Destructive sampling of 20 trees of four tree species in a mixed New England conifer/hardwood stand shows that leaf area comprises 72, 77, and 78 percent of plant area as measured with digital hemispherical photography of the stand in (1) leaf-off, (2) leaf-out and pre-harvest, and (3) leaf-out and post-harvest conditions. Leaf area index values for the stand, estimated through destructive sampling, were 4.42, 5.98, and 5.08 respectively, documenting the progression of leaf growth through post-harvest. Terrestrial lidar scans (TLS) of the stand in (1) leaf-off and (2) leaf-out and pre-harvest conditions provided leaf area index values of 4.49 and 6.00 using the correction applied to observed plant area index, showing good agreement. The method relies on destructive sampling to relate the weight of foliage removed from sample trees to leaf area and fine twig area within the foliage as measured by a flatbed scanner. Two conifer species, eastern hemlock and white pine, and two deciduous species, red maple and red oak, in five diameter-size classes, were harvested from the 50 x 50-m stand in late summer. Leaf and twig areas of these trees provided species-specific allometric equations relating stem basal area to leaf and twig area, and a stand map provided species, counts and diameters of all trees in the plot. These data then allowed estimation of the leaf area of the stand as a whole for comparison with optical methods. The ratios of leaf to fine-branch area for each species vary, with values of 5.33, 25.38, 260.88 and 140.35 for eastern hemlock, white pine, red maple, and red oak respectively. This variance shows that woody-to-total area constants, which are used for calculating leaf area index from plant area index values determined by optical gap probability methods, will be quite dependent on stand composition and questions the common usage of literature constants for this purpose. This study shows how destructive sampling can lead to better estimation of forest leaf area index and wood area index from hemispherical photography and terrestrial lidar scanning, which has the potential to improve modeling of nutrient cycling and carbon balance in ecosystem models.

As coberturas de hidroxiapatita (HAp) são utilizadas para aumentar a osteointegração em implantes de titânio (Ti), devido à sua capacidade de promover a biomineralização para corrigir defeitos esqueléticos e craniofaciais. O objetivo desta pesquisa foi avaliar a influência dos revestimentos sobre culturas primárias de osteoblastos. Na primeira fase de estudos, desenvolvemos uma nova abordagem de revestimento baseada em filmes Langmuir-Blodgett (LB) de dihexadecilfosfato (DHP) e ácido octadecilfosfônico (OPA) depositados em discos Ti, e crescimento subsequente de cristais de HAp. Analisamos a viabilidade dos osteoblastos, a atividade da fosfatase alcalina (ALP) e a formação da matriz mineralizada por métodos colorimétricos e a morfologia das culturas por microscopia eletrônica de varredura e microscopia confocal. Os resultados revelaram que o revestimento DHP/HAp aumentou a viabilidade dos osteoblastos até 150% em comparação com o controle em todos os dias testados. O revestimento OPA/HAp promoveu a maior viabilidade ao 14 dias (190%). A atividade de ALP foi aumentada apenas pelo revestimento de DHP/HAp ao 14º dia em comparação com o controle e Ti limpo. A microscopia eletrônica de varredura e as microfotografias confocais revelaram diferenças morfológicas entre os osteoblastos cultivados em ambos os revestimentos, aumentando o seu número e o espalhamento. O revestimento de DHP/HAp aumentou a produção de nódulos biomineralizados. O ensaio de biomineralização pela técnica do Vermelho de Alizarina mostrou que o revestimento de OPA/HAp possuía uma concentração de cálcio (Ca2+) 1,88 vezes superior à cobertura de DHP/HAp. Uma vez que a literatura relata que o Ca2+ pode estimular ou inibir a atividade da ALP e, consequentemente, o processo de biomineralização, as diferenças no comportamento desses dois revestimentos podem estar relacionadas às diferenças de concentração de superfície de Ca2+. O bom desempenho do revestimento de DHP/HAp pode estar relacionado às características da composição química, adicionada à técnica de deposição LB. Na segunda fase da pesquisa, as monocamadas de Langmuir de DHP e dipalmitoil fosfatidilcolina (DPPC) foram testadas e utilizadas para incorporar o paratormônio 1-34 (PTH 1-34) (DHP/Ca+PTH e DPPC/Ca+PTH, respectivamente). Também foram testadas as ações dos revestimentos DHP/HAp com PTH em solução (DHP/HAp+PTH S) e gotejado (DHP/HAp+PTH G) em culturas de osteoblastos. Um potencial zeta negativo