Made available in DSpace on 2014-12-17T14:10:20Z (GMT). No. of bitstreams: 1 Julliane Tamara Araujo de Melo.pdf: 3651115 bytes, checksum: d9a05a183487ffcfb8bd75edd16acd9d (MD5) Previous issue date: 2010-06-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Riboflavin is a vitamin very important in aerobic organisms, as a precursor of many coenzymes involved in the electron transporter chain. However, after photosensitization of riboflavin with UV or visible light, it generates reactive oxygen species (ROS), which can oxidize the DNA. The repair of oxidative lesions on DNA occurs through the base excision repair pathway (BER), where APE1 endonuclease plays a central role. On the other hand, the nucleotide excision repair pathway (NER) repairs helix-distorting lesions. Recently, it was described the participation of NERproteins in the repair of oxidative damage and in stimulation of repair function fromAPE1. The aim of this research was to evaluate the cytotoxic effects of photosensitized riboflavin (RF*) in cells proficient and deficient in NER, correlating with APE1 expression. For this propose, the cells were treated with RF* and it was performed the cell viability assay, extraction of whole proteins, cells fractionation, immunoblotting, indirect immunofluorescence and analysis of polymorphisms of BER gens. The results evidenced that cells deficient in XPA and CSB proteins were more sensitive to RF*. However, XPC-deficient cells presented similar resistance to MRC5- SV cells, which is proficient in NER. These results indicate that XPA and CSB proteins have an important role on repair of oxidative lesions induced by RF*. Additionally, it was evidenced that single nucleotide polymorphisms (SNPs) in BER enzymes may influence in sensitivity of NER-deficient cell lines. Concerning the APE1 expression, the results showed that expression of this protein after treatment with RF* only changed in XPC-deficient cells. Though, it was observed that APE1 is recruited and is bound to chromatin in MRC5-SV and XPA cells after treatment with RF*. The results also showed the induction of DNA damage after treatment with RF*, through the analysis of-H2AX, since the treatment promoted an increase of endogenous levels of this phosphorylated protein, which acts signaling double strand-break on DNA. On the other hand, in XPC-deficient cells, regardless of resistance of RF*, the endogenous levels of APE1 are extremely reduced when compared with other cell lines and APE1 is not bound to chromatin after treatment with RF*. These results conclude that RF* was able to induce cell death in NERdeficient cells, where XPA and CSB cells were more sensitive when compared with MRC5-SV and XPC-deficient cells. This last result is potentially very interesting, since XPC-deficient cell line presents low levels of APE1. Additionally, the results evidenced that APE1 protein can be involved in the repair of oxidative damage induced by RF*, because APE1 is recruited and bound strongly to chromatin after treatment. / A riboflavina ? uma vitamina de fundamental import?ncia em organismos aer?bios, sendo precursora de importantes coenzimas que participam da cadeia transportadora de el?trons. Contudo, ap?s a sensibiliza??o da riboflavina com luz UV ou luz vis?vel, observou-se a forma??o de esp?cies reativas de oxig?nio (EROs), as quais podem oxidar o DNA. O reparo de les?es oxidativas no DNA ocorre principalmente atrav?s da via de reparo por excis?o de bases (BER), na qual a endonuclease APE1 exerce um papel central. Por sua vez, a via de reparo por excis?o de nucleot?deos (NER) atua reparando les?es no DNA que causam distor??es na dupla h?lice. Recentemente tem sido descrito a participa??o da via NER na remo??o de danos oxidativos e na estimula??o da fun??o de reparo de APE1. Desta forma, o objetivo desta pesquisa foi analisar os efeitos citot?xicos da riboflavina fotossensibilizada (RF*) em c?lulas proficientes e deficientes na via NER, correlacionando ? express?o de APE1. Para tanto, as linhagens proficientes e deficientes no NER foram submetidas ao tratamento com RF* e em seguida foram realizados os ensaios de viabilidade celular, extra??o de prote?nas totais, fracionamento celular, immunoblotting, imunofluoresc?ncia indireta e a an?lise de polimorfismos em genes da via BER. Os resultados evidenciaram perfis de sensibilidade distintos ao estresse oxidativo induzido pela RF*, onde as linhagens XPA e CSB foram mais sens?veis, enquanto a linhagem XPC mostrou resist?ncia similar ? linhagem MRC5-SV, a qual ? proficiente na via NER. Esses resultados indicam que as prote?nas XPA e CSB possuem um importante papel no reparo das les?es oxidativas induzidas pela RF*. Al?m disso, foi demonstrado que polimorfismos em um ?nico nucleot?deo (SNPs) em enzimas do BER podem influenciar na sensibilidade dessas linhagens. Em rela??o ?s an?lises dos n?veis de express?o de APE1, os resultados mostraram que houve altera??o na express?o dessa prote?na ap?s o tratamento com RF* somente na linhagem deficiente em XPC. Por?m, observou-se que APE1 ? recrutada e se torna ligada ? cromatina ap?s o tratamento nas linhagens MRC5-SV e XPA. Os resultados tamb?m comprovaram a indu??o de danos ap?s o tratamento com RF* atrav?s do estudo da prote?na-H2AX, pois o tratamento provocou um aumento nos n?veis end?genos desta prote?na fosforilada, a qual atua na sinaliza??o de quebras de fita dupla no DNA. Por?m, na linhagem XPC, al?m de ter sido observado uma curva de sobreviv?ncia semelhante ? linhagem MRC5-SV, os n?veis end?genos de APE1 s?o significativamente reduzidos quando comparados com as outras linhagens e APE1 n?o se encontra ligada ? cromatina ap?s tratamento com RF*. Conclui-se que a RF* foi capaz de induzir a morte celular em linhagens deficientes no sistema de reparo por excis?o de nucleot?deos, onde as linhagens XPA e CSB foram mais sens?veis quando comparadas ? linhagem normal MRC5-SV e ? linhagem XPC. Este ?ltimo resultado ? potencialmente interessante, considerando que a linhagem XPC apresenta baixos n?veis prot?icos de APE1. Adicionalmente, os resultados comprovaram que a prote?na APE1 pode estar envolvida no reparo de danos oxidativos causados pela RF*, j? que APE1 ? recrutada na cromatina e se liga fortemente a esta ap?s o tratamento.

Orientadores: Carmen Verissima Ferreira, Silvia Mika Shishido / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T19:46:07Z (GMT). No. of bitstreams: 1 Machado_Daisy_M.pdf: 1450123 bytes, checksum: e8dc961f855a7ea88daa10bb8ee00685 (MD5) Previous issue date: 2009 / Resumo: A riboflavina (RF) é uma vitamina hidrossolúvel pertencente ao complexo vitamínico B2 e precursora das coenzimas FMN e FAD. Além da função biológica como componente de coenzimas, a RF apresenta atividade antitumoral e fotossensibilizante. A proposta principal desse estudo se baseou na avaliação da fototoxicidade da RF, bem como na determinação de marcadores moleculares da inflamação, senescência e morte celular, ações deletérias normalmente desencadeadas pela radiação UVA. Os resultados obtidos indicam que a RF tem potencial aplicação na terapia fotodinâmica, levando se em conta os modelos celulares utilizados, fibroblastos (BALB/c 3T3) e queratinócitos humanos (HaCaT). As células BALB/c 3T3 tratadas com RF 6,0 mM e dose de 5 J/cm² de radiação UVA, apresentaram indução de apoptose principalmente pela via intrínseca. A indução de senescência foi evidenciada pelo aumento da p-caveolina e aumento da atividade da MMP-2 (principal protease responsável pela degradação de colágeno). De acordo com os níveis de NFkB e p- IKKa/b, a RF não alterou significativamente o processo de inflamação desencadeado pela radiação UVA. Nas células HaCaT, o tratamento com 5,0 mM de RF e dose de 5 J/cm² de radiação UVA levou a ativação da apoptose induzida tanto pela via extrínseca como pela intrínseca. O aumento nos níveis de p-caveolina, p21 e da atividade das MMPs-2 e -9 sugerem à indução de processo de senescência precoce também nos queratinócitos. Além disto, a diminuição da expressão do NF?B indica que o mesmo esteja se translocando para o núcleo e, portanto, regulando a resposta inflamatória. Outro aspecto avaliado foi a influência do copolímero F-127 na fototoxicidade da RF. A irradiação do hidrogel contendo F-127 e riboflavina manteve a propriedade fototóxica da mesma. Nossos dados sugerem que a RF apresenta potencial para uso em terapia fotodinâmica, uma vez que a mesma se mostrou fototóxica quando irradiada com doses subtóxicas de radiação UVA. / Abstract: Riboflavin (RF) is a water soluble vitamin which belongs to vitamin B2 complex, an essential precursor of FMN and FAD coenzymes. Besides being a component of coenzymes, RF displays antitumoral and photossensitizing activities. The main proposal of this study was to evaluate the RF phototoxicity, as well as to determine molecular markers of inflammation, senescence and cellular death, deleterious actions normally triggered by the UVA radiation. Taking into consideration both cell lines used as models, fibroblasts (BALB/c 3T3) and human keratinocytes (HaCaT), the results obtained indicate that RF has potential application in photodynamic therapy. BALB/c 3T3 cells treated with 6.0 µM RF associate with UVA radiation (5 J/cm²) showed apoptosis induction mainly via intrinsic pathway. An increase of p-caveolin and MMP-2 activity (major protease responsible for degradating collagen) evidenced senescence induction. According to NF?B and p-IKKa/ß levels, RF did not significantly change the process of inflammation triggered by UVA radiation, in fibroblast cells. In HaCaT cells 5.0 µM RF associated with UVA radiation (5 J/cm ²) was observed apoptosis induction thorough extrinsic and intrinsic pathways. Senescence process was also observed in keratinocytes as indicated by an increase of pcaveolin, p21 and MMPs-2 and -9 activities. Besides, the decrease of NF?B expression indicates that this transcription factor translocates into the nucleus and in turn, regulates inflammatory response. Other aspect evaluated in this work was the influence of the F-127 in the RF phototoxicity. The irradiation of hydrogel containing F-127 and RF remained RF phototoxicity property. Our findings suggest that RF displays potential for use in photodynamic therapy, once it was phototoxic when irradiated with subtoxic UVA dose / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Carmen Verissima Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-14T15:16:22Z (GMT). No. of bitstreams: 1 ChavesNeto_AntonioHernandes_D.pdf: 8240508 bytes, checksum: 293949f75ca619cdc0fa61d29d1703ba (MD5) Previous issue date: 2009 / Resumo: Riboflavina (Rb - Vitamina B2) é o precursor das flavocoenzimas essenciais flavina mononucleotídeo (FMN) e flavina adenina dinucleotídeo (FAD). Estas coenzimas participam de processos enzimáticos dependentes das reações de transferências de elétrons, que ocorrem nas vias de produção de energia, biossíntese, desintoxicação e sequestro de elétrons. O aumento dietético da riboflavina e piridoxina foi associado com maiores densidades minerais em mulheres e homens idosos. Fotoderivados da riboflavina demonstraram efeitos citotóxicos em células cancerosas de próstata e leucemias, entretanto, o efeito direto da Rb e seus fotoderivados em osteoblastos não foram examinados. Neste trabalho os efeitos biológicos da Rb e riboflavina irradiada (IRb) foram investigados na linhagem de pré-osteoblastos MC3T3-E1, um modelo bem aceito de osteogênese in vitro caracterizado pela indução de genes específicos associados com o fenótipo osteoblástico quando tratados com ácido ascórbico e ß-glicerofosfato. A viabilidade celular foi avaliada através da redução do MTT, da incorporação do corante vermelho neutro e do conteúdo de ácidos nucléicos. Marcadores de diferenciação osteoblástica foram analisados através do RT-PCR semi-quantitativo (osteopontina e osteocalcina) e através de análises colorimétricas de atividade da fosfatase alcalina (FAL) e síntese de colágeno pela coloração de picrosirius. As atividades das metaloproteinases (MMP) -9 e -2 foram avaliadas pela zimografia de gelatina. Microarranjos de peptídeos com subtratos específicos para quinases e imunoblotting foram usados para identificar os efeitos na sinalização celular. As atividades de enzimas antioxidantes (superóxido dismutase, catalase, glutationa peroxidase e glutationa S-transferase) foram determinadas em lisados celulares usando métodos espectrofotométricos. As atividades das caspases-8, -9 e -3 foram analisadas através de métodos colorimétricos. Na primeira análise Rb e IRb causaram a parada do ciclo celular na fase G0/G1 e também a inibição da quinase AKT, um mediador da proliferação. Flavinas causaram a diferenciação de pré-osteoblastos, evidenciada pelo aumento da expressão de osteocalcina, osteopontina e BMP-2. Atividades mais elevadas de MMP-9 e MMP-2 também foram observadas. A capacidade das flavinas em engatilhar a diferenciação de osteoblastos foi reforçada pelo aumento da conexina 43, diminuição da caveolina-1 e repressão da sinalização Notch. Na segunda análise, nós encontramos que as interações entre Rb, em sua forma irradiada e não-irradiada, e indutores osteogênicos (ácido ascórbico e ß-glicerofosfato) afetaram significativamente a proliferação de osteoblastos, a atividade de FAL, biossíntese de colágeno, expressão de osteocalcina e osteopontina, a atividade das MMP-2 e MMP-9 e a expressão de fatores osteoclastogênicos (RANKL e osteoprotegerina). Nós também encontramos que os efeitos das flavinas em osteoblastos nesta segunda etapa foram independentes das suas propriedades antioxidantes. A atividade biológica da combinação de indutores osteogênicas com Rb e seus fotoprodutos foi associada com a ativação de diferentes vias de sinalização (AKT, FAK, CaMKII), caspases -8, -9 e -3 e aumento da expressão e/ou estabilização de fatores de transcrição osteoblásticos (Runx2 e ß-catenin). Este estudo nos trouxe fortes evidências que altas concentrações de Rb e IRb geraram um microambiente osteogênico através da modulação de diferentes vias de sinalização, além de promover um efeitos aditivo durante a diferenciação das células pré-osteoblasticas MC3T3 induzida por ácido ascórbico e ß- glicerofosfato. Em resumo, este estudo aponta para uma potencial aplicação da Rb e seus fotoprodutos no desenvolvimento do fenótipo osteoblástico e, consequentemente, uma alternativa terapêutica coadjuvante para osteoporose. / Abstract: Riboflavin (Rb-Vitamin B2) is the precursor of essential flavocoenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes participate in numerous enzymatic processes dependent on electron transfer reactions that occur in energyproducing, biosynthetic, and detoxifying and electron-scavenging pathways. Increase dietary riboflavin and pyridoxine intake has been associated with higher bone mineral density in elderly men and women. Photoderivatives of riboflavin have been shown strong activity in haematological malignancy and prostate cancer cells, however, the direct effect of Rb and its photoderivatives on osteoblast has not been examined. In this work, the biologic effects of Rb and irradiated riboflavin (IRb) were investigated in the MC3T3-E1 pre-osteoblastic cell line, a well-accepted model of osteogenesis in vitro characterized for the induction of specific genes associated with the osteoblastic phenotype when treated with ascorbic acid and ß-glycerophosphate. Cell viability was assessed by MTT reduction, neutral red uptake and nucleic acids content. Osteoblastic differentiation markers were analyzed by semiquantitative RT-PCR (osteopontin and osteocalcin), alkaline phosphatase (ALP) activity measured colorimetrically and collagen synthesis by Sirius red staining. Metalloproteinases (MMP) -9 and -2 activities were assayed by gelatin zymography. Peptide microarray of substrate specificity to kinases and immunoblotting were used to identify the effects on signal transduction pathways. Antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase and glutathione Stransferase) were determined in cellular lysate using spectrophotometric methods. Caspase-8, -9 and -3 activation were measured by a colorimetric assay. In the first analysis Rb and IRb caused cell cycle arrest at G0/G1 phase and accordingly inhibited AKT kinase, a proliferation mediator. Flavins caused differentiation of preosteoblast cells as evidenced by increase of osteocalcin, osteopontin and BMP2 expressions. In addition, higher MMP-9 and -2 activities were observed. Importantly, the capacity of flavins to trigger osteoblasts differentiation was also reinforced by upregulation of connexin 43, down regulation of caveolin-1 and negative modulation of Notch cascade. In the second analysis, we found that the interaction between Rb and IRb and osteogenic inductors (ascorbic acid and ß-glycerophosphate) significantly affected the osteoblast proliferation, alkaline phosphatase activity, collagen biosynthesis, osteopontin and osteocalcin mRNA expression, MMP-2 and MMP-9 activities and the expression of osteoclastogenesis factors (RANKL and OPG). We also showed that the effects of flavins in osteoblasts cells were independent on flavins antioxidant property. The biological activity of the combination of osteogenic medium with riboflavin and its photoderivatives was associated with the activation of different signaling pathways (AKT, FAK, CaMKII), caspases -8, -9 and -3, and up-regulation and/or stabilization of osteoblastic transcription factors (Runx2 and ß-catenin). This study brought out strong evidences that high concentration of Rb and IRb generates an osteogenic microenvironment through modulating different mediators of signaling pathways, besides of the additive effect of riboflavin and its photoproducts during the ascorbate and ß-glycerophosphateinduced osteoblast differentiation of MC3T3-E1 cells. In summary, this study pointed out the potential application of Rb and its photoproducts in osteoblasts phenotype development and, consequently, it is possible use as an alternative therapeutic adjuvant of osteoporosis. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Orientador: Walkiria Hanada Viotto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-23T15:48:33Z (GMT). No. of bitstreams: 1 Urzedo_AnaCarolinaBorgesde_D.pdf: 4425198 bytes, checksum: ca9f39f7b13a21e55c2d558014da6722 (MD5) Previous issue date: 2013 / Resumo: A foto-oxidação do leite é o fator determinante na vida de prateleira do leite microfiltrado pasteurizado. A exposição do leite à luz, nas gôndolas dos supermercados, desencadeia a foto-oxidação da riboflavina e das proteínas do leite, provocando mudanças na cor e formação de off-flavors. O objetivo do trabalho foi verificar, em condições reais, a influência do tipo de embalagem e da intensidade da luz na degradação da riboflavina, formação de lumicromo, oxidação de proteína, cor e vida de prateleira do leite desnatado microfiltrado pasteurizado. Leite desnatado microfiltrado pasteurizado foi acondicionado em garrafas de vidro e de polietileno de alta densidade, e armazenados no escuro (controle) e sob incidência de luz (500 e 1200 lux), durante 14 dias de estocagem refrigerada. Análises de espectroscopia de fluorescência da riboflavina, lumicromo, triptofano e ditirosina e análise de cor instrumental foram realizadas para acompanhar a foto-oxidação dos componentes do leite. Para estimar a vida de prateleira do leite microfiltrado pasteurizado, sob diferentes condições de luz e embalagem, contagens de micro-organismos mesófilos aeróbios e análise sensorial com assessores treinados, teste de aceitação e intenção de compra foram realizados durante o tempo de armazenamento refrigerado. Assessores foram treinados para avaliação da cor, aroma e sabor característicos de leite desnatado e de leite desnatado oxidado. Aos 14 dias de estocagem refrigerada, 76,1% da riboflavina foi degradada no leite exposto à radiação de 500 lux. Já a 1200 lux, esse valor foi de 86,4%. A degradação da riboflavina, a formação de lumicromo e a fotodegradação do triptofano foram maiores e mais rápidas quando a intensidade de luz foi mais intensa (1200 lux). Durante o armazenamento refrigerado, a oxidação das proteínas resultou em desnovelamento da estrutura terciária, e consequentemente, em exposição e posterior degradação do triptofano. No período estudado, houve somente formação de ditirosina para os leites submetidos à intensidade de luz mis intensa (1200 lux). A vida de prateleira dos leites armazenados no escuro (controle) foi de 10 a 14 dias. O aparecimento do sabor oxidado, proveniente da foto-oxidação dos componentes do leite, foi o parâmetro determinante para o fim da vida de prateleira dos leites armazenados sob luz. O tipo de embalagem somente influenciou a vida de prateleira do leite, quando a intensidade de exposição à luz foi mais baixa (500 lux). Nessa intensidade de radiação luminosa, a vida de prateleira do leite pasteurizado aumentou de 4-6 dias para 10-13 dias, quando a embalagem de vidro foi substituída pela de polietileno / Abstract: Photo-oxidation of milk is probably the main cause for the end of shelf life of a microfiltered pasteurized milk. Milk is inevitably exposed to light on the supermarket shelves, which triggers the photo-oxidation of riboflavin and milk proteins, affecting the sensory quality with changes in color and formation of off-flavors. The objective was to verify, in real conditions, the influence of the type of packaging and the light intensity on the riboflavin degradation, protein oxidation, color, shelf life of microfiltered pasteurized skim milk. After processing, milk was packaged in glass and high density polyethylene bottles and stored in the dark (control) and under influence of light (500 and 1200 lux), during 14 days of refrigerated storage. Analyses of fluorescence spectroscopy of riboflavin, lumicrome, tryptophan and dityrosine and instrumental color were performed to monitor the photo-oxidation of milk components. The shelf life of pasteurized microfiltered skim milk, under different light conditions and packaging was estimated by standard plate count of aerobic mesophilic and sensory analysis with trained assessors, acceptance testing, and purchase intent, during refrigerated storage time. Assessors were trained to evaluate sensorially the color, aroma and flavor of skim milk and oxidized skim milk. At 14 days of refrigerated storage, 76.1% of riboflavin was degraded in milk exposed to radiation of 500 lux. However, at 1200 lux, degradation of riboflavin reached 86.4% of its initial content in milk. Riboflavin degradation, lumicrome and tryptophan formation were higher and faster when light intensity was more intense (1200 lux). During storage time, the oxidation of proteins resulted in the tertiary structure unfolding, and exposure and subsequent degradation of tryptophan. During this period of time, there was formation of dityrosine only for the milks exposed to more intense light radiation (1200 lux). The shelf life of milk stored in the dark (control) was 10-14 days. The development of oxidized flavor, derived from the photo-oxidation of milk components, was the main parameter for determining the ending of the shelf life of milk stored under light. Packaging material influenced the milk shelf life when the intensity of light was lower (500 lux). In this condition, the shelf life of pasteurized milk increased from 10-13 days to 4-6 days when the glass container was replaced by polyethylene bottle / Doutorado / Tecnologia de Alimentos / Doutora em Tecnologia de Alimentos

La riboflavine (RF) est une vitamine essentielle pour la croissance et le développement cellulaire. Elle possède des propriétés physico-chimiques intéressantes et est internalisée dans les cellules par des transporteurs spécifiques. Le premier objectif de ce projet était de synthétiser des dérivés amphiphiles de la RF (RFA) et d'étudier leurs capacités d'auto-assemblages. Le second objectif était d'insérer les RFA dans des liposomes et d'évaluer leur efficacité de ciblage tumoral in vitro et in vivo. La préparation des différents RFA repose sur l'ajout d'un lipide en différentes positions de la RF. L’un d'eux, de type phospholipide (RfdiC14) a été capable de former des objets tridimensionnels de taille μm constitués de lamelles multicouches dont l’architecture et la dynamique sont très différentes de celles des phospholipides classiques. L’insertion de RfdiC14 dans des liposomes est efficace et n’influence pas leurs propriétés physico-chimiques. Les liposomes fonctionnalisés ont montré une internalisation cellulaire spécifique dans les lignées A431, PC3 et HUVECs. Afin de tester l’efficacité du ciblage tumoral in vivo, un analogue de RfdiC14 portant un espaceur PEG a été préparé puis inséré dans des liposomes péguylés. Grâce à un marquage adéquat (ICG et DiR), leur accumulation tumorale a été suivie par imagerie photoacoustique dans un modèle A431 et leur biodistribution évaluée par imagerie μCT/FMT dans un modèle PC3. Les résultats montrent une légère amélioration de l’accumulation tumorale dans les xénogreffes A431 et une augmentation du ciblage vasculaire dans le modèle tumoral PC3. La biodistribution globale des liposomes marqués est comparable à celle des contrôles. / Riboflavin (RF) is an essential vitamin for cell growth and development. It possesses interesting physicochemical properties and is internalized by the cells through specific transporters. The first aim of this study was to prepare amphiphile derivatives of RF (RFA) and study their auto-assembly. The second aim was to insert RFA into established drug delivery systems and test their tumour-targeting potential in vitro and in vivo. RFA were prepared by the molecule functionalization with lipid moieties in different positions. One of them, a phospholipid-like derivative (RfdiC14) was able to self-assembly in aqueous solutions into μm-sized 3D objects constituted from slightly curved multilayer lamellas. The bilayer architecture and dynamics were very different from ordinary phospholipids. In contrast, the insertion of small amount of RfdiC14 in a liposome did not influence membrane dynamics and physicochemical characteristics. RfdiC14-functionalised liposomes displayed high and specific uptake in vitro in A431, PC3 cells and HUVECs. The efficiency of RF targeting was also tested in vivo. For that purpose, liposome composition was optimized and a new RF amphiphile with a PEG spacer between RF and lipid was prepared. The tumour accumulation of the liposomes labelled with ICG was studied by photoacoustic imaging in A431 tumour model. The biodistribution of DiR labelled liposomes was accessed by combined μCT/FMT imaging in PC3 tumour model. The results show slight improvement of the tumour accumulation in A431 xenographts and the enhancement of vascular targeting in PC3 tumour model. The overall biodistribution of the RF-targeted liposomes was comparable to control.

The aim of this project was to analyse the cellular and biomechanical changes after collagen cross-linking (CXL) treatment on postmortem eye-banked human corneas using different UVA intensities and repeated treatments, and to explore the effects of standard collagen cross-linking on keratoconic corneal buttons, in-vitro. Preliminary studies were conducted to assess the feasibility of using eye-banked corneas to assess the effects of collagen cross-linking, and the possibility of applying scanning acoustic microscopy (SAM) to measure the speed of sound/elasticity of corneal tissue. Eye-banked human corneas were successfully cross-linked allowing the effects of CXL to be studied in-vitro and SAM was used effectively to determine the mechanical properties of corneal tissue at different depths. The results of two experiments comparing UVA intensity suggested that no statistically significant difference was found in the histological changes or in the induced stiffness after applying low and high intensity cross-linking on normal human corneas. However, the number of apoptotic cells was found to be significantly less but deeper into the posterior stroma in the high intensity cross-linked corneas. Collectively, these results confirmed the safety and efficacy of both techniques with the advantage of reducing the treatment time using the higher-intensity treatment. In another in-vitro study, keratoconic corneal tissue was used. Different histological and biomechanical outcomes were found between the cross-linked and control keratoconic tissue. The effects of cross-linking were found to penetrate deeper in the keratoconic tissue compared to in the normal corneal tissue found in previous studies. This could be due to the altered collagens and extracellular matrix of the keratoconic corneas, as they were taken from patients in advanced stages of the disease. This study confirmed the importance of having corneal thickness of at least 400μm after epithelial debriding to maintain the endothelial cell density and integrity. Finally, further cross-links were induced when collagen cross-linking treatment was repeated. However, repeating cross-linking three times a deeper cell death close to the endothelium was noticed which suggests that multiple treatments could be unsafe. Additionally, lower speed of sound than the cross-linking twice. This could be due to elimination of the induced cross-links by longer exposure to UVA irradiation. In conclusion, eye-banked human corneas were successfully used to evaluate the effects of cross-linking treatment and repeated treatment. Additionally, keratoconic corneal buttons were used to study the effects of collagen cross-linking in-vitro. This model of using eye-banked human corneas and keratoconic corneal tissue enabled us to study the effects of cross-linking treatment using different protocols and the effects of repeated treatment, and it could ultimately be used to compare the results with in-vivo studies.

Entende-se por pólen apícola o resultado da aglutinação do pólen das flores, efetuado pelas abelhas operárias, mediante néctar e substâncias salivares, o qual é recolhido no ingresso da colmeia. A literatura descreve que esse alimento contém proteínas, carboidratos, lipídeos, vitaminas e minerais. De acordo com estudo prévio, amostras de pólen apícola in natura e desidratado, da cidade de Pariquera-Açu (São Paulo), apresentaram teores significativos de vitamina B1(tiamina) e B2 (riboflavina), além da presença dos vitâmeros da vitamina B3 (ácido nicotínico e nicotinamida) e B6 (piridoxal, piridoxol e piridoxamina) em sua composição o que foi associado à flora local explorada pelas abelhas. A região Sul do Brasil possui clima, relevo e vegetação diferenciados de outras regiões, necessitando-se assim da verificação do potencial vitamínico deste produto local. Destaca-se, ainda, o fato de que nesta região encontra-se um dos dois maiores produtores nacionais de pólen apícola (estado de Santa Catarina). O presente trabalho teve como objetivo principal quantificar os teores das vitaminas do complexo B: vitaminas B1 e B2, assim como os vitâmeros das vitaminas B3 e B6. Foram coletados 28 lotes de pólen apícola desidratado de diferentes localidades da região Sul durante o período de agosto de 2011 a dezembro de 2012 que posteriormente foram armazenados, a -18 °C até o momento das análises. As vitaminas do complexo B foram analisadas por cromatografia liquida de alta eficiência (CLAE) na matriz pólen apícola desidratado e os resultados foram expressos em base seca. Entre as amostras analisadas foram verificados teores de vitamina B1 variando entre 0,46 e 1,83 mg / 100 g de pólen apícola; vitamina B2 de 0,40 à 1,86 mg / 100 g e quanto à vitamina B6 apenas os vitâmeros piridoxal e piridoxamina puderam ser quantificados em todos os lotes analisados. O piridoxal teve variação entre as amostras de 0,42 à 6,70 mg / 100 g e a piridoxamina de 0,26 à 0,95 mg / 100g. Em relação à vitamina B3, o vitâmero ácido nicotínico apresentou-se nos diferentes lotes variando de 0,68 à 3,93 mg / 100 g e a nicotinamida de 0,27 à 5,54 mg / 100 g de produto. Tomando-se como porção sugerida para consumo diário 25 g de pólen apícola, verificou-se que num total de 28 amostras, 15 foram consideradas fontes e 2 como ricas em tiamina; 19 lotes foram fontes e 3 ricos em riboflavina, e; 2 lotes foram fontes e 26 ricos em piridoxina segundo à Ingestão Diária Recomendada (IDR) para adultos como disponibilizado na Resolução de Diretoria Colegiada (RDC) nº. 269, de 22 de setembro de 2005. / Bee pollen is understood to be the result of agglutination of pollen from flowers, made by worker bees, and nectar through salivary substances, which is collected at the hive entrance. The literature describes that this product contains proteins, carbohydrates, lipids, vitamins, minerals. Previous study with fresh and dehydrated bee pollen, from the city of Pariquera-Açu (São Paulo) showed significant levels of vitamin B1 (thiamine), B2 (riboflavin), presence of B3 (nicotinic acid and nicotinamide) and B6 (pyridoxal, pyridoxamine, piridoxol) vitamins vitamers in its composition which was associated with the local flora explored by bees. Southern Brazil has a differentiated climate, topography and vegetation from other regions, thus requiring verification of vitamin potential of this local product. Also stands out the fact that this region is one of the two largest national producers of bee pollen (Santa Catarina state). This study aimed to quantify the levels of B complex vitamins: vitamins B1, B2, as well as the vitamers of vitamins B3 and B6. Thus, it was collected 28 batches of dehydrated bee pollen from different locations in the South during the period from August 2011 to December 2012. Samples were obtained and subsequently stored at -18 ° C until the analysis time. B vitamins were analyzed by high performance liquid chromatography (HPLC) in bee pollen dehydrated matrix and results were expressed on a dry basis. Among the samples it levels of vitamin B1 varied from 0.46 to 1.83 mg / 100 g; vitamin B2 from 0.40 to 1.86 mg / 100 g; and for vitamin B6, only the pyridoxal and pyridoxamine vitamers could be quantified in all analyzed batches. The pyridoxal had variation between samples from 0.42 to 6,70 mg / 100 g and pyridoxamine from 0.26 to 0.95 mg / 100g. Taking 25 g of bee pollen as suggested for daily intake portion, it was found in a total of 28 samples that 15 were considered sources and 2 rich in thiamine; 19 lots were sources and 3 rich in riboflavin, and; 2 lots were sources and 26 rich in pyridoxine in relation to the Reference Daily Intake (RDI) for adults as provided in Resolução de Diretoria Colegiada (RDC) nº 269, de setembro de 2005.

In this doctoral thesis, we have studied the oxidation of carboxylic acids to obtain the corresponding acyloxy radicals, using visible light and non-toxic and inexpensive organic dyes, as photocatalysts. On the one hand, we study the photooxidation of aromatic carboxylic acids to obtain acyloxy radicals, whose decarboxylation is relatively slow (Chapter I and Chapter II). On the other hand, we describe the photooxidation of aliphatic carboxylic acids, to take advantage of the rapid decarboxylation of the corresponding acyloxy radicals, to generate nucleophilic radicals that were trapped by different reagents (Chapter III to Chapter V). It should be noted that all the protocols are free of expensive and toxic noble metals, the reactions were promoted with visible light at room temperature and the scalability of some reactions was demonstrated in batch conditions or using flow chemistry. In addition, mechanistic studies were carried out to propose plausible photocatalytic routes to all the reactions studied.

A simple and economical fluorometer using blue LEDs excitation sources and simple PMT detection had been built, assembled, optimized, and employed for measurement of fluorescence from riboflavin (vitamin B2). Surfactants have been known to enhance the intensity of fluorescence of fluorescent compounds. Fluorescence analysis of riboflavin in the presence of various anionic, cationic, and nonionic surfactants was also conducted to determine if they could improve analysis. However, the surfactants employed did not seem to have any meaningful enhancement; in fact, some actually diminished the fluorescence intensity of riboflavin. The procedure was linear for riboflavin from 0.01 to 2.5 μg/mL. Reproducibility expressed as relative standard deviation was about 2%. The recoveries obtained range from 91.3% to 100.21% for the samples determined. The proposed method was successfully applied to the analysis of riboflavin in commercial vitamin tablets and cereal products. The results obtained were consistent with expected values as provided by the manufacturers. The method is simple, sensitive, economical, and rapid.

Two studies were conducted to determine the relative effect of various combinations of antioxidants on vitamin A oxidation and isomerization in nonfat dry milk (NDM). In the first study, one lot of pasteurized unfortified skim milk was divided, fortified with vitamins A and D and one of 11 antioxidant treatments, and spray dried. A control batch from the same lot was also fortified with vitamins A and D and spray dried. Samples were analyzed for total vitamin A bioactivity after zero, one, and two weeks of storage. After two weeks at 50°C, the only NDM samples that did not experience significant vitamin A loss were those treated with butylated hydroxytoluene, either alone at 0.57 ppm or at 0.29 ppm in combination with 250 ppm ascorbic acid. The control sample was significantly different from both of these treatments, and retained only 17% of its original retinol activity equivalents. Isomer composition changed over the two weeks of storage, with an increase of the 13-cis, 9,13-di-cis and the 9-cis isomers as well as a decrease in the all-trans isomer. In the second study, two lots of pasteurized, vitamin A & D fortified, condensed skim milk were divided into four batches, three of which were spiked with an antioxidant treatment: 250 ppm ascorbic acid + 1 ppm propyl gallate, 250 ppm ascorbic acid + 1 ppm butylated hydroxyanisole, or 2 ppm propyl gallate; the fourth batch was a control. Each of the eight batches was homogenized, spray dried and stored in the absence of light at 30°C for 3 months. Vitamin A and riboflavin were analyzed before spray drying, and after 0, 1, 2, 4, 6, 8, 10, and 12 weeks. Two treatments had significant higher vitamin A than the control, the 250 ppm ascorbic acid + 1 ppm butylated hydroxyanisole treatment and the 2 ppm propyl gallate treatment. Limited vitamin A degradation occurred in all samples during the study timeframe, although the overall degree of retinol isomerization began and remained high in all samples, with cis isomers accounting for approximately 23% of the total μg of retinol after 12 weeks. There were no significant differences in riboflavin content between any of the antioxidant treatments and no significant degradation in riboflavin over time. Antioxidants appear to be an effective means of reducing vitamin A oxidation and isomerization in nonfat dry milk. Butylated hydroxytoluene in combination with ascorbic acid was the most effective antioxidant blend observed. Antioxidants may be less effective at protecting against degradation of vitamin A if isomerization has already taken place.