Neste projeto estudou-se a interação da enzima peroxidase de raiz forte (HRP) em interfaces nanoestruturadas e sua possível aplicação em biossensores de peróxido de hidrogênio. Foram utilizadas as técnicas de Langmuir, Langmuir-Blodgett (LB) e automontagem por adsorção física para formar filmes nanoestruturados. A interação da enzima com espécies em interfaces foi investigada com materiais que serviram de matrizes de adsorção, ou seja, a quitosana (Ch) e o fosfolipídio 1,2-dipalmitoil-sn-glicero-3-[fosfatidil-rac-(1-glicerol)] (sal de sódio) (DPPG). Os filmes de Langmuir foram caracterizados com medidas de pressão e potencial de superfície, espectroscopia no infravermelho, e tensão superficial dinâmica. Para os filmes LB e automontados, empregaram-se espectroscopias de fluorescência, ultravioleta-visível e infravermelho e microgravimetria por cristal de quartzo. A peroxidase de raiz forte apresentou forte interação com DPPG, confirmada em filmes de Langmuir por medidas de pressão de superfície, elasticidade dinâmica e de espectroscopia de reflexão e absorção no infravermelho, com modulação por polarização (PM-IRRAS). A massa de peroxidase transferida em filmes Langmuir-Blodgett (LB) mistos com DPPG foi de aproximadamente 200 ng, de acordo com medidas com uma microbalança de cristal de quartzo. A atividade da HRP foi mantida no filme LB, inclusive com atividade catalítica maior do que em meio homogêneo e nos filmes automontados com quitosana. As medidas de atividade não afetaram a morfologia dos filmes LB, estudada com microscopia de força atômica (AFM), ao contrário dos filmes automontados. Conclui-se que a imobilização de HRP é mais eficiente num filme LB, com matriz fosfolipídica, apresentando boas perspectivas de emprego em biossensores de peróxido de hidrogênio. / A study has been performed on the interaction of the enzyme horseradish peroxidase (HRP) in nanostructured interfaces and their possible application in biosensors for hydrogen peroxide. The nanostructured films were obtained with the Langmuir, Langmuir-Blodgett (LB) and layer-by-layer (LbL) methods. The interaction between HRP and species at interfaces was investigated using materials that served as matrix for immobilization, viz. chitosan (Ch) and the phospholipid 1,2-dipalmytoil-sn-glycero-3-[phosphatidyl-rac-(1-glycerol)] (sodium salt) (DPPG). The Langmuir films were characterized with surface pressure, surface potential, elasticity measurements and polarization-modulation reflection and absorption infrared spectroscopy (PM-IRRAS). For LB and LbL films, use was made of fluorescence, absorption in the UV-vis. and infrared spectroscopy. HRP displayed strong interaction with DPPG, which was confirmed in Langmuir films with measurements of surface pressure, dynamic elasticity and PM-IRRAS. The mass of HRP transferred onto a solid support in a mixed LB film with DPPG was 200 ng, according to data from a quartz crystal microbalance. The HRP activity was preserved in the mixed LB film, with a catalytic activity that was even higher than in solution or in LbL films of HRP/Ch. The catalytic activity measurements did not affect the morphology of the LB films, studied with atomic force microscopy (AFM), in contrast to the LbL films. The main conclusion is that HRP immobilization is more efficient in an LB film with a phospholipid matrix, with good prospects for developing biosensors for hydrogen peroxide.