em pH 7,4 foi encontrado (-14,9 mV) para o PTH 1-34. A isoterma de DPPC mostrou um aumento da área mínima ocupada por molécula lipídica após a injeção de PTH na subfase de água (50 ?L de solução 0,5 mg/mL) em 10,97 Å2, o que pode ser devido à inserção de PTH neste filme. A área mínima de DHP foi alterada em 2,3 Å2, o que não é estatisticamente significativo. A análise de QCM mostrou um depósito de 72,5 ng de PTH em filme de DPPC e 29,3 ng de PTH em filme de DHP para cada 25 ?g de PTH injetado na cuba de Langmuir. A viabilidade celular e a formação da matriz mineralizada de culturas de osteoblastos crescidas em DHP/Ca+PTH e revestimentos DPPC/Ca+PTH diminuíram quando comparadas com Ti limpo. Os revestimentos DHP/HAp+PTH S e DHP/HAp+PTH G mostraram ser tão eficientes quanto o Ti DHP/HAp para estimular o processo de biomineralização. Mas a cobertura de DHP/HAp+PTH G aumentou a viabilidade dos osteoblastos e a formação de matriz mineralizada quando comparada com Ti DHP/HAp. Esta é uma cobertura inovadora que abre precedentes para o uso da técnica de gotejamento em HAp para outros hormônios e drogas que agem sobre o tecido ósseo. / Due to their ability to promote biomineralization, Hydroxyapatite (HAp) coatings are used to increase the osteointegration in titanium (Ti) implants, in order to correct skeletal and craniofacial defects. The objective of the research was to evaluate the influence of the coatings on osteoblasts primary cultures. In the first phase of the research we developed a new coating approach based on Langmuir-Blodgett (LB) films of dihexadecyl phosphate (DHP) and octadecylphosphonic acid (OPA) deposited on Ti discs and subsequent growth of HAp crystals. We analyzed the osteoblast viability, alkaline phosphatase (ALP) activity and mineralized matrix formation by colorimetric methods, and the morphology of the cultures by scanning electron microscopy and confocal micrographies. The results revealed that the DHP/HAp coating increased osteoblast viability up to 150% compared to the control at all days tested. The OPA/HAp coating promoted the highest viability on the 14th day (190%). The ALP activity was enhanced only by the DHP/HAp coating on the 14th day compared to control, and clean Ti. To explore the morphology of the cells, the scanning electron microscopy and confocal micrographies were obtained, and revealed morphological differences between osteoblasts grown on both coated Ti compared to clean Ti. Both coatings increased the number and spreading of osteoblasts, while the DHP/HAp coating enhanced the production of biomineralized nodules. The Alizarin Red assay showed that OPA/HAp coating has 1.88 times higher calcium (Ca2+) concentration than DHP/HAp. The same test confirmed the increase of mineralization only by DHP/HAp coating compared to clean Ti. Since literature reports that Ca2+ can stimulate or inhibit the ALP activity and consequently, the biomineralization process, the differences on the behavior of these two coatings could be related to the Ca2+ surface concentration differences. The good performance of the DHP/HAp coating can be explained due to the characteristics of the chemical composition, added to the LB deposition technique. In the second phase of the research, Langmuir monolayers of DHP and dipalmitoyl phosphatidylcholine (DPPC) was tested and used to incorporate 1-34 parathyroid hormone (PTH 1-34) (DHP/Ca+PTH, and DPPC/Ca+PTH, respectively). DHP/HAp coatings with PTH in solution (DHP/HAp+PTH S), and dropped (DHP/HAp+PTH G) also were tested on osteoblasts cultures. A negative zeta-potential at pH 7.4 was found (-14.9 mV) to PTH 1-34. The Langmuir isotherm of DPPC showed an increase of the minimum area occupied per lipid molecule after the PTH injection into the water subphase (50 ?L of 0.5 mg/mL solution) by 10.97 Å2, which could be due to the insertion of PTH in this film. The DHP minimum area changed by 2.3 Å2, which is not statistically significant. The QCM analysis showed the deposit of 72.5 ng of PTH on DPPC film, and 29.3 ng of PTH on DHP film for each 25 ?g of PTH injected into the Langmuir trough. The cell viability and matrix mineralization of osteoblasts cultures grown on DHP/Ca+PTH, and DPPC/Ca+PTH coatings decreased when compared to clean Ti. DHP/HAp+PTH S and DHP/HAp+PTH G coatings proved to be as efficient as Ti DHP/HAp to stimulate the biomineralization process. But DHP/HAp+PTH G increased the osteoblast viabilitiy and formation of mineralized matrix when compared to Ti DHP/HAp. This is an innovative coating that sets the precedent for the use of the drip technique on HAp for other hormones and drugs that act on bone tissue.

As coberturas de hidroxiapatita (HAp) são utilizadas para aumentar a osteointegração em implantes de titânio (Ti), devido à sua capacidade de promover a biomineralização para corrigir defeitos esqueléticos e craniofaciais. O objetivo desta pesquisa foi avaliar a influência dos revestimentos sobre culturas primárias de osteoblastos. Na primeira fase de estudos, desenvolvemos uma nova abordagem de revestimento baseada em filmes Langmuir-Blodgett (LB) de dihexadecilfosfato (DHP) e ácido octadecilfosfônico (OPA) depositados em discos Ti, e crescimento subsequente de cristais de HAp. Analisamos a viabilidade dos osteoblastos, a atividade da fosfatase alcalina (ALP) e a formação da matriz mineralizada por métodos colorimétricos e a morfologia das culturas por microscopia eletrônica de varredura e microscopia confocal. Os resultados revelaram que o revestimento DHP/HAp aumentou a viabilidade dos osteoblastos até 150% em comparação com o controle em todos os dias testados. O revestimento OPA/HAp promoveu a maior viabilidade ao 14 dias (190%). A atividade de ALP foi aumentada apenas pelo revestimento de DHP/HAp ao 14º dia em comparação com o controle e Ti limpo. A microscopia eletrônica de varredura e as microfotografias confocais revelaram diferenças morfológicas entre os osteoblastos cultivados em ambos os revestimentos, aumentando o seu número e o espalhamento. O revestimento de DHP/HAp aumentou a produção de nódulos biomineralizados. O ensaio de biomineralização pela técnica do Vermelho de Alizarina mostrou que o revestimento de OPA/HAp possuía uma concentração de cálcio (Ca2+) 1,88 vezes superior à cobertura de DHP/HAp. Uma vez que a literatura relata que o Ca2+ pode estimular ou inibir a atividade da ALP e, consequentemente, o processo de biomineralização, as diferenças no comportamento desses dois revestimentos podem estar relacionadas às diferenças de concentração de superfície de Ca2+. O bom desempenho do revestimento de DHP/HAp pode estar relacionado às características da composição química, adicionada à técnica de deposição LB. Na segunda fase da pesquisa, as monocamadas de Langmuir de DHP e dipalmitoil fosfatidilcolina (DPPC) foram testadas e utilizadas para incorporar o paratormônio 1-34 (PTH 1-34) (DHP/Ca+PTH e DPPC/Ca+PTH, respectivamente). Também foram testadas as ações dos revestimentos DHP/HAp com PTH em solução (DHP/HAp+PTH S) e gotejado (DHP/HAp+PTH G) em culturas de osteoblastos. Um potencial zeta negativo em pH 7,4 foi encontrado (-14,9 mV) para o PTH 1-34. A isoterma de DPPC mostrou um aumento da área mínima ocupada por molécula lipídica após a injeção de PTH na subfase de água (50 ?L de solução 0,5 mg/mL) em 10,97 Å2, o que pode ser devido à inserção de PTH neste filme. A área mínima de DHP foi alterada em 2,3 Å2, o que não é estatisticamente significativo. A análise de QCM mostrou um depósito de 72,5 ng de PTH em filme de DPPC e 29,3 ng de PTH em filme de DHP para cada 25 ?g de PTH injetado na cuba de Langmuir. A viabilidade celular e a formação da matriz mineralizada de culturas de osteoblastos crescidas em DHP/Ca+PTH e revestimentos DPPC/Ca+PTH diminuíram quando comparadas com Ti limpo. Os revestimentos DHP/HAp+PTH S e DHP/HAp+PTH G mostraram ser tão eficientes quanto o Ti DHP/HAp para estimular o processo de biomineralização. Mas a cobertura de DHP/HAp+PTH G aumentou a viabilidade dos osteoblastos e a formação de matriz mineralizada quando comparada com Ti DHP/HAp. Esta é uma cobertura inovadora que abre precedentes para o uso da técnica de gotejamento em HAp para outros hormônios e drogas que agem sobre o tecido ósseo. / Due to their ability to promote biomineralization, Hydroxyapatite (HAp) coatings are used to increase the osteointegration in titanium (Ti) implants, in order to correct skeletal and craniofacial defects. The objective of the research was to evaluate the influence of the coatings on osteoblasts primary cultures. In the first phase of the research we developed a new coating approach based on Langmuir-Blodgett (LB) films of dihexadecyl phosphate (DHP) and octadecylphosphonic acid (OPA) deposited on Ti discs and subsequent growth of HAp crystals. We analyzed the osteoblast viability, alkaline phosphatase (ALP) activity and mineralized matrix formation by colorimetric methods, and the morphology of the cultures by scanning electron microscopy and confocal micrographies. The results revealed that the DHP/HAp coating increased osteoblast viability up to 150% compared to the control at all days tested. The OPA/HAp coating promoted the highest viability on the 14th day (190%). The ALP activity was enhanced only by the DHP/HAp coating on the 14th day compared to control, and clean Ti. To explore the morphology of the cells, the scanning electron microscopy and confocal micrographies were obtained, and revealed morphological differences between osteoblasts grown on both coated Ti compared to clean Ti. Both coatings increased the number and spreading of osteoblasts, while the DHP/HAp coating enhanced the production of biomineralized nodules. The Alizarin Red assay showed that OPA/HAp coating has 1.88 times higher calcium (Ca2+) concentration than DHP/HAp. The same test confirmed the increase of mineralization only by DHP/HAp coating compared to clean Ti. Since literature reports that Ca2+ can stimulate or inhibit the ALP activity and consequently, the biomineralization process, the differences on the behavior of these two coatings could be related to the Ca2+ surface concentration differences. The good performance of the DHP/HAp coating can be explained due to the characteristics of the chemical composition, added to the LB deposition technique. In the second phase of the research, Langmuir monolayers of DHP and dipalmitoyl phosphatidylcholine (DPPC) was tested and used to incorporate 1-34 parathyroid hormone (PTH 1-34) (DHP/Ca+PTH, and DPPC/Ca+PTH, respectively). DHP/HAp coatings with PTH in solution (DHP/HAp+PTH S), and dropped (DHP/HAp+PTH G) also were tested on osteoblasts cultures. A negative zeta-potential at pH 7.4 was found (-14.9 mV) to PTH 1-34. The Langmuir isotherm of DPPC showed an increase of the minimum area occupied per lipid molecule after the PTH injection into the water subphase (50 ?L of 0.5 mg/mL solution) by 10.97 Å2, which could be due to the insertion of PTH in this film. The DHP minimum area changed by 2.3 Å2, which is not statistically significant. The QCM analysis showed the deposit of 72.5 ng of PTH on DPPC film, and 29.3 ng of PTH on DHP film for each 25 ?g of PTH injected into the Langmuir trough. The cell viability and matrix mineralization of osteoblasts cultures grown on DHP/Ca+PTH, and DPPC/Ca+PTH coatings decreased when compared to clean Ti. DHP/HAp+PTH S and DHP/HAp+PTH G coatings proved to be as efficient as Ti DHP/HAp to stimulate the biomineralization process. But DHP/HAp+PTH G increased the osteoblast viabilitiy and formation of mineralized matrix when compared to Ti DHP/HAp. This is an innovative coating that sets the precedent for the use of the drip technique on HAp for other hormones and drugs that act on bone tissue.

