Estabilidade de géis de amido de milho normal, ceroso e com alto teor de amilose adicionados de gomas guar e xantana durante os processos de congelamento e descongelamento

O objetivo do presente trabalho foi estudar os efeitos das gomas guar e xantana sobre a estabilidade dos géis de amido de milho normal, ceroso e com alto teor de amilose submetidos aos processos de congelamento e descongelamento. Os géis desses amidos, com concentração total de sólidos de 10% e adicionados das gomas (0,15; 0,50; 0,85 e 1%), foram submetidos a 5 ciclos de congelamento (20 horas a -18 °C) e descongelamento (4 horas a 25 °C), com exceção dos géis com alto teor de amilose, que foram submetidos a apenas 1 ciclo, devido à perda da estrutura de gel. A determinação da sinérese (porcentagem de água liberada) foi realizada pela diferença entre a massa inicial e a massa final das amostras. O gel de amido de milho normal liberou 74,45% de água, sendo que a adição de 1% da goma xantana reduziu significativamente a sinérese para 66,43%. A adição de 0,85 e 1% da goma xantana também reduziu a sinérese dos géis de amido ceroso. O menor teor de sinérese foi obtido com a utilização de 1% de goma xantana ao gel de amido de milho com alto teor de amilose, evidenciando a ação crioprotetora desta goma.

Avaliação físico-química de bolo de chocolate com coberturas comestíveis à base de gelatina, ácido esteárico, amido modificado ou cera de carnaúba

Coberturas comestíveis biodegradáveis são uma alternativa às embalagens sintéticas, que causam preocupações ambientais. Este trabalho avaliou o efeito de diferentes tipos de coberturas sobre propriedades físico-químicas de bolo de chocolate durante a estocagem, em comparação com bolo sem cobertura (CO) e bolo sem cobertura embalado em polipropileno (EMB). As seguintes coberturas foram aplicadas sobre os bolos: 10% gelatina (GE), 10% gelatina com 10% ácido esteárico (GE + AE), 18% cera de carnaúba (CE), 10% amido modificado (AM) e fondant (FO). Os bolos foram avaliados durante 10 dias de estocagem. FO e EMB apresentaram menor perda de massa, enquanto todos os demais tratamentos apresentaram valores superiores a CO. GE, GE + AE e EMB apresentaram a menor redução da atividade de água, enquanto CE e CO apresentaram a maior redução. As superfícies dos bolos recobertos estavam mais duras que as de CO e EMB. Os maiores valores para dureza e mastigabilidade foram encontrados para CE e CO e os menores, para EMB, GE e GE + AE. Em relação à cor, GE + AE foi diferente dos demais tratamentos, devido à presença do ácido esteárico. Os resultados indicam que a perda de massa dos bolos pode ser atribuída também à perda de água das coberturas.

Tratamento térmico do amido de batata-doce (Ipomoea batatas L.) sob baixa umidade em micro-ondas

O objetivo deste trabalho foi avaliar o efeito do tratamento térmico sob baixa umidade (TTBU) aplicado por forno micro-ondas sobre as propriedades estruturais e funcionais do amido de batata-doce e compará-las com as propriedades de amido tratado pelo método convencional. O amido extraído dessa raiz foi submetido à modificação física, nas umidades de 25 e 35%, em forno convencional (90 °C/16 horas) e em microondas (35 a 90 °C/1 hora). O tratamento térmico sob baixa umidade resultou em alterações significativas no teor de amilose e em características como a cristalinidade, suscetibilidade enzimática, fator de expansão e propriedades de pasta. Tais variações evidenciam modificações na estrutura granular interna dos amidos, tanto em áreas cristalinas como amorfas do grânulo. As alterações conferidas pelo TTBU foram variáveis com o tipo de tratamento térmico e com o teor de umidade. A umidade das amostras também foi determinante na modificação da maioria das características do amido, como maior digestibilidade enzimática e redução da expansão, menores picos de viscosidade e quebras de viscosidade, independentemente do tipo de tratamento térmico aplicado. Considerando-se o tipo e a intensidade da modificação física do amido tratado pelo método convencional como referência, a utilização da energia de micro-ondas para esse mesmo fim precisa ser melhor estudada.

Propriedades termodinâmicas de adsorção de água do amido de rizomas do lírio-do-brejo (Hedychium coronarium)

Determinaram-se as propriedades termodinâmicas (entalpia diferencial, entropia diferencial, entalpia integral e entropia integral) do amido de rizomas do lírio-do-brejo (Hedychium coronarium) por meio de isotermas de adsorção de água. As isotermas foram determinadas em atividades de água no intervalo de 0,11 a 0,84, sob temperaturas que variaram de 30 a 50 °C. A Equação de GAB, que se ajustou bem às isotermas experimentais, foi utilizada para estimar as propriedades termodinâmicas de adsorção. As isotermas apresentaram ligeira inversão, indicando a precença de amido danificado. A entalpia diferencial e a entropia diferencial aumentaram com a diminuição da umidade de equilíbrio e correlacionaram entre si confirmando a compensação química linear. Um modelo exponencial do tipo Y = b.e(a/Xe) descreveu adequadamente a dependência destas propriedades diferenciais ao teor de umidade de equilíbrio. A entalpia integral e a entropia integral aumentaram continuamente com o teor de umidade de equilíbrio, porém com valores negativos para a entropia integral. Estas propriedades termodinâmicas de adsorção de água demonstraram que o amido extraído dos rizomas do lírio-do-brejo possui baixa higrocopicidade apesar da ocorrência de grânulos de amido danificados.

Viscosidade extensional e em cisalhamento de suspensões acidificadas de amido de amaranto e caseinato de sódio

Foram avaliadas as viscosidades extensional e em cisalhamento de suspensões acidificadas de amido de amaranto-caseinato de sódio. Sistemas mistos de amido de amaranto-caseinato de sódio acidificados com glucona-delta-lactona (GDL) foram estudados por ensaios reológicos em compressão biaxial e cisalhamento. Os efeitos da velocidade de acidificação (lenta e rápida) e pH final (neutro e no ponto isoelétrico da caseína) foram avaliados considerando as interações entre os biopolímeros e sua consequente influência nos parâmetros reológicos. Todas as amostras apresentaram comportamento pseudoplástico, no entanto, a adição de caseinato de sódio nas suspensões de amido, em pH neutro, promoveu um efeito negativo sobre a viscosidade aparente. Amostras acidificadas apresentaram um aumento na complexidade do sistema devido à formação da rede de amido e caseína, observando que a força necessária para o escoamento foi sempre maior para as amostras contendo concentrações maiores de caseinato. Isso mostra que a agregação e gelificação da proteína promovidas pela acidificação, impediram a microsseparação de fases. Esta rede foi mais forte em sistemas gelificados lentamente, devido à formação de uma rede de proteína mais organizada. Apesar da técnica de compressão biaxial imperfeita ser limitada para avaliação de determinados sistemas, neste estudo, mostrou ser um modo prático e eficiente de se mensurar o comportamento reológico.

Propriedades físico-químicas do amido de aveia da variedade brasileira IAC 7

Este estudo caracterizou o amido de aveia da variedade IAC-7 quanto às suas características químicas, reológicas, funcionais e térmicas. O amido de aveia apresentou 1,36% de lipídios, 32,23% de amilose e baixa capacidade retrogradante (9,19% após 30 dias de armazenagem). Embora o amido de aveia tenha apresentado alto teor de amilose, sua baixa retrogradação pode ser devida à presença dos lipídios que, por impedimento estérico, dificultariam a reaproximação das cadeias poliméricas. O comportamento reológico das pastas de amido de aveia foi caracterizado como sendo pseudoplático. A baixa temperatura de gelatinização (64,71 °C) do amido de aveia também pode estar relacionada ao maior teor de lipídio deste amido.

VAP-1 as drug target in inflammation : structural features determining ligand interactions

Vascular adhesion protein-1 (VAP-1), which belongs to the copper amine oxidases (CAOs), is a validated drug target in inflammatory diseases. Inhibition of VAP-1 blocks the leukocyte trafficking to sites of inflammation and alleviates inflammatory reactions. In this study, a novel set of potent pyridazinone inhibitors is presented together with their X-ray structure complexes with VAP-1. The crystal structure of serum VAP-1 (sVAP-1) revealed an imidazole binding site in the active site channel and, analogously, the pyridazinone inhibitors were designed to bind into the channel. This is the first time human VAP-1 has been crystallized with a reversible inhibitor and the structures reveal detailed information of the binding mode on the atomic level. Similarly to some earlier studied inhibitors of human VAP-1, the designed pyridazinone inhibitors bind rodent VAP-1 with a lower affinity than human VAP-1. Therefore, we made homology models of rodent VAP-1 and compared human and rodent enzymes to determine differences that might affect the inhibitor binding. The comparison of the crystal structures of the human VAP-1 and the mouse VAP-1 homology model revealed key differences important for the species specific binding properties. In general, the channel in mouse VAP-1 is more narrow and polar than the channel in human VAP-1, which is wider and more hydrophobic. The differences are located in the channel leading to the active site, as well as, in the entrance to the active site channel. The information obtained from these studies is of great importance for the development and design of drugs blocking the activity of human VAP-1, as rodents are often used for in vivo testing of candidate drugs. In order to gain more insight into the selective binding properties of the different CAOs in one species a comprehensive evolutionary study of mammalian CAOs was performed. We found that CAOs can be classified into sub-families according to the residues X1 and X2 of the Thr/Ser-X1-X2-Asn-Tyr-Asp active site motif. In the phylogenetic tree, CAOs group into diamine oxidase, retina specific amine oxidase and VAP-1/serum amine oxidase clades based on the residue in the position X2. We also found that VAP-1 and SAO can be further differentiated based on the residue in the position X1. This is the first large-scale comparison of CAO sequences, which explains some of the reasons for the unique substrate specificities within the CAO family.

Unconventional integrin-ligand interactions

Integrins are cell surface adhesion and signaling receptors. Cells use integrins to attach to the extracellular matrix and to other cells, as well as for sensing their environment. In addition to adhesion and migration, integrins have been shown to be important for many biological processes including apoptosis, cell proliferation, and differentiation into specific tissues. Many important next generation biological drugs inhibit integrin functions. Thus, research into interactions between integrins and their ligands under different physiological and pathological conditions is not only of academic interest, but is also important for the field of drug discovery. In this Ph.D. project, the functions of integrin-ligand interactions were studied under different physiologically interesting conditions including 1) human echovirus 1 binding to integrin α2β1, 2) integrin α2β1 binding to collagen under flow conditions, 3) integrin α2β1 binding to a ligand in the presence of the angiogenesis inhibitor histidine rich glycoprotein (HRG) and 4) integrin binding to posttranslationally citrullinated ligands. As a result of the project, we could show that for each condition the integrin-ligand interaction is somewhat unconventional. 1) Echovirus 1 binds only to non-activated conformations of integrin α2β1. 2) Surprisingly, the non-activated conformation is also the primary conformation of integrin α2β1 when it binds to collagen under flow conditions, like when platelets adhere to subendothelial collagen in vascular injuries. In addition, the pre-activation of integrin α2β1 does not increase adhesion under flow. 3) HRG binds to integrin α2β1 through a low-affinity interaction that inhibits integrin binding to collagen. This shows that low affinity interactions could be biologically relevant and possibly regulate angiogenesis. 4) The citrullination of collagen, a posttranslational modification reported to occur in rheumatoid arthritis, specifically inhibits the binding of integrin α10β1 and α11β1, but does not affect the binding of α1β1 ja α2β1. On the other hand, the citrullination of isoDGR in fibronectin and RGD in pro-TGF- β:n inhibit integrin binding completely. Citrullination seems to be an inflammation related process and integrin ligands become citrullinated frequently in vivo. This Ph.D. thesis suggests that unconventional interaction mechanisms between integrins and their ligands, such as posttranslational modifications, low affinity interactions, and non-activated integrin conformations, can have an important role in pathological processes. The study of these kinds of integrin-ligand interactions is important for understanding biological phenomena more deeply. The research might also be beneficial for the development of integrin based therapies for treating diseases.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

The design, synthesis and characterization of new building blocks for the preparation of molecule-based magnetic materials /

Two new families of building blocks have been prepared and fully characterized and their coordination chemistry exploited for the preparation of molecule-based magnetic materials. The first class of compounds were prepared by exploiting the chemistry of 3,3'-diamino-2,2'-bipyridine together with 2-pyridine carbonyl chloride or 2-pyridine aldehyde. Two new ligands, 2,2'-bipyridine-3,3'-[2-pyridinecarboxamide] (Li, 2.3) and N'-6/s(2-pyridylmethyl) [2,2'bipyridine]-3,3'-diimine (L2, 2.7), were prepared and characterized. For ligand L4, two copper(II) coordination compounds were isolated with stoichiometrics [Cu2(Li)(hfac)2] (2.4) and [Cu(Li)Cl2] (2.5). The molecular structures of both complexes were determined by X-ray crystallography. In both complexes the ligand is in the dianionic form and coordinates the divalent Cu(II) ions via one amido and two pyridine nitrogen donor atoms. In (2.4), the coordination geometry around both Cu11 ions is best described as distorted trigonal bipyramidal where the remaining two coordination sites are satisfied by hfac counterions. In (2.5), both Cu(II) ions adopt a (4+1) distorted square pyramidal geometry. One copper forms a longer apical bond to an adjacent carbonyl oxygen atom, whereas the second copper is chelated to a neighboring Cu-Cl chloride ion to afford chloride bridged linear [Cu2(Li)Cl2]2 tetramers that run along the c-axis of the unit cell. The magnetic susceptibility data for (2.4) reveal the occurrence of weak antiferromagnetic interactions between the copper(II) ions. In contrast, variable temperature magnetic susceptibility measurements for (2.5) reveal more complex magnetic properties with the presence of ferromagnetic exchange between the central dimeric pair of copper atoms and weak antiferromagnetic exchange between the outer pairs of copper atoms. The Schiff-base bis-imine ligand (L2, 2.7) was found to be highly reactive; single crystals grown from dry methanol afforded compound (2.14) for which two methanol molecules had added across the imine double bond. The susceptibility of this ligand to nucleophilic attack at its imine functionality assisted via chelation to Lewis acidic metal ions adds an interesting dimension to its coordination chemistry. In this respect, a Co(II) quaterpyridine-type complex was prepared via a one-pot transformation of ligand L2 in the presence of a Lewis acidic metal salt. The rearranged complex was characterized by X-ray crystallography and a reaction mechanism for its formation has been proposed. Three additional rearranged complexes (2.13), (2.17) and (2.19) were also isolated when ligand (L2, 2.7) was reacted with transition metal ions. The molecular structures of all three complexes have been determined by X-ray crystallography. The second class of compounds that are reported in this thesis, are the two diacetyl pyridine derivatives, 4-pyridyl-2,6-diacetylpyridine (5.5) and 2,2'-6,6'-tetraacetyl-4,4'-bipyridine (5.15). Both of these compounds have been designed as intermediates for the metal templated assembly of a Schiff-base N3O2 macrocycle. From compound (5.15), a covalently tethered dimeric Mn(II) macrocyclic compound of general formula {[Mn^C^XJCl-FkO^Cl-lO.SFbO (5.16) was prepared and characterized. The X-ray analysis of (5.16) reveals that the two manganese ions assume a pentagonal-bipyramidal geometry with the macrocycle occupying the pentagonal plane and the axial positions being filled by a halide ion and a H2O molecule. Magnetic susceptibility data reveal the occurrence of antiferromagnetic interactions between covalently tethered Mn(II)-Mn(II) dimeric units. Following this methodology a Co(II) analogue (5.17) has also been prepared which is isostructural with (5.16).

A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta™ and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Fast atom bombardment mass spectrometry of the coordination complexes of the ligand 5, 5, 7, 12, 12, 14- hexamenthyl-1, 4, 8, 11-tetraazacycloteradecane

A number of metal complexes containing the ligand 5,5,7,12,12,14-hexamethyl-l,4,8,11-tetra-azatetradecane were synthesized and analyzed using electron impact (EI) and fast atom bombardment (FAB). The FAB mass spectra were obtained in positive and negative ion mode. FAB in the positive ion mode proved to be the most successful technique for the identification of these compounds. In the majority of cases the spectra obtained using positive ion FAB were structurally informative, although not all showed molecular (M+) or quasimolecular ([M+H]+) ions. The fragmentations observed were characteristic of the ligands, and were interpreted based on the chemistry of these compounds.