Thesis (M.Sc. (Anatomy))--Mahidol University, 1982.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-19T13:34:44Z No. of bitstreams: 5 jus.pdf: 3394180 bytes, checksum: 71d0ff7761a41fcaa36b37cfba79f687 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-20T10:26:30Z (GMT) No. of bitstreams: 5 jus.pdf: 3394180 bytes, checksum: 71d0ff7761a41fcaa36b37cfba79f687 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) / Made available in DSpace on 2015-02-20T10:26:30Z (GMT). No. of bitstreams: 5 jus.pdf: 3394180 bytes, checksum: 71d0ff7761a41fcaa36b37cfba79f687 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2110 bytes, checksum: b4c884761e4c6c296ab2179d378436d4 (MD5) Previous issue date: 2013-12-17 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Atualmente os nanotubos de carbono (NTC) representam um importante grupo de nanomateriais com atrativas propriedades qu?micas, eletr?nicas e mec?nicas. Estes materiais podem ser combinados a pol?meros, de maneira a moldar as propriedades f?sicas desses compostos (nanotubos-pol?mero), aumentando sua condutividade el?trica, estabilidade qu?mica e resist?ncia mec?nica. O presente trabalho apresenta o desenvolvimentoum material comp?sitonanoestruturado ? base de polianilina (PANI) e nanotubos de carbono de paredes m?ltiplas(MWCNT), para tal, os NTC foram funcionalizados com agrupamentos amina (PEG-NH2). A inser??o do grupamento amina junto aos NTC (PEG-MWCNT) foi confirmada atrav?s da caracteriza??o deste material utilizando as t?cnicas de Microscopia Eletr?nica de Varredura (MEV), Espectroscopia de infravermelho com transformada de fourrier(FT-IR) e Espectroscopia de Energia dispersiva de Raios-X. Foi produzido um eletrodo modificado atrav?s da polimeriza??o do mon?mero anilinaem presen?a dos NTC funcionalizados denominado PANI/PEG-MWCNT. Foram analisadas as propriedades do comp?sito PANI/PEG-MWCNT pelas t?cnicas de Voltametria c?clica e Espectroscopia de Imped?ncia Eletroqu?mica, atrav?s das quais foi verificado que a presen?a combinada dos NTC e da PANI favorece forma??o de um material comp?sito com propriedades sin?rgicas. Al?m disso, atrav?s da Microbalan?a Eletroqu?mica de Cristal de Quartzo pode-se constatarque a dopagem da PANI com PEG-MWCNT favorece o transporte de pr?tons significativamente. Atrav?s da t?cnica de microscopia eletroqu?mica de varredura foi poss?vel evidenciar que existem mais s?tios ativos na superf?cie do filme de PANI/PEG-MWCNT do que na superf?cie da PANI devido ? incorpora??o uniforme dos MWCNT. Finalmente, o eletrodo modificado com o material comp?sito foi utilizado como plataforma no desenvolvimento de um biossensor enzim?tico, pela imobiliza??o da enzima HRP (HRP/PANI/PEG-MWCNT). O biossensor HRP/PANI/PEG-MWCNT apresentou um ?timo suporte para aredu??o de per?xido de hidrog?nio a baixo potencial (-100 mV) vs Ag/AgCl, bem como alta sensibilidade (1.01) ?Acm-2 ?M?1e baixo limite de detec??o de 0,5 ?M L-1. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2013. / ABSTRACT Currently carbon nanotubes (NTC) represent an important group of nanomaterials with attractive properties (chemical, electronic and mechanical). These materials can be combined with polymers in order to shape their physical properties of these compounds (nanotube-polymer), increasing its electrical conductivity, chemical stability and mechanical strength. In fact, this study shows the production of a nanostructured composite material based on polyaniline (PANI) and carbon nanotubes multi-walled (MWCNT), for this, the NTC were functionalized with amine groups (PEG-NH2). The insertion of the amine grouping together to NTC (MWCNT-PEG) was confirmed by the characterization of this material using the techniques of Scanning Electron Microscopy (SEM) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and energy dispersive X-Ray. A modified electrode was produced through polymerization in the presence of PANI called NTC functionalized PANI/PEG-MWCNT. The properties of the composite PANI/MWCNT-PEG were examined by cyclic voltammetry and Electrochemical Impedance Spectroscopy, it was found that the combined presence of the NTC and polyaniline in the modified electrode provides a formation of a composite material with synergistic properties. Furthermore, through the technique of Electrochemical Microbalance Quartz Crystal can be seen that doping of PANI with PEG-MWCNT facilitates significantly the transport of protons. Finally, through the technique of scanning electrochemical microscopy was possible to show that there are more active sites on the surface of the film of PANI / MWCNT-PEG than on the surface of PANI due to the incorporation of MWCNT uniform. Lastly, the modified electrode composite material was used as a support for a biosensor for immobilization of the enzyme HRP (HRP/PANI/PEG-MWCNT). The biosensor HRP/PANI/MWCNT-PEG showed a great support for the reduction of hydrogen peroxide at a low potential (-100mv) vs Ag / AgCl as well as high sensitivity (1.01) ?A cm-2 mM-1 and low detection limit 0.5 ?m.

Neste projeto estudou-se a interação da enzima peroxidase de raiz forte (HRP) em interfaces nanoestruturadas e sua possível aplicação em biossensores de peróxido de hidrogênio. Foram utilizadas as técnicas de Langmuir, Langmuir-Blodgett (LB) e automontagem por adsorção física para formar filmes nanoestruturados. A interação da enzima com espécies em interfaces foi investigada com materiais que serviram de matrizes de adsorção, ou seja, a quitosana (Ch) e o fosfolipídio 1,2-dipalmitoil-sn-glicero-3-[fosfatidil-rac-(1-glicerol)] (sal de sódio) (DPPG). Os filmes de Langmuir foram caracterizados com medidas de pressão e potencial de superfície, espectroscopia no infravermelho, e tensão superficial dinâmica. Para os filmes LB e automontados, empregaram-se espectroscopias de fluorescência, ultravioleta-visível e infravermelho e microgravimetria por cristal de quartzo. A peroxidase de raiz forte apresentou forte interação com DPPG, confirmada em filmes de Langmuir por medidas de pressão de superfície, elasticidade dinâmica e de espectroscopia de reflexão e absorção no infravermelho, com modulação por polarização (PM-IRRAS). A massa de peroxidase transferida em filmes Langmuir-Blodgett (LB) mistos com DPPG foi de aproximadamente 200 ng, de acordo com medidas com uma microbalança de cristal de quartzo. A atividade da HRP foi mantida no filme LB, inclusive com atividade catalítica maior do que em meio homogêneo e nos filmes automontados com quitosana. As medidas de atividade não afetaram a morfologia dos filmes LB, estudada com microscopia de força atômica (AFM), ao contrário dos filmes automontados. Conclui-se que a imobilização de HRP é mais eficiente num filme LB, com matriz fosfolipídica, apresentando boas perspectivas de emprego em biossensores de peróxido de hidrogênio. / A study has been performed on the interaction of the enzyme horseradish peroxidase (HRP) in nanostructured interfaces and their possible application in biosensors for hydrogen peroxide. The nanostructured films were obtained with the Langmuir, Langmuir-Blodgett (LB) and layer-by-layer (LbL) methods. The interaction between HRP and species at interfaces was investigated using materials that served as matrix for immobilization, viz. chitosan (Ch) and the phospholipid 1,2-dipalmytoil-sn-glycero-3-[phosphatidyl-rac-(1-glycerol)] (sodium salt) (DPPG). The Langmuir films were characterized with surface pressure, surface potential, elasticity measurements and polarization-modulation reflection and absorption infrared spectroscopy (PM-IRRAS). For LB and LbL films, use was made of fluorescence, absorption in the UV-vis. and infrared spectroscopy. HRP displayed strong interaction with DPPG, which was confirmed in Langmuir films with measurements of surface pressure, dynamic elasticity and PM-IRRAS. The mass of HRP transferred onto a solid support in a mixed LB film with DPPG was 200 ng, according to data from a quartz crystal microbalance. The HRP activity was preserved in the mixed LB film, with a catalytic activity that was even higher than in solution or in LbL films of HRP/Ch. The catalytic activity measurements did not affect the morphology of the LB films, studied with atomic force microscopy (AFM), in contrast to the LbL films. The main conclusion is that HRP immobilization is more efficient in an LB film with a phospholipid matrix, with good prospects for developing biosensors for hydrogen peroxide.

Doctor of Philosophy / Department of Civil Engineering / Alok Bhandari / Human activities generate large amounts of chlorinated phenolic chemicals that are often introduced into the soil environment during pesticide and insecticide application, industrial releases, and accidental spills. For example, 2,4-dichlorophenol (DCP), a derivative of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) can been found in soil within 24 hours of 2,4- D application. Horseradish peroxidase (HRP)-mediated polymerization has been proposed as an approach to remediate soils and groundwater contaminated by phenolic pollutants. Treatment with HRP results in the transformation of phenols into polyphenolic oligomers that sorb strongly or precipitate on soils surfaces. Although HRP-mediated chlorophenol stabilization has been studied extensively in surface soils, very limited scientific data is available that supports the application of this technology in subsurface materials. Hence, the focus of this study was to evaluate sorption and binding of DCP and products of HRP-mediated polymerization of DCP to model geosorbents representing subsurface geomaterials. These sorbents included two humin-mineral geomaterials and one mineral geosorbent derived from surface soils. Soil-water phase distribution of total solute in the HRP-amended systems was observed to reach equilibrium within 7 days in woodland humin-mineral soil (WHM), and within 1 day in agricultural humin-mineral (AHM) and model mineral geomaterials. For all the geomaterials used, water extraction data indicated the development of contact time-dependent resistance to extraction/dissolution of soil-associated DCP and DPP. Solute associated with WHM geomaterial was higher at the end of the study than that associated with AHM. Contact time increased DCP stabilization at all initial aqueous DCP concentrations studied. Results of this study suggest that DCP stabilization in organic geosorbents results from a combination of sorption and cross-coupling of DCP and precipitation of DPP; in inorganic soils, precipitation of DPP macromolecules is the dominant process. HRP-mediated stabilization of DCP in soils was effective and independent of the solution ionic concentration. The amount of DCP stabilized in the mineral soil was comparable to that stabilized in humin-mineral geomaterials. The research reported in this dissertation demosntrates the potential of HRP enzyme to stabilize DCP in subsurface geomaterials under variable contaminant and salt concentrations, thereby restricting its transport in the environment.

Moringa oleifera Lam. is a tree with great potential as it is fast growing and drought tolerant. Amongst the tree’s multitude of benefits, it can also be used to produce a biodiesel fuel. However, prior to the establishment of commercial plantations, all cultivation aspects of this promising tree have to be understood. Temperature is a significant climatic factor influencing both geographical plant distribution and growth, and since M. oleifera trees are naturally found in tropical climates around the world, the extent of their adaptability to cooler climates was the main objective of this study. Trees were cultivated from seed and germinated in a controlled greenhouse environment at the Experimental Farm of the University of Pretoria. After germination, 50% of the seedlings were hardened-off by placing them outside under ambient growing temperatures, while the rest remained inside the greenhouse. With trial commencement, 132 of both the hardened and non-hardened seedlings were planted into 10 ℓ plastic bags and randomly placed into three temperature-controlled greenhouses, each with a different fluctuating night/day temperature regime namely; 10/20°C ± 2°C, 15/25°C ± 2°C and 20/30°C ± 2°C. In addition, half the trees within each temperature regime were treated with the growth regulator paclobutrazol to determine its effect on growth/flowering at different temperatures. During the 224-day trial period, biweekly measurements of tree height, stem diameter and leaf area estimates of each individual tree within all three temperature regimes were taken. Despite germination percentages being slightly higher at the low 10/20°C regime, the MGT, germination rate, uniformity and seedling growth were superior at the higher 20/30°C regime. The temperature induced seed dormancy at the 20/30°C regime, could be overcome by an incubation period at lower temperatures, as fewer instances of seed dormancy were observed at the 10/20°C regime. The increase in temperature resulted in significant (P≤0.05) growth rate increases of over 650% between the 10/20°C and 20/30°C and 250% between the 10/20°C and 15/25°C night/day temperature regimes. In addition, the 20/30°C temperature treatment, although fluctuative, consistently had the highest leaf area over the entire trial period. Hardening-off of trees during the seedling stage, significantly (P≤0.05) increased the final tree height by 3.09X, 1.44X and 1.23X, compared to their non hardened-off counterparts under the 10/20°C, 15/25°C and 20/30°C temperature regimes respectively. Leaf thickness decreased by a significant (P≤0.05) 43.1% with increase in temperature between the 10/20°C and 20/30°C regime, mostly due to a thinner mesophyll layer. The efficacy of paclobutrazol on M. oleifera growth was found to be temperature dependant, reducing growth at 10/20°C, while increasing growth at both the higher 15/25°C and 20/30°C regimes. Flowering however remained unaffected by paclobutrazol. The highest instances of flowering and pollen viability were observed at the 15/25°C regime. The absence of inflorescence induction at the 20/30°C regime was responsible for the reduced flowering, signifying the necessity of vernalization prior to flowering. Even though all the results confirm the preference of M. oleifera trees towards a tropical climate, satisfactory growth with possibly improved flowering during the hot summer months in certain sub-tropical climates is achievable. Copyright / Dissertation (MSc(Agric))--University of Pretoria, 2010. / Plant Production and Soil Science / unrestricted

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care diagnostic tests for use by untrained users with potential applications in healthcare, environmental monitoring, and food safety. These devices can be developed for a multitude of different tests, many of which employ enzymes as catalysts. Without specialized treatment, some enzymes tend to lose their activity when stored on microPADs within 48 hours, which is a major hurdle for taking these types of devices out of the laboratory and into the real world. This work focused on the development of simple methods for stabilizing enzymes by applying polymers to chromatography paper. The longterm stabilization was exlored and SU-8 of various concentrations was found to stabilize horseradish peroxidase for times in excess of two weeks. A variety of microPAD fabrications, enzyme dispensing methods, and substrate delivery techniques were explored.

Neurons of the nucleus reuniens thalami stained with Golgi methods are compared to cells in this nucleus labelled in retrograde fashion after hippocampal injections of horseradish peroxidase. The cellular morphology ranges from fusiform to multiangular with most cells showing radiating processes characteristic of neurons in the reticular core. Dendrites are long and relatively smooth, with a few sparsely distributed spinous processes. These cells are comparable to the cholinergic cells of the median septal/diagonal band area which also project into the hippocampal formation.

The digastric muscle of the rabbit consists of a single anterior belly which inserts onto the lower jaw. Horseradish peroxidase was injected into the muscle and into subcutaneous regions overlying the lower jaw to determine the sites of origin of the motor innervation to both the digastric muscle and the platysma muscles. After digastric muscle injection, labelled cells were found in the ipsilateral retrotrigeminal nucleus as well as in the intermediate subnucleus of the main facial nucleus on both sides. Subcutaneous injections produced labelling which was found bilaterally in the intermediate subnucleus and in the ventromedial portion of the medial subnucleus. These results are interpreted in relation to the common embryological origin of these two muscles and their innervation.

The anterograde horseradish peroxidase method was used to determine if prenatal exposure to ethanol affected the development of the characteristic afferent lamination pattern of the commissural projection to the dentate gyrus. Mean ethanol consumption for the ethanol-consuming dams was 12.7 g/kg ± 0.3 g per day. Adult offspring of rats that consumed a liquid diet containing 35% ethanol-derived calories during days 1-21 of gestation, and both pair-fed and normal controls were examined. Brain weights and volumes of the ethanol and pair-fed control rats did not differ significantly from normal controls. However, body weights of ethanol-exposed rats were significantly reduced compared to normal controls. Computer-assisted image analysis of the HRP-labeling revealed that in spite of the heavy ethanol exposure there was no evidence of alterations in the spatial distribution of the commissural terminal field.