Lignins are produced by all vascular plants and they represent one of the most abundant groups of biopolymers in nature. Lignin chemistry research, which has been of great importance for the progress of pulping technologies, has been plagued by the difficulties of its isolation and characterization. The pioneering work of Karl Freudenberg in the 1950’s with synthetic models of lignin paved the way for a detailed structural characterization of many lignin substructures. His work with the so-called “synthetic lignins” or dehydrogenative polymers (DHP) also laid a foundation for understanding how different lignin substructures are formed, reinforcing the already existing theory of lignin polymerization. However, subsequent structural characterizations of DHPs and lignins have repeatedly put this theory to the test. In the past decade, even a new radically different hypothesis for lignin polymerization has emerged and is sustained by a few researchers in the field. In this work, DHPs were produced from phenolic monomers, mostly coniferyl alcohol, a common lignin monomer, in a variety of reaction conditions. This was done in order to establish how different chemical factors, potentially active in the plant cell wall during lignin polymerization, influence the polymer’s final properties. In the presence of nicotine amide adenine dinucleotide (NADH), a quinone methide model, which is an intermediate formed during lignin polymerization, was effectively reduced. An equivalent reduced structure was produced during DHP synthesis in the presence of NADH. These studies showed that reduction might take place during oxidative polymerization, possibly explaining how reduced lignin structures are formed in the plant cell wall. Another reductive agent, ascorbic acid, was also tested during synthesis of DHPs. It displayed a totally different effect than NADH, probably due to its anti-oxidant nature, by altering the final amounts of certain inter-unit substructures, in favour of β-O-4′ structures, which are so prominent in natural lignins. Furthermore, the new suggested model for lignin polymerization, stating that lignin itself possesses the ability for template replication, was tested by synthesizing DHPs in the presence of a simple β-β′ substructure model. The DHPs produced the same amounts of β-β′ substructures as a control synthesis without the model structure, indicating that no replication had occurred. Finally, the role of the monolignol γ-carbon oxidation state in lignin polymerization, was studied. Hypothetically, lignin- like polymers could be produced by the plant, using monolignol biosynthetic precursors which exhibit γ-carbonyl groups instead of an alcohol group, like the common lignin monomer. Synthetic lignins produced with ferulic acid, coniferaldehyde and the normal monolignol, coniferyl alcohol, displayed important differences in chemical and physical properties. Both the ferulic acid and coniferaldehyde polymers exhibited almost no saturated inter-unit substructures and very few cyclic structures, both of which are very common in coniferyl alcohol dehydrogenative polymers and natural lignins. This could have significant implications for the formation of an important type of lignin carbohydrate complexes (LCC). Also the hydrophobicity of the alcohol-type polymer was lower than the other two. The biological implications of all these findings are discussed and some suggestions are made to explain how all these factors might affect lignin polymerization and structure in nature. / QC 20100811

A series of cis- and trans-bis(ethynyl)platinum complexes containing dimethyldihydropyrene (DHP) photochromic compounds were synthesized from RDHPCCH (R = H, CH3CO, PhCO, 1-naphthoyl and benzo[e]) and platinum chloride with appropriate ancillary ligands (PEt3, PPh3, dppe, bipy and phen). The complexes were studied using mass spectrometry, NMR spectroscopy (1H, 13C, 31P and 195Pt) and IR. The X-ray structural information for bis(DHP-ethynyl) platinum complexes 44, 45, 47, 49, and 52 revealed that the Pt complexes possessed a square planar geometry at the metal centers. These platinum complexes are T type photochromic compounds. The BDHP-derived platinum complexes 48, 49, and 52 open completely when irradiated by visible light (λ > 590 nm). The photoopening rates for the platinum complexes are about 4 times slower than the corresponding DHP-alkynes. All the alkynyl platinum complexes close thermally faster than the corresponding free alkynes, and the closing rate is not affected substantially by changing the ancillary phosphine ligands. The BDHP-ethynyl platinum complexes with PEt3, PPh3 and dppe ligands have similar thermal closing half lives at 25°C of τ1/2 = 42 h, 38 h and 33 h, respectively, in contrast to the half life τ1/2 = 62 h of the BDHP-ethyne 40. The first cyclobutadienyl cobalt substituted dihydropyrenes were prepared by CpCo(CO)2 cyclization of a series of dihydropyrenyl ethynes. When the other alkyne substituent was small (methyl or carboxyethyl) only cis (head-to-head) isomers were obtained, but with larger sized groups, mixed head-to-head and head-to-tail isomers were obtained. The crystal structure of complex 21 indicated some unusually short bond distances were present. By comparison of the NMR and bond length data for complexes 21 or 69 with those for phenyl-DHP 60, the aromaticity of the cyclobutadienyl cobalt fragment was estimated quantitatively to be at least as large as that of benzene. The organometallic fragment [Cbd-Co-Cp] substantially slowed the DHP photoopening reaction of complex 72 relative to precursors 67 or 35. DHP[e]tropone, 17, DHP[e]tropylium cation, 18 and DHP[e]cyclooctatetraene dianion, 20 were synthesized to study the relative bond localizing abilities of COT dianion 80 (105 % relative to benzene), tropylium cation 15 (55 % relative to benzene), tropone 79 (13 % relative to benzene) using DHP as the NMR probe. The internal methyl protons of DHP resonated at δ -3.56 for 17, δ -2.61 for 18 and δ -1.38 for 20. Cycloheptatrienyl anions 111, 112 and 19 were made from cyclohepta-2,4,6-triene (CHT) isomers 107 and 108 (for two anions, 112 and 19). The internal methyl protons of their DHP resonated at δ -2.52, 2.67 for 111, δ -0.80, -0.84 for 112 and δ +2.10 for 19. The anion 19 was best taken as a 20π electron paratropic system. The homo-aromaticity of the CHT isomers 107, 108 and 117 was estimated based on the NMR data and X-ray structural data. Obvious anisotropic effects existed and the bond localization ability of the CHT (24% based on NMR data for 108) may have a large error. The ring inversion barriers of the COT 82 and CHT 77 were measured using variable temperature NMR spectroscopy as 13.9 and 8.7 kcal/mol, respectively. Five X-ray crystallography structures were obtained for 99B, 17, 108, 117 and 125 and the information was used in estimating the bond localization abilities and in establishing absolute stereochemistry / Graduate

Made available in DSpace on 2016-06-02T20:34:43Z (GMT). No. of bitstreams: 1 5218.pdf: 2403909 bytes, checksum: d82fc82071edd00ee14aaa70f939364d (MD5) Previous issue date: 2013-03-21 / Universidade Federal de Sao Carlos / In this work, the development of electroanalytical methods for the determination of methotrexate (MTX), propylthiouracil (PTU), propanolol (PROP) and verapamil (VPM) in pharmaceutical formulations is described. In these methods it was employed a glassy carbon electrode (GC) modified with dihexadecyl hydrogenphosphate (DHP) or polyallylamine hydrochloride (PAH) film containing immobilized multi-walled carbon nanotubes (MWCNTs). Initially, the electrochemical behavior of each modified electrode was studied by cyclic voltammetry at different scan rates for a 1.0 × 103 mol L1 potassium hexacyanoferrate (III) in 0.1 mol L1 potassium chloride solution. Applying the Randles-Sevcik equation, it was observed an increase of the active area of MWCNTs-DHP/GC and MWCNTs-PAH/GC electrodes of 1.5 and 3.1 times respectively in comparision with the GC electrode. The square wave voltammetry, differential pulse voltammetry and linear sweep voltammetry parameters were studied and optimized for each analyte and then it was selected the best technique for the development of the analytical method. In the developed method for MTX determination in pharmaceutical formulations, it was used the GC electrode modified with MWCNTs and DHP (MWCNTs-DHP/GC), employing differential pulse adsorptive anodic stripping voltammetry (DPAdASV) technique after the optimization of experimental conditions. The analytical curve obtained was linear in the MTX concentration range from 5.0 × 10-8 to 5.0 × 10-6 mol L-1, with a limit of detection (LOD) of 1.2 × 10-8. The MTX was quantified in commercial samples and the results compared with an HPLC method, and the relative errors ranged from -4.0 to 4.0%. An analytical method for determination of PTU in pharmaceutical formulations was also developed using a modified GC electrode with MWCNTs within a PAH film (MWCNTs-PAH/GC) and linear sweep adsorptive cathodic stripping voltammetry (LSAdCSV). After the optimization of experimental conditions, the analytical curve was linear in the PTU concentration range from 5.0 × 10-6 to 5.8 × 