Neste trabalho realizou-se estudos comparativos da fototransformação da protoporfirina IX (PpIX) sintética e endógena, extraída de glândulas Harderianas dos ratos da espécie Rattus Novergicus Albinus, quando irradiada pela luz visível. A fototransformação foi analisada através de monitoramento dos espectros de absorção ótica e de fluorescência em soluções aquosas homogêneas de tampão fosfato e na presença de micelas e albumina de soro bovino (ASB) e em soluções não aquosas de Acetonitrila e Dimetilsulfóxido na proporção 3:1 e em acetona, todos na presença e ausência de oxigênio. Além disso, foram obtidos através de técnica de flash fotólise as curvas cinéticas da absorção do Tripleto da PpIX e da fosforescência do oxigênio singleto. Observamos que sob ação de luz visível, a PplX sobre fototransformação cujas características dependem tanto da natureza da PplX qunto do ambiente onde a PplX se encontra. Devido a sua alta hidrofobicidade a PplX sintética em solução aquosa está presente em forma de agregados o que diminui a velocidade da sua fototransformação. Já a PplX endógena, quando em soluções aquosas, não sofre agregação, pois continua ligada com algumas estruturas biológicas de natureza anfifílica, que sobraram após sua extração de glândulas, e sua velocidade de fototransformação é elevada. A ligação com essas estruturas aumenta a afinidade da PplX endógena com micelas e dificulta sua ligação com a ASB. Os processos de fototransformação de ambas as PplX na interação com as micelas, com ASB e em tecidos glandulares in vivo são semelhantes. Isso mostra que o processo de fototransformação em meio micelar e na interação com ASB modela com sucesso o processo dentro de tecidos.Baseando-se nos fatos que a taxa de fototransformação da PplX aumenta na presença do oxigênio e que foi observada a formação do oxigênio singleto pela transferência de energia do estado tripleto da PplX para o oxigênio molecular, podemos dizer que o mecanismo tipo II contribui na fototransformação da PplX e esse mecanismo é responsável pela formação de fotoproduto, caracterizado pelos novos picos nos espectros de absorção óptica e de fluorescência. Entretanto, a existência de fototransformação da PplX na ausência do oxigênio mostra a participação de outros mecanismos de fototransformação. / In this work we carried out comparative studies of phototransformation of protoporphyrin IX (PpIX) both synthetic and endogenous, extracted from the Harderian glands of the Rattus Novergicus Albinus, when irradiated by visible light. The phototransformation was analyzed by monitoring the PpIX optical absorption and fluorescence spectra in homogeneous aqueous solution (phosphate buffer) and in the presence of bovine serum albumin (BSA), micelles, formed from different surfactants, in non-aqueous acetonitrile and dimethyl sulfoxide 3:1 solutions, and in acetone, both in the presence and absence of oxygen. Besides, the kinetic curves of the PpIX Triplet state absorption and the singlet oxygen phosphorescence were obtained using the flash photolysis technique. It was observed that when irradiated with visible light PplX suffers phototransformation with characteristics depending on its nature and on the environment. Due to high hydrophobicity the synthetic PplX forms aggregates, which reduces its phototransformation rate, while the endogenous PplX is not aggregated due to its interaction with the residues of some biological amphiphilic structures, which continue after it extraction from glands, and its phototransformation rate is elevated. Besides, the interaction with these structures increases the endogenous porphyrin affinity with micelles and reduces that with BSA. The characteristics of both PplX phototransformation in micellar media, at the interaction with BSA and in the gland tissues in vivo are similar. This demonstrates that the phototransformation process in micellar media and at the interaction with BSA may successfully simulate the process in the tissue.Basing on the facts that the phototransformation rate increases in the presence of molecular oxygen and that the singlet oxygen formation due to energy transfer from the PplX triplet state to oxygen was observed we can conclude that the type II mechanism contributes on the PplX phototransformation and is responsible on the formation of a photoproduct with observed new optical absorption and fluorescence peaks. At the same time, the PplX phototransformation in the oxygen absence shows the participation of other mechanisms on the PplX phototransformation.

Cytoglobin (Cygb) and neuroglobin (Ngb) are among the newest members of vertebrate globin family characterized by a classical 3-over-3 α-helical fold and a heme prosthetic group capable of reversibly binding small ligands such as O2, CO and NO. The physiological functions of Cygb and Ngb remain to be determined; however, current data suggest that both proteins have a significant role in cytoprotection in hypoxic and genotoxic conditions. Cytoglobin and Ngb are distinct from their better-known counterparts, hemoglobin (Hb) and myoglobin (Mb), in several structural features. First, in the absence of an external ligand, the sixth coordination site of the heme iron in Cygb and Ngb is occupied by a distal histidine residue, leading to a complex ligand rebinding mechanism dependent on the rate of distal His dissociation from the heme iron. Although hexacoordination was observed before in plant and bacterial hemoglobins, the physiological role of this feature remains unknown. Second, both Ngb and Cygb are capable of forming an intraprotein disulfide bond, which has been shown to regulate ligand binding affinity, leading to a hypothesis that intracellular function of these proteins is redox-dependent. Lastly, Cygb contains 20 amino acid long extensions on both N- and C- termini, a unique feature among vertebrate globins with unknown physiological function. The work presented in the dissertation reveals that hexacoordinate heme reactivity is distinct from that of pentacoordinate heme and is strongly influenced by the distal histidine residue and the disulfide bond. In the case of human Cygb, experimental and computational approaches demonstrated that the disulfide bond regulates the flexibility of the N terminus and the accessibility of the 1,8-ANS binding site. Furthermore, molecular dynamics of the hexa- and pentacoordinate human Ngb were probed computationally to elucidate structural requirements that govern signal transmission between CD loop and the distal pocket. Lastly, Ngb and Cygb were reconstituted with a fluorescent analog of the native heme group to produce hexacoordinate variants with favorable photophysical properties that can be used to characterize protein-protein interactions.

Porphyria is a rare metabolic disease which occurs as a result of accumulation of endogenous porphyrins due to specific enzyme deficiency in the biosynthetic pathway of heme. Chloroquine is currently used in the treatment of cutaneous porphyria, although its mechanism of action is not yet well understood. It is believed that chloroquine works in porphyria by forming complexes with excess porphyrin molecules and thus enhancing their elimination from the body. Previous reports of porphyrin-chloroquine complexes have been done mostly in aqueous models. In this study, UV/Visible optical absorbance difference spectroscopy was used to study the complexation of protoporphyrin IX with chloroquine and a range of acceptor molecules in hydrophobic models. The results show that chloroquine, mefloquine, amodiaquine, quinacrine, and pyronaridine formed relatively stronger complexes compared to other molecules such as quinine, duroquinone and caffeine. Therefore, relative to chloroquine, some of the molecules with comparable or greater binding affinity to protoporphyrin IX might also be useful in the treatment of porphyria.

Porphyria is a rare metabolic disease which occurs as a result of accumulation of endogenous porphyrins due to specific enzyme deficiency in the biosynthetic pathway of heme. Chloroquine is currently used in the treatment of cutaneous porphyria, although its mechanism of action is not yet well understood. It is believed that chloroquine works in porphyria by forming complexes with excess porphyrin molecules and thus enhancing their elimination from the body. Previous reports of porphyrin-chloroquine complexes have been done mostly in aqueous models. In this study, UV/Visible optical absorbance difference spectroscopy was used to study the complexation of protoporphyrin IX with chloroquine and a range of acceptor molecules in hydrophobic models. The results show that chloroquine, mefloquine, amodiaquine, quinacrine, and pyronaridine formed relatively stronger complexes compared to other molecules such as quinine, duroquinone and caffeine. Therefore, relative to chloroquine, some of the molecules with comparable or greater binding affinity to protoporphyrin IX might also be useful in the treatment of porphyria.

Neste trabalho realizou-se estudos comparativos da fototransformação da protoporfirina IX (PpIX) sintética e endógena, extraída de glândulas Harderianas dos ratos da espécie Rattus Novergicus Albinus, quando irradiada pela luz visível. A fototransformação foi analisada através de monitoramento dos espectros de absorção ótica e de fluorescência em soluções aquosas homogêneas de tampão fosfato e na presença de micelas e albumina de soro bovino (ASB) e em soluções não aquosas de Acetonitrila e Dimetilsulfóxido na proporção 3:1 e em acetona, todos na presença e ausência de oxigênio. Além disso, foram obtidos através de técnica de flash fotólise as curvas cinéticas da absorção do Tripleto da PpIX e da fosforescência do oxigênio singleto. Observamos que sob ação de luz visível, a PplX sobre fototransformação cujas características dependem tanto da natureza da PplX qunto do ambiente onde a PplX se encontra. Devido a sua alta hidrofobicidade a PplX sintética em solução aquosa está presente em forma de agregados o que diminui a velocidade da sua fototransformação. Já a PplX endógena, quando em soluções aquosas, não sofre agregação, pois continua ligada com algumas estruturas biológicas de natureza anfifílica, que sobraram após sua extração de glândulas, e sua velocidade de fototransformação é elevada. A ligação com essas estruturas aumenta a afinidade da PplX endógena com micelas e dificulta sua ligação com a ASB. Os processos de fototransformação de ambas as PplX na interação com as micelas, com ASB e em tecidos glandulares in vivo são semelhantes. Isso mostra que o processo de fototransformação em meio micelar e na interação com ASB modela com sucesso o processo dentro de tecidos.Baseando-se nos fatos que a taxa de fototransformação da PplX aumenta na presença do oxigênio e que foi observada a formação do oxigênio singleto pela transferência de energia do estado tripleto da PplX para o oxigênio molecular, podemos dizer que o mecanismo tipo II contribui na fototransformação da PplX e esse mecanismo é responsável pela formação de fotoproduto, caracterizado pelos novos picos nos espectros de absorção óptica e de fluorescência. Entretanto, a existência de fototransformação da PplX na ausência do oxigênio mostra a participação de outros mecanismos de fototransformação. / In this work we carried out comparative studies of phototransformation of protoporphyrin IX (PpIX) both synthetic and endogenous, extracted from the Harderian glands of the Rattus Novergicus Albinus, when irradiated by visible light. The phototransformation was analyzed by monitoring the PpIX optical absorption and fluorescence spectra in homogeneous aqueous solution (phosphate buffer) and in the presence of bovine serum albumin (BSA), micelles, formed from different surfactants, in non-aqueous acetonitrile and dimethyl sulfoxide 3:1 solutions, and in acetone, both in the presence and absence of oxygen. Besides, the kinetic curves of the PpIX Triplet state absorption and the singlet oxygen phosphorescence were obtained using the flash photolysis technique. It was observed that when irradiated with visible light PplX suffers phototransformation with characteristics depending on its nature and on the environment. Due to high hydrophobicity the synthetic PplX forms aggregates, which reduces its phototransformation rate, while the endogenous PplX is not aggregated due to its interaction with the residues of some biological amphiphilic structures, which continue after it extraction from glands, and its phototransformation rate is elevated. Besides, the interaction with these structures increases the endogenous porphyrin affinity with micelles and reduces that with BSA. The characteristics of both PplX phototransformation in micellar media, at the interaction with BSA and in the gland tissues in vivo are similar. This demonstrates that the phototransformation process in micellar media and at the interaction with BSA may successfully simulate the process in the tissue.Basing on the facts that the phototransformation rate increases in the presence of molecular oxygen and that the singlet oxygen formation due to energy transfer from the PplX triplet state to oxygen was observed we can conclude that the type II mechanism contributes on the PplX phototransformation and is responsible on the formation of a photoproduct with observed new optical absorption and fluorescence peaks. At the same time, the PplX phototransformation in the oxygen absence shows the participation of other mechanisms on the PplX phototransformation.

Kolorektale Karzinome weisen eine Rotfluoreszenz auf. Die Art des zugrunde liegenden Fluorophors und sein diagnostisches Potential waren Gegenstand der Arbeit. Protoporphyrin IX (PpIX) wurde als das prädominant vorkommende Fluororphor in Primärtumoren und ihren Metastasen identifiziert. Das Fluorophor wurde in Abwesenheit von Nekrose und in sterilen Lokalisationen nachgewiesen. Affymetrix-GeneChip und quantitative PCR untersuchungen der Enzyme der Häm-Synthese machen eine Minderexpression als Ursache der PpIX Akkumulation wahrscheinlich. In Nicht-vorbehandelten Fällen erlaubt die PpIX-Fluoreszenz eine Diskrimination der metastatisch befallenen Lymphknoten mit einer Sensitivität von 62% bei einer Spezifität von 78% (p / Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. Affymetrix-GeneChip and quantitative PCR investigations of the heme metabolic pathway enzymes suggest a reduced expression of the enzyme ferrochelatase to cause the PpIX accumulation. In untreated cases, PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (p

The in ovo sexing of chicken eggs is a current task and a prerequisite to overcome the mass killing of male day-old chicks from laying lines. Although various methods have been developed and tested in recent years, practicable methods for sex determination are still missing which can be applicated in poultry hatcheries before the chicken embryo is capable of nociception and pain sensation. Optical spectroscopic methods enable an early determination of the sex. In this study, a novel method based on two-wavelength in ovo fluorescence excitation is described. More than 1600 eggs were examined. In ovo fluorescence was sequentially excited at 532 nm and 785 nm. The fluorescence intensities of the spectral regions behave inversely with respect to sex. It is shown that the observed sex-related differences in the fluorescence intensities are based on the embryonic hemoglobin synthesis. The accuracy of sex determination is 96% for both sexes. The hatching rate is not reduced compared to an equivalent reference group.

<p>I. Gallium-hemoglobin Coated Silver Nanoparticles for Antimicrobial Photodynamic Therapy Against Bacterial Pathogens One of the mechanisms for bacterial pathogens’ hemin acquisition is through cell-surface hemin receptors (CSHRs), which are responsible for rapid hemin recognition. GaPpIX, as a hemin analog, can be rapidly taken up by CSHR-expressing bacteria, such as <i>Staphylococcus aureus</i> (<i>S.aureus)</i>. Previous works shown that GaPpIX has aPDI activity at micromolar level of concentration following 10 seconds of 405-nm light exposure using LED array. The photosensitizing ability of GaPpIX can be further enhanced by incorporating with hemoglobin (GaHb) and 10 nm silver nanoparticles (AgNP). The results suggested a higher aPDI activity of GaHb-AgNP than any of its components against MRSA strains and neglectable cytotoxicity against keratinocytes. GaHb-AgNPs were also found having aPDI activity against intracellular MRSA and <i>Mycobacterium abscessus </i>but not effective against <i>S. aureus</i> biofilm. GaHb-AgNPs have no significant toxicity toward macrophages with concentrations lower than 22.64 μg/mL.</p> <p> </p> <p>II. Synthesis and Properties of Magnetic Gold Nanoparticles</p> <p> Superparamagnetic gold nanoparticles support hybrid magnetic and plasmonic properties that can be exploited for a variety of applications. In this paper we present new insights on the synthesis of magnetic gold nanoparticles (MGNPs) with an emphasis on efficiency, scalability, and waste reduction, supported by a comprehensive analysis of their physical and materials properties. Aqueous suspensions of colloidal Fe₃O₄ are conditioned with 5-kDa polyethylene glycol and L-histidine to mediate the nucleation and growth of gold by a mild reducing agent. Isotropic MGNPs on the order of 100 nm can be synthesized using scalable reaction conditions with Au:Fe mole ratios as low as 1:2 and cleansed with generally regarded as safe (GRAS) chemicals for the removal of residual iron oxide. High-resolution energy-dispersive x-ray imaging of individual MGNCs revealed these to be ultrafine composites of gold and SPIO rather than core–shell structures. The attenuated total reflectance infrared (ATR-IR) spectroscopy and Raman spectroscopy indicated that the cleansing step does change the optical properties of the synthesized MGNPs. Magnetometry of MGNCs in bulk powder form confirmed their superparamagnetic nature, with bulk moments between 6 to 7 emu/g.</p>

A cor vermelha brilhante característica do presunto Parma é resultante, principalmente, do pigmento Zinco-protoporfirina IX (ZnPP). A ZnPP é formada a partir da mioglobina por uma reação de transmetalação, catalisada pela enzima ferroquelatase (FECH), em que o íon de Fe(II) coordenado ao grupo heme é substituído pelo íon Zn(II). O presunto Parma apresenta uma maior estabilidade oxidativa em relação aos demais produtos cárneos curados além de não conter nitrito e nitrato, portanto, são considerados mais saudáveis. A utilização da FECH no processamento de carnes curadas pode permitir a produção de produtos cárneos curados mais saudáveis e em menor tempo. No presente trabalho a proteína ferroquelatase de Bacillus subtilis (BsFECH) foi expressa em células de E. coli BL21(DE3), purificada por cromatografia de afinidade ao níquel e exclusão por tamanho e caracterizada por dicroísmo circular, emissão de fluorescência do triptofano e cromatografia de exclusão por tamanho analítico. Em termos de estabilidade foi encontrado que altas concentrações de sal aumentam a estabilidade da proteína frente aos agentes denaturantes ureia e temperatura. A BsFECH produzida é capaz de ligar-se ao substrato modelo de porfirina (TPPS), conforme verificado por espectroscopia de UV-Vis, com uma Ka = 3,8x105 M-1 e é capaz de se associar à metamioglobina, conforme verificado por reação de cross-linking com dissuccinimidil suberato e avaliado por SDS-PAGE. A BsFECH aumenta significativamente a taxa de inserção de íons de zinco na TPPS e mostra uma cinética de saturação com uma constante de ligação aparente de Zn(II) ao complexo [BsFECH-TPPS] de 1,3x104 M e uma constante de primeira ordem de 6,6x10-1 h-1 para a dissociação do complexo ternário. A reação de troca ferro/zinco na mioglobina catalisada pela BsFECH é facilitada pela proteólise limitada da mioglobina com pepsina que abre um caminho para a reação de troca metálica com base na interação proteína-proteína entre o fragmento globina da mioglobina e a BsFECH. / The bright red color, characteristic of the Parma ham, results mainly of the pigment Zinc-Protoporphyrin IX (ZnPP). The ZnPP is formed from myoglobin by the reaction, catalyzed by ferrochelatase enzyme (FECH), in which Fe(II) ions coordinated to the heme group is replaced by Zn(II) ions. Parma ham shows greater oxidative stability when compared to others cured meat products besides do not contain nitrite and nitrate and, therefore, is considered healthier. The use of FECH in the processing of cured meats may allow the production of healthier cured meat products in a shorter period of time. In this work, the ferrochelatase protein from Bacillus subtilis was expressed in E. coli BL21(DE3) cells, purified by nickel affinity chromatography and size exclusion, and characterized by circular dichroism, fluorescence emission of tryptophan and analytical size exclusion chromatography. In terms of stability, it was found that the high salt content enhances the protein stability against the denaturation agents urea and temperature. The BsFECH produced is able to bind to the porphyrin model substrate (TPPS), as verified by UV-Vis spectroscopy, with Ka = 3.8x105 M-1 and is capable to associate to metamyoglobin as verified by cross-linking reaction with dissuccinimidil suberato, as observed by SDS-PAGE. The BsFECH increases, significantly, the zinc ions insertion rate in TPPS and shows a saturation kinetics behavior with an apparent biding constant of Zn(II) to the [BsFECH-TPPS] complex of 1.3x104 M and a first order rate constant for the dissociation of ternary complex of 6.6x10-1 h-1. The Fe/Zn exchange reaction in the myoglobin as catalyzed by BsFECH is facilitated by myoglobin-limited proteolysis with pepsin that opens a reaction channel for the metallic exchange based on protein-protein interaction between the globin moiety of myoglobin and BsFECH.

Made available in DSpace on 2014-06-11T19:22:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-06-18Bitstream added on 2014-06-13T18:07:56Z : No. of bitstreams: 1 barberis_lrm_me_botfca.pdf: 692407 bytes, checksum: e9fd31bfed979387901d4ea3d7ea99a0 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O presente trabalho teve como objetivo desenvolver tecnologias para o uso de plantas como unidades de produção ou diretamente como fontes de agentes fotossensibilizantes do Ácido 5-Aminolevulinato (5-ALA), Protoporfirina IX (Proto IX) e precursores destes compostos em plantas. Os experimentos foram conduzidos no Núcleo de Pesquisas Avançadas em Matologia (NUPAM) – FCA – UNESP - Botucatu/SP, durante o ano de 2007. Foram realizados ensaios para seleção do melhor conjunto de condições para o acúmulo de protoporfirina IX e seus precursores em alface, milho e cana-de-açúcar, cujas variáveis analisadas foram: 1) seleção de comprimentos de onda; 2) duração dos períodos com e sem luz; 3) variedades (11 genótipos); 4) aplicação de antioxidantes (vitaminas C e E); 5) compostos que interferem na síntese de Proto IX (oxyfluorfen, carfentrazone e ácido levulênico); 6) adição de precursores da síntese de Proto IX (glutamato); 7) substituição da atmosfera com supressão do Oxigênio. Os resultados foram analisados sobre a dispersão das médias dos tratamentos em relação às parcelas testemunhas para os tratamentos e espécies estudadas. Utilizou-se a técnica de HPLC-MS para identificação e quantificação dos compostos. A partir dos resultados dos ensaios realizados foram selecionados três experimentos: I) Acúmulo de Proto IX e 5-ALA a partir de inibidores da protoporfirinogênio oxidase (Protox) e fontes seletivas de luz (Escuro, Sombrite 75%, Claro+Vitamina C+E, Claro, Filtro Azul, Filtro vermelho, Filtro Amarelo, Filtro Verde, Testemunha) em plantas de alface, pulverizadas com Oxyfluorfen + Glutamato monossódico + Vitamina C e E, portanto 9 tratamentos com 4 repetições. II) Seleção de genótipos de cana-de-açúcar (Saccharum officinarum) para acúmulo de Proto IX com uso de herbicidas inibidores da Protox (Oxyfluorfen e Carfentrazone)... / The present work had as objective to develop technologies for the use of plants about units of production or directly as sources of agents photosensitizers, Acid 5- Aminolevulinate (5-ALA), Protoporphyrin IX (Proto IX) and precursors this compounds in plants. The experiments were conducted at the Center for Advanced Research in Weed Plant (NUPAM) – FCA – UNESP - Botucatu/SP, during the year 2007. Tests were conducted to select the best set of conditions for the accumulation of protoporphyrin IX and precursors in lettuce, corn and sugar cane, whose variables were analyzed: 1) selection of a wavelength, 2) duration of periods with and without light, 3) varieties (11 genotypes), 4) application of antioxidants to reduce the action of Proto IX (vitamins C and E), 5) compounds that interfere with the synthesis of Proto IX (oxyfluorfen, carfentrazone and levulênic acid), 6) addition of precursors for synthesis of Proto IX (glutamate), 7) Replacement of the atmosphere with suppression of the Oxygen. The results were analyzed on the dispersion of the averages of the treatments in relation to the portions witness for the treatments and studied species. It was used technique of HPLC-MS for identification and quantification of compounds. From the results of tests conducted three experiments were selected: I) Accumulation of Proto IX and 5- ALA from protoporphyrinogen oxidase (Protox) inhibitor selective and sources of light (Dark, Shade 75%, Light + Vitamin C + E, Light, Blue filter, Red filter, Yellow filter, Green filter, Witness) in lettuce plants, sprayed with Monosodium Glutamate + Oxyfluorfen + Vitamin C and E, so 9 treatments with 4 repetitions. II) Selection of genotypes of sugarcane for accumulation of Proto IX with the use of herbicides Protox inhibitors (Oxyfluorfen and Carfentrazone), precursors of Proto IX (Glutamate and Levulenic Acid) and antioxidants (vitamin C and E) in 8 genotypes... (Complete abstract click electronic access below)