Studies of some cocondensations of nickel atoms with unsaturated organic ligands and with mixed ligand systems

The cocondensation of nickel with a number of unsaturated ligands was studied, as was the cocondensation with a number of mixed ligand systems. Enamines were found not to react with nickel while acrylonitrile was polymerized. In the mixed ligand syst.ems different products were obtained than when the ligands were cocondensed individually. Cocondensations of benzyl halide/allyl halide mixtures gave unstable products that were not observed when the halides were cocondensed individually. The effect of Kao-Wool insulation on nickel/benzyl halide cocondensations was found to be significant. Kao-Wool caused the bulk of the benzyl halide to be polymeri zed to a number of poly-benzylic species. An alkali metal reactor was designed for the evaporation of sodium and potassium atoms into cold solutions of metal halide and an or ganic substrate. This apparatus was used to synthesize Ni(P¢3 )3' but proved unsuccessful for synthesizing a nickel-enamine compound.

Synthesis and characterization of BODIPY-α-tocopherol : a new ligand to explore the intracellular transfer of Vitamin E

Since its discovery nearly a century ago, a-tocopherol (vitamin E) research has been mainly focused on its ability to terminate the cycle of lipid peroxidation in membranes. Nitrobenzoxadiazole fluorescent analogues were made previously to study the intracellular transfer of vitamin E in cells. However, these molecules were reportedly susceptible to photobleaching while under illumination for transfer assays and microscopy. Here is reported the synthesis of a series of fluorescent analogues of vitamin E incorporating the more robust dipyrrometheneboron difluoride fluorophore (BDP-a-Tocs; Aex = 507 nm, Aem = 511 nm). C8-BDP-a-Toc 42c, having an eight-carbon chain between the chromanol and fluorophore, wa<; shown to bind specifically to a-tocopherol transfer protein with a dissociation constant of approximately 100 nM. Another fluorescent analogue of vitamin E with a thienyl derivative of BODIPY that is excited and fluoresces at longer wavelengths (Aex = 561 nm, Aem = 570 nm) is in development.

Ligand design for molecule-based magnetic and/or conducting materials

Work in the area of molecule-based magnetic and/or conducting materials is presented in two projects. The first project describes the use of 4,4’-bipyridine as a scaffold for the preparation of a new family of tetracarboxamide ligands. Four new ligands I-III have been prepared and characterized and the coordination chemistry of these ligands is presented. This project was then extended to exploit 4,4’-bipyridine as a covalent linker between two N3O2 macrocyles. In this respect, three dimeric macrocycles have been prepared IV-VI. Substitution of the labile axial ligands of the Co(II) complex IV by [Fe(CN)6]4- afforded the self-assembly of the 1-D polymeric chain {[Co(N3O2)H2O]2Fe(CN)6}n•3H2O that has been structurally and magnetically characterized. Magnetic studies on the Fe(II) complexes V and VI indicate that they undergo incomplete spin crossover transitions in the solid state. Strategies for the preparation of chiral spin crossover N3O2 macrocycles are discussed and the synthesis of the novel chiral Fe(II) macrocyclic complex VII is reported. Magnetic susceptibility and Mössbauer studies reveal that this complex undergoes a gradual spin crossover in the solid state with no thermal hysteresis. Variable temperature X-ray diffraction studies on single crystals of VII reveal interesting structural changes in the coordination geometry of the macrocycle accompanying its SCO transition. The second project reports the synthesis and characterization of a new family of tetrathiafulvalene derivatives VIII – XII, where a heterocyclic chelating ligand is appended to a TTF donor via an imine linker. The coordination chemistries of these ligands with M(hfac)2.H2O (M( = Co, Ni, Mn, Cu) have been explored and the structural and magnetic properties of these complexes are described.