10-5 mol L-1, with a LOD equal to 1.0 × 10-6 mol L-1. The PTU was quantified in commercial samples and the results were compared with those results obtained using the British Pharmacopoeia method. The relative errors obtained were between -1.0 and 2.0%. Using DPAdASV technique and a MWCNTs-PAH/GC electrode, PROP was determined in pharmaceutical formulations. The analytical curve was linear in the concentrations range from 7.5 × 10-8 to 1.0 × 10-6 mol L-1 PROP, with LOD equal to 6.3 × 10-8 mol L-1 and the results were in close agreement whit those obtained using the Brazilian Pharmacopoeia method at a confidence level of 95 %. The relative standard deviation ranged from 2.5 and 1.2%. Finally, as a fourth electroanalytical method, a square wave adsorptive anodic stripping voltammetry (SWAdASV) was proposed for determination of VPM in pharmaceutical formulations using a MWCNTs-PAH/GC electrode. The analytical curve was linear over the VPM concentration range 5.0 × 10-9 - 1.0 × 10-7 mol L-1, with an LOD of 3.6 × 10-9 mol L-1 . The VPM was quantified in pharmaceutical samples and the results compared with results obtained using the method of the Brazilian Pharmacopoeia. / Neste trabalho descreve-se o desenvolvimento de métodos eletroanalíticos para a determinação de metotrexato (MTX), propiltiouracil (PTU), propranolol (PROP) e verapamil (VPM) em formulações farmacêuticas. Nestes métodos foi empregado um eletrodo de carbono vítreo (GC) modificado com filme dihexadecil hidrogênofosfato (DHP) ou hidrocloreto de poli(alilamina) (PAH), contendo, imobilizados, nanotubos de carbono de paredes múltiplas (MWCNTs) tratados quimicamente. Inicialmente verificou-se o comportamento eletroquímico desses eletrodos modificados empregando-se voltametria cíclica, em diferentes velocidades de varredura, para uma solução de hexacianoferrato (III) de potássio 1,0 x 10-3 mol L-1 em cloreto de potássio 0,1 mol L-1. Aplicando-se a equação de Randles-Sevcik, observa-se um aumento da área ativa para os eletrodos MWCNTs-DHP/GC e MWCNTs-PAH/GC de 1,5 e 3,1 vezes respectivamente quando comparado com o eletrodo de GC. Os parâmetros da voltametria de onda quadrada, voltametria de pulso diferencial e voltametria linear foram estudados e otimizados para cada analito e selecionada a melhor técnica para o desenvolvimento do método analítico. No método desenvolvido para a determinação de MTX em formulações farmacêuticas, utilizouse o eletrodo de GC modificado com MWCNTs e DHP (MWCNTs-DHP/GC), empregando-se a Voltametria Adsortiva de Redissolução Anódica por Pulso Diferencial (DPAdASV), após a otimização das condições experimentais. A curva analítica obtida foi linear no intervalo de concentração de MTX de 5,0  108 a 5,0  106 mol L-1, com um limite de detecção (LD) igual a 1,2  108. O MTX foi quantificado em amostras comerciais e os resultados comparados com um método de HPLC, sendo que os erros relativos variaram de -4,0 e 4,0 %. O segundo método analítico desenvolvido foi para a determinação de PTU em formulações farmacêuticas, utilizando como eletrodo o GC modificado com MWCNTs e PAH (MWCNTs-PAH/GC). A técnica eletroanalítica selecionada foi a Voltametria Linear Adsortiva de Redissolução (LSAdCSV), após a otimização das condições experimentais. A curva analítica obtida foi linear para o PTU nas concentrações de 5,0  106 a 5,8  105 mol L-1 com LD igual a 1,0  106 mol L-1. O PTU foi quantificado em amostras comerciais, os resultados comparados com método preconizado pela Farmacopéia Britânica e os erros relativos obtidos ficaram entre 1,0 e 2,0 %. O terceiro método analítico desenvolvido foi para a determinação de PROP em formulações farmacêuticas, utilizando como eletrodo o MWCNTs- PAH/GC, sendo a técnica selecionada a DPAdASV. A curva analítica obtida foi linear no intervalo de concentração do PROP de 7,5  108 a 1,0  106 mol L-1, com LD igual a 6,3  108 mol L1.. O PROP foi quantificado em amostras comerciais e os resultados comparados com o método proposto pela Farmacopéia Brasileira. Os erros relativos obtidos ficaram entre 2,5 e 1,2 %. Na sequência, o quarto método desenvolvido foi para a determinação de VPM em formulações farmacêuticas, utilizando como eletrodo o MWCNTs-PAH/GC, sendo a técnica selecionada a voltametria de redissolução anódica de onda quadrada (SWAdASV). A curva analítica obtida foi linear no intervalo de concentrações de VPM de 5,0  109 a 1,0  107 mol L-1, com um LD igual a 3,6  109 mol L1. O VPM foi quantificado em amostras farmacêuticas e os resultados comparados com os resultados obtidos empregando o método espectrofotométrico preconizado pela Farmacopéia Brasileira.

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Determinações de tensão superficial (γ) na interface ar-água e espessuras médias elipsométricas in situ (d), foram usadas para estudar as interações entre dispersões de lípides (em forma de bicamadas) e filmes de poliestireno sulfato produzidos por revestimento rotacional sobre placas de óxido de silício. A adição de NaCl para uma concentração final de 50 mM em uma dispersão de 0.2 mM de lípide catiônico sintético produziu instantaneamente uma camada de 6 nm de espessura que permaneceu estável com o tempo, indicando uma cinética de adsorção muito rápida, determinada possivelmente, pela atração hidrofóbica entre defeitos sobre a bicamada, induzidos pelo sal, e a superfície do filme. Contudo, em água a adsorção de DODAB cresceu monotonicamente alcançando no máximo uma espessura de 1.6-1.8 nm (após 15 horas de interação) em função do tempo, a qual não foi consistente com deposição de bicamada. Nos estágios iniciais em água pura, a adsorção do anfifílico aumentou linearmente com a raiz quadrada do tempo, mostrando um processo controlado por difusão de vesículas, com coeficiente de difusão(D), aproximadamente igual a 1.0 x 10-11 m22s-1 em boa aproximação com valores determinados para vesículas de outros lípides por outros autores. Medidas elipsométricas sob o ar (ex- situ) de adsorção de DODAB, DHP e PC foram difíceis de realizar devido ao aumento da espessura média do filme polimérico durante o ciclo de lavagem e secagem. Na faixa de concentração de 0.1- 1.0 mM o filme adsorvido de DODAB em ar foi mais hidrofóbico (ângulo de contato de avanço ΘA= 84° ± 3°) do que aquele para o filme puro de polímero (ΘA=71 ° ± 4°). Nenhum efeito do filme de DHP sobre a molhabilidade do filme de PSA foi observado, apenas um aumento de rugosidade revelado pela mudança em ΔΘ de 2° para 11° (tabela 3). Filmes planos de PSA são hidrofóbicos(ΘA= 86° ± 5°). Após interação com PC, as superfícies tornam-se menos hidrofóbicas (ΘA= 75° ± 3°). A tensão superficial em ar-água para uma dispersão lipídica em água pura diminuiu rapidamente sob adição de sal (7-50 mM de NaCl), sugerindo a ocorrência de fusão de vesículas, induzida por sal, com a interface ar-água.O estudo da interação entre sais de DODA e partículas poliméricas por medidas de tamanho e potencial-zeta das partículas permitiu observar deposição de bicamadas optimizada por adição de sal a baixas concentrações (0.05-5.0 mM NaCl). / Determinations of surface tension (γ) at the air--water interface, contact angles (Θ), and in and ex-situ ellipsometric mean thickness (d) were used to study the interaction between dioctadecyldimethylammonium bromide (DODAB) small vesicles and spin-coated polystyrene sulfate (PSS) films on silicon wafers. Upon the adition of NaCl (50 mM final concentration) to a 0.2 mM DODAB dispersion, adsorption from vesicles on PSS films immediatly yielded a DODAB layer 6.0 nm thick which remained stable as a function of time. However, in water, in situ DODAB adsorption monotonically increased reaching at most 1.6--1.8 nm as a function of time (from 15 mm of interaction), which were not values consistent with bilayer deposition. At early stages in pure water, DODAB adsorption linearly increased with the square root of time, indicating a vesicle difision controlled process with ca. 1.0 x 10-11 m2s-1 as the vesicle diffusion coeficient (D) in nice agreement with reported D for similar vesicles. In contrast, adding 50 mM salt, resulted in a very fast adsorption kinetics determined by the hydrophobic atraction between salt-induced defects on lhe bilayer and the film surface. Ex situ measurements of DODAB, DHP e PC adsorption were difficult because wetting/drying cycles of the polimeric fiIms increased its mean thickness. From 0.1 up to 1.0 mM DODAB, the adsorbed film in air was more hydrophobic (advancing contact angle, ΘA = 84 ± 3°) than the bare PSS film (ΘA = 71 ± 4°). No effect of DHP film on the wettability of PSA film could be observed, just a increase of rougness revealed by the change of ΔΘ from2° to 11°( table 3). Pure PSA films are hydrophobic, as revealed by the mean advancing angle ΘA = 86 ± 5°. After interaction with PC, the surfaces turn less hydrophobic with a mean ΘA = 75 ± 3°. Air-water surface tension for a DODAB dispersion in pure water rapidly decreased upon salt addition (7-50 mM NaCI), suggesting salt-induced vesicle fusion wilh the air--water interface occurred, in nice agreement with saIt-induced vesicle fusion at the hydrophobic polymer-water surface.The investigation of the interaction between DODA salts and polymeric particles from diameter and zeta- potential measurements, allowed us to detect bilayer deposition optimized by salt adition in low concentration(0.05-5.0 mM NaCl).

Determinações de tensão superficial (γ) na interface ar-água e espessuras médias elipsométricas in situ (d), foram usadas para estudar as interações entre dispersões de lípides (em forma de bicamadas) e filmes de poliestireno sulfato produzidos por revestimento rotacional sobre placas de óxido de silício. A adição de NaCl para uma concentração final de 50 mM em uma dispersão de 0.2 mM de lípide catiônico sintético produziu instantaneamente uma camada de 6 nm de espessura que permaneceu estável com o tempo, indicando uma cinética de adsorção muito rápida, determinada possivelmente, pela atração hidrofóbica entre defeitos sobre a bicamada, induzidos pelo sal, e a superfície do filme. Contudo, em água a adsorção de DODAB cresceu monotonicamente alcançando no máximo uma espessura de 1.6-1.8 nm (após 15 horas de interação) em função do tempo, a qual não foi consistente com deposição de bicamada. Nos estágios iniciais em água pura, a adsorção do anfifílico aumentou linearmente com a raiz quadrada do tempo, mostrando um processo controlado por difusão de vesículas, com coeficiente de difusão(D), aproximadamente igual a 1.0 x 10-11 m22s-1 em boa aproximação com valores determinados para vesículas de outros lípides por outros autores. Medidas elipsométricas sob o ar (ex- situ) de adsorção de DODAB, DHP e PC foram difíceis de realizar devido ao aumento da espessura média do filme polimérico durante o ciclo de lavagem e secagem. Na faixa de concentração de 0.1- 1.0 mM o filme adsorvido de DODAB em ar foi mais hidrofóbico (ângulo de contato de avanço ΘA= 84° ± 3°) do que aquele para o filme puro de polímero (ΘA=71 ° ± 4°). Nenhum efeito do filme de DHP sobre a molhabilidade do filme de PSA foi observado, apenas um aumento de rugosidade revelado pela mudança em ΔΘ de 2° para 11° (tabela 3). Filmes planos de PSA são hidrofóbicos(ΘA= 86° ± 5°). Após interação com PC, as superfícies tornam-se menos hidrofóbicas (ΘA= 75° ± 3°). A tensão superficial em ar-água para uma dispersão lipídica em água pura diminuiu rapidamente sob adição de sal (7-50 mM de NaCl), sugerindo a ocorrência de fusão de vesículas, induzida por sal, com a interface ar-água.O estudo da interação entre sais de DODA e partículas poliméricas por medidas de tamanho e potencial-zeta das partículas permitiu observar deposição de bicamadas optimizada por adição de sal a baixas concentrações (0.05-5.0 mM NaCl). / Determinations of surface tension (γ) at the air--water interface, contact angles (Θ), and in and ex-situ ellipsometric mean thickness (d) were used to study the interaction between dioctadecyldimethylammonium bromide (DODAB) small vesicles and spin-coated polystyrene sulfate (PSS) films on silicon wafers. Upon the adition of NaCl (50 mM final concentration) to a 0.2 mM DODAB dispersion, adsorption from vesicles on PSS films immediatly yielded a DODAB layer 6.0 nm thick which remained stable as a function of time. However, in water, in situ DODAB adsorption monotonically increased reaching at most 1.6--1.8 nm as a function of time (from 15 mm of interaction), which were not values consistent with bilayer deposition. At early stages in pure water, DODAB adsorption linearly increased with the square root of time, indicating a vesicle difision controlled process with ca. 1.0 x 10-11 m2s-1 as the vesicle diffusion coeficient (D) in nice agreement with reported D for similar vesicles. In contrast, adding 50 mM salt, resulted in a very fast adsorption kinetics determined by the hydrophobic atraction between salt-induced defects on lhe bilayer and the film surface. Ex situ measurements of DODAB, DHP e PC adsorption were difficult because wetting/drying cycles of the polimeric fiIms increased its mean thickness. From 0.1 up to 1.0 mM DODAB, the adsorbed film in air was more hydrophobic (advancing contact angle, ΘA = 84 ± 3°) than the bare PSS film (ΘA = 71 ± 4°). No effect of DHP film on the wettability of PSA film could be observed, just a increase of rougness revealed by the change of ΔΘ from2° to 11°( table 3). Pure PSA films are hydrophobic, as revealed by the mean advancing angle ΘA = 86 ± 5°. After interaction with PC, the surfaces turn less hydrophobic with a mean ΘA = 75 ± 3°. Air-water surface tension for a DODAB dispersion in pure water rapidly decreased upon salt addition (7-50 mM NaCI), suggesting salt-induced vesicle fusion wilh the air--water interface occurred, in nice agreement with saIt-induced vesicle fusion at the hydrophobic polymer-water surface.The investigation of the interaction between DODA salts and polymeric particles from diameter and zeta- potential measurements, allowed us to detect bilayer deposition optimized by salt adition in low concentration(0.05-5.0 mM NaCl).

This study was conducted to identify and characterize rumen bacteria that are able to degrade the toxic compound, 3-hydroxy-4(lH)-pyridone (3,4 DHP), that is produced in the rumen from mimosine. Mimosine is a non-protein amino acid that is found in leaves and seeds of Leucaena leucocephala, a leguminous tree used as a forage crop for ruminants in the tropics, and degradation of 3,4 DHP by ruminal microbes is critical for protection of animals from leucaena toxicity. Microbes with this capacity are, however, not ubiquitous and microbial populations in the rumens of animals in some parts of the world are unable to metabolize 3,4 DHP. Four strains of obligately anaerobic, gram-negative, rod-shaped bacteria that degrade 3,4 DHP were isolated from rumen contents from a goat in Hawaii. The isolates do not ferment carbohydrates, but are able to use both 3,4 DHP and its isomer, 3-hydroxy-2(lH)-pyridone (2,3 DHP), as well as arginine and histidine as substrates for growth. Comparisons of the 16S rRNA sequence from one of these isolates with sequences from a widely diverse group of bacteria agree with other information indicating that these isolates do not fit into any existing taxon. Thus, we are hereby proposing a new genus and species designation, Synergistes jonesii, for these organisms.