Background: A mouse model of combined UVA/riboflavin irradiation to eliminate stromal cells and other antigen-presenting cells in the cornea provides the basis for a probably low risk of corneal transplantation. Methods: After abrasion of the epithelium, the central corneas of mouse eyes were treated with UVA/riboflavin in vitro. Histological studies of hematoxylin-eosin and immunohistochemical staining with caspase 3 were performed. Dissected mouse corneas were analyzed by Western blot. Results: Apoptotic cells were shown on the central corneal stroma; a cell-free zone was displayed in the cornea. Numbers of dead cells increased according to cultivation time. However, the endothelium survived due to the adjustment of the irradiation dose. Conclusions: A cell-free zone in the stroma of the mouse cornea was produced by UVA/riboflavin irradiation in vitro. The technique makes possible to prevent or reduce immunