrolases by examining two related phosphate ester-degrading enzymes. Since one of the main features of catalysis addressed in this thesis are the differential metal binding affinities of GpdQ, Chapter 1 also canvasses a range of other binuclear metallohydrolases with similar behaviour. Chapter 2 examines the structural and evolutionary relationship between GpdQ and a number of other related enzymes. Using genome database searches, the two most closely related enzymes are identified. In performing these searches, a novel, putative binuclear metallohydrolase from Homo sapiens is also discovered. This enzyme, Hsa_aTRACP, is most closely related to PAPs, however construction of a homology model indicates that the active site tyrosine residue of PAP is replaced by histidine. In this respect, it may represent an evolutionary link to Ser/Thr protein phosphatases and GpdQ. The biology and chemistry of this putative enzyme is discussed. PAPs are the only binuclear enzymes with an established heterovalent active site of the type Fe(III)-M(II) (where M=Fe, Zn or Mn) whereas the majority of enzymes in this family have homovalent metal centres, including GpdQ and Ser/Thr protein. This is brought about due to the nature of the coordination sphere imposed by the enzyme. The activity of GpdQ can be reconstituted in the presence of Co(II), Zn(II), Mn(II) and Cd(II). Chapter 3 examines the kinetic properties of a binuclear homovalent system by studying the kinetic properties of Cd(II)-substituted GpdQ and a corresponding model complex. This comparative study leads to the identification of a terminal hydroxide molecule as the likely reaction-initiating nucleophile in Cd(II)-GpdQ with a pKa of 9.4. In Chapter 4, a detailed study of the structural, kinetic and spectroscopic behaviour of Co(II)-substituted GpdQ is presented. This chapter specifically probes the formation of the binuclear active site, the role of the metal ions in catalysis, the identity of the nucleophile and the potential role of any first or second coordination sphere residues in the regulation of enzyme activity, proton donation and metal ion coordination. Based on these findings, a detailed reaction mechanism is proposed in which the substrate itself promotes the formation of the catalytically competent binuclear centre and phosphorolysis occurs following nucleophilic attack by a terminal hydroxide molecule. A potential role of Asn80 (a ligand of one of the metal ions) in regulating both substrate and metal binding, and the role of the bridging hydroxide molecule in the activation of the terminal nucleophile is proposed. Chapter 5 employs a combination of kinetic and spectroscopic techniques to probe the proposed catalytic mechanism of GpdQ in depth. The formation of the catalytically competent binuclear centre is observed in pre-steady state studies, an integral first step in the catalytic mechanism. The dissociation and rate constants associated with formation of the binuclear centre are quantified. The rate of substrate turnover in GpdQ is relatively modest but is enhanced by a structural rearrangement involving the flexible Asn80 ligand. This structural change fine-tunes the reaction mechanism, leading to optimal reactivity. The steady-state kinetic properties of a series of metal ion derivatives (Co(II), Cd(II) and Mn(II)) of GpdQ and their reactivity towards a number of substrates are also compared. These findings lead to the conclusion that the reaction mechanism of GpdQ is modulated by both substrate and metal ion. In this respect, GpdQ is adaptive to the environmental conditions to which it is exposed by employing a flexible mechanistic strategy to achieve catalysis. Chapter 6 correlates the electronic and geometric structure of the binuclear centre in GpdQ as a means to probe specific aspects of the mechanism. This study uses the wild type enzyme and a site-directed mutant (Asn80Asp) to examine the structure of the metal ions at two stages of catalysis. The role of the bridging hydroxide molecule in nucleophilic activation is specifically addressed by monitoring changes in the electronic exchange interaction and other structural parameters as a result of phosphate binding. Also, the coordination environment of the metal ions in both the free enzyme and the phosphate-bound enzyme of wild type and Asn80Asp GpdQ were assessed against the currently proposed structures. The findings in this chapter corroborate the proposed catalytic mechanism of GpdQ. In summary, this project led to a detailed understanding of the mechanism of GpdQ, and provided insight into how both the metal ion composition and the identity of the substrate may modulate this mechanism. The knowledge gained may lead to the design of catalytically more efficient derivatives (mutants) of GpdQ for application in bioremediation.

Neste trabalho, apresentamos resultados da caracterização Magneto-Óptica de íons terras-raras (Nd3+ e Yb3+) em monocristais de niobato de lítio (LiNbO3). Medidas de Dicroísmo Circular Magnético (MCD) e de Emissão Circularmente Polarizada em Presença de Campo Magnético (MCPE) foram realizadas pela primeira vez nesses sistemas. Os resultados foram obtidos à temperatura de 2K, e em campos magnéticos de até 5 T. Através desses estudos, foi possível identificar os números quânticos cristalinos (&#956) dos subníveis Zeeman desses íons. A partir da dependência do sinal de MCD com a intensidade de campo magnético, determinou¬se o fator giromagnético efetivo g// do estado fundamental de cada íon, obtendo-se os valores: g//Nd = (1,4 &#177 0,1) e g//Yb = (4,7 &#177 0,1). Esses valores foram confirmados através de medidas de espectroscopia de Ressonância Paramagnética Eletrônica (EPR), realizadas a baixa temperatura (4-8 K), em banda X. Os espectros de EPR foram tomados em função da orientação relativa do campo magnético externo com o eixo c cristalino dos cristais, em 3 planos perpendiculares entre si. Os espectros de EPR mostram a existência de diferentes sítios ocupados pelos íons terras-raras. O sítio mais populado tem simetria axial, e para esse centro determinaram-se os fatores g efetivos g//Nd = (1,440 &#177 0,005) e g//Nd = (2,959 &#177 0,004), para o íon Nd3+, e g//Yb = (4,705 &#177 0,008) e g//Yb = (2,693 &#177 0,005) para o íon Yb3+. Espectros de MCD e MCPE obtidos para um cristal de rubi ilustram as convenções utilizadas e atestam que o sistema experimental funciona adequadamente. Os espectros obtidos nessa amostra também são originais, tendo sido resolvidas as transições permitidas com luz circularmente polarizada entre os subníveis Zeeman correspondentes aos níveis de energia 4A2 e &#8254E (2E) do íon Cr3+. / In this work we present Magneto-Optícal characterizations of rare-earth ions (Nd3+ e Yb3+) in lithium niobate (LiNbO3) single crystals. Magnetic Circular Dichroism (MCD) and Magnetic Circularly Polarized Emission (MCPE) measurements were performed for the first time on those systems. Spectra were obtained at 2K and at magnetic field strength up to 5T. From these studies, it was possible to assign the crystal quantum number (&#956) of the Zeeman sublevels of these ions, so that the sign and allowance of the electronic transitions could be predicted. From the dependence of suitable MCD spectral lines on the magnetic field strength, the effective parallel gyromagnetic factor (g//) of the ground state for each of the rare earth ions has been determined to be: g//Nd = (1,4 &#177 0,1) e g//Yb = (4,7 &#177 0,1). These values are in dose agreement to those obtained by means of Electron Paramagnetic Resonance (EPR) spectroscopy, at 4-8 K, and at X-band frequency. EPR spectra were recorded as a function of the external magnetic field orientation relative to the c crystalline axis in three mutual perpendicular planes. These spectra show evidence of multiple sites occupied by the rare-earth ions. For the most intense line seen in the spectra of each ion, it could be clearly assigned a site with axial symmetry, with effective g factors of g//Nd= (1,440 &#177 0,005) and g//Nd = (2,959 &#177 0,004), for the Nd3+3+ ion, and g//Yb = (4,705 &#177 0,008) and g//Yb= (2,693 &#177 0,005) for the Yb3+ ion. MCD and MCPE spectra recorded for a ruby crystal shows the experimental conventions used so far in this work, as well as assure that the experimental system works properly. These results are original ones, by means of which, the spectral transitions between the Zeeman sublevels of the 4A2 and &#8254E (2E) of the Cr3+ ions in ruby could be resolved.

Neste trabalho, descrevemos um espectrômetro ótico para medidas de Dicroismo Circular Magnético (DCM), utilizado nas seguintes experiências: 1) Medidas de tempo de relaxação spin-rede (T1) para centros F em NaCl, NaBr, CsBr e CsCl, a temperatura de 1,8&#176K em campos magnéticos até 17000Gs. Verificamos a validade da teoria da referência (8) para explicar as diferenças observadas, no comportamento de \'T IND.1\', para halogenetos com diferentes íons alcalinos, bem como diferentes estruturas. Comprovamos que a interação hiperfina ainda continua a ser o mecanismo mais importante para esses centros. Verificamos também que, para temperaturas entre 6&#176K e l5&#176K, os valores de experimentais para T1, em KBr, concordam razoavelmente com a teoria da referência (21). Esta terapia é uma extensão daquela da referência (8). 2) Espectros de DCM para KCl: Co++ e CaF2: Co++ foram obtidos para campos magnéticos ate 56KGs e temperaturas entre 1,8&#176K e 4,2 &#176K. Os resultados obtidos mostraram ser concordantes com a hipótese dos centros Co++ ocuparem sítios intersticiais na rede de KCl. / In this work we describe a Magnetic Circular Dicroism Spectrometer wich was used in the following experiments: 1) We measured the spin-lattice relaxation time (T1) for F centers in NaCl, NaBr, CsBr and CsCl, at 1,8&#176K in magnetic fields up to 15000Gs. We verified the suitability of the theory of ref.(O8) to explain the differences observed for halides of differents alkali ions as well as for different structures. This proves that the hyperfine interaction is the most important mechanism for this kind of centers. We also verified that, for temperatures between 6&#176K and l5&#176K, T1, the T1 experimental values fits the theory of ref.(21) reasonably well ,for F centers in KBr. This theory is an extension of that of ref.(8). 2) We obtained the MCD spectra for KCl: Co++ and CaF2: Co++ in different magnetic fields up to 56KGs, and in temperature range between 1,8 &#176K and 4,2&#176K. Our results are consistent with the assumption that Co++ centers are intersticial in KCl lattice.

Neste trabalho, descrevemos um espectrômetro ótico para medidas de Dicroismo Circular Magnético (DCM), utilizado nas seguintes experiências: 1) Medidas de tempo de relaxação spin-rede (T1) para centros F em NaCl, NaBr, CsBr e CsCl, a temperatura de 1,8&#176K em campos magnéticos até 17000Gs. Verificamos a validade da teoria da referência (8) para explicar as diferenças observadas, no comportamento de \'T IND.1\', para halogenetos com diferentes íons alcalinos, bem como diferentes estruturas. Comprovamos que a interação hiperfina ainda continua a ser o mecanismo mais importante para esses centros. Verificamos também que, para temperaturas entre 6&#176K e l5&#176K, os valores de experimentais para T1, em KBr, concordam razoavelmente com a teoria da referência (21). Esta terapia é uma extensão daquela da referência (8). 2) Espectros de DCM para KCl: Co++ e CaF2: Co++ foram obtidos para campos magnéticos ate 56KGs e temperaturas entre 1,8&#176K e 4,2 &#176K. Os resultados obtidos mostraram ser concordantes com a hipótese dos centros Co++ ocuparem sítios intersticiais na rede de KCl. / In this work we describe a Magnetic Circular Dicroism Spectrometer wich was used in the following experiments: 1) We measured the spin-lattice relaxation time (T1) for F centers in NaCl, NaBr, CsBr and CsCl, at 1,8&#176K in magnetic fields up to 15000Gs. We verified the suitability of the theory of ref.(O8) to explain the differences observed for halides of differents alkali ions as well as for different structures. This proves that the hyperfine interaction is the most important mechanism for this kind of centers. We also verified that, for temperatures between 6&#176K and l5&#176K, T1, the T1 experimental values fits the theory of ref.(21) reasonably well ,for F centers in KBr. This theory is an extension of that of ref.(8). 2) We obtained the MCD spectra for KCl: Co++ and CaF2: Co++ in different magnetic fields up to 56KGs, and in temperature range between 1,8 &#176K and 4,2&#176K. Our results are consistent with the assumption that Co++ centers are intersticial in KCl lattice.

No description available.

No description available.

Structural characterization is essential for the understanding of the magnetic properties of thin films and multilayers. In this thesis, both crystalline and amorphous thin films and multilayers were analyzed utilizing transmission electron microscopy (TEM). High resolution TEM and electron diffraction studies emphasize on the growth of amorphous Fe91Zr9 and Co68Fe24Zr8 on both Al2O3 and Al70Zr30 in multilayer structures by magnetron sputtering. The properties of the growth surfaces were found to strongly influence the formation of nano-crystallites of the magnetic material at interfaces. Field induced uniaxial magnetic anisotropy was found to be possible to imprint into both fully amorphous and partially crystallized Co68Fe24Zr8 layers, yielding similar magnetic characteristics regardless of the structure. These findings are important for the understanding of both growth and magnetic properties of these amorphous thin films. As magnetic systems become smaller, new analysis techniques need to be developed. One such important step was the realization of electron energy-loss magnetic circular dichroism (EMCD) in the TEM, where information about the ratio of the orbital to spin magnetic moment (mL/mS) of a sample can be obtained. EMCD makes use of angular dependent inelastic scattering, which is characterized using electron energy-loss spectroscopy. The work of this thesis contributes to the development of EMCD by performing quantitative measurements of the mL/mS ratio. Especially, methods for obtaining energy filtered diffraction patterns in the TEM together with analysis tools of the data were developed. It was found that plural inelastic scattering events modify the determination of the mL/mS ratio, wherefore a procedure to compensate for it was derived. Additionally, utilizing special settings of the electron gun it was shown that EMCD measurements becomes feasible on the nanometer level through real space maps of the EMCD signal.

In order to understand the properties of thin film devices, knowledge of the material's structure is essential. The work presented here combines magnetic and structural characterization of the systems studied to gain a deeper physical understanding. The magnetic properties have been studied with a combination of x-ray magnetic circular dichroism, SQUID magnetometry and magneto-optical Kerr effect. For the structural characterization, x-ray reflectivity and diffraction have been used, complemented by neutron diffraction and transmission electron microscopy. One structural property that affects the magnetic moment in metal-on-metal superlattices is interdiffusion between the layers. This is discussed for bcc Fe/Co(001) and bcc Fe81Ni19/Co(001) superlattices. The effect of interdiffusion was seen as a large region of enhanced magnetic moments as compared to theoretical calculations, which assume perfectly sharp interfaces. For the Fe81Ni19/Co(001) superlattices the chemical interface region, as revealed by neutron diffraction, was in good agreement with the region of magnetic enhancement. Another structural property that has been investigated is the strain in the magnetic layers. This does not affect the spin magnetic moment to a large extent. However the magnetocrystalline anisotropy and the orbital moment are affected by the presence of strain. The effects on the orbital moment from strain and interfaces for Fe in Fe/V superlattices was studied, and it was found that the two contributions were separable. In this context the effect of strain on the out-of-plane magnetocrystalline anisotropy in FeCo/Pt has also been studied. The latter system is interesting from a technological perspective since tetragonally distorted FeCo alloys have the potential to be suitable new materials in computer hard drives. Finally, a computer program, based on the Differential Evolution algorithm, to refine primarily x-ray reflectivity data, is presented.

Orientadores: Abner de Siervo e Flavio Garcia / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin. / Made available in DSpace on 2018-08-12T11:00:44Z (GMT). No. of bitstreams: 1 Figueiredo_JoseJadsomSampaiode_M.pdf: 4194889 bytes, checksum: 489e23ac30c4fa90808ef2de00ae46d4 (MD5) Previous issue date: 2008 / Resumo: Nanopartículas (NPs) metálicas, filmes ultrafinos e sistemas nano-estruturados têm motivado vários estudos teóricos e experimentais devido a novas propriedades eletrônicas, óticas e magnéticas destes materiais. Nas NPs, tais propriedades estão diretamente relacionadas aos efeitos de tamanho (size effect) e superfície, os quais afetam suas estruturas eletrônica e cristalográfica, concentrando um maior número de átomos na superfície em relação ao seu volume. Estudos recentes têm sugerido que alguns metais nobres sem nenhum tipo de ordenamento ferromagnético espontâneo podem apresentá-lo, mesmo a temperatura ambiente, quando segmentados em forma de nanopartículas. Exemplos desses metais são Au e Pd. Apesar dos esforços para provar a existência de comportamento ferromagnético nestes materiais, não existem medidas que demonstrem de forma não ambígua a polarização ferromagnética nos átomos de Pd. Na maioria dos trabalhos mostrados na literatura, o ferromagnetismo é quantificado por magnetometria SQUID. Faz-se necessário, portanto, verificar a existência de ferromagnetismo nestes materiais utilizando-se de técnicas elemento específicas, tais como XMCD (dicroísmo circular magnético de raios X). Esta técnica é muito importante para determinação quantitativa e seletiva dos momentos magnéticos de spin e orbital para cada elemento separadamente. O nosso trabalho se diferencia em relação a estes, pois ao invés de estudarmos cada elemento individualmente trabalhamos com nanopartículas do composto bimetálico Aux Pd1-x (x =0,00, 0,25, 0,50, 0,75) passivadas por tiol. Medidas de SQUID indicaram claramente um sinal magnético nestas nanopartículas, inclusive à temperatura ambiente. Neste trabalho apresentaremos os resultados preliminares da caracterização destas nanopartículas por medidas de SQUID, XANES, XPS, bem como XMCD nas bordas L2,3 do Pd e L3 do Au . Ressaltamos também, o desenvolvimento de uma instrumentação para medidas de XMCD em ambientes de UHV onde é possível submeter às amostras a altos campos magnéticos e condições de baixas temperaturas. Com base nos resultados obtidos discutiremos a possibilidade de ferromagnetismo nestas nanopartículas e sua origem. / Abstract: Metallic Nanoparticles (NP's), ultra-thin films and nano-structured systems have motivated several theoretical and experimental works because of their new electronic optical and magnetic properties of these materials. In NP's, such properties are directly related to the size and surface effects, which affect its electronic and crystallographic structures, by concentrating a larger number of atoms on the surface in relation to their volume. Recent studies have suggested that some noble metals without any kind of spontaneous ferromagnetic order can exhibit it, even at room temperature, when segmented as nanoparticles. Examples of these metals are Au and Pd. Despite the efforts to prove the existence of a ferromagnetic behavior in such materials, there are no measurements that demonstrate unambiguously the ferromagnetic polarization in Pd atoms, for example. Most of the works shown in the literature have used SQUID measurements to characterize the ferromagnetism in these materials. It is therefore necessary to verify the existence of ferromagnetism in these materials by using element specific techniques, such as XMCD (X-ray magnetic circular dichroism). XMCD is a very important technique, which is element selective and performs quantitative determination of the spin and orbital magnetic contributions for each element separately. Our work has a particular difference when compared to others in literature because instead of studying each element individually we have worked with NP's of the bimetallic compound Aux Pd1-x (x = 0.00, 0.25, 0.50, 0.75) capped by thiol. SQUID measurements clearly showed ferromagnetic behavior in these nanoparticles, even at room temperature. In this thesis we will present results of a systematic characterization of these nanoparticles by a multi-technique approach which involves measurements with SQUID, XANES, XPS and XMCD in the L2,3 edges of the Pd and Au L3 edge. We also emphasize the development of a new instrumentation for XMCD measurements at UHV where samples can be submitted to high magnetic fields and conditions of low temperatures. Based on these findings we discuss the possibility of ferromagnetism in these nanoparticles and its origin. / Mestrado / Física da Matéria Condensada / Mestre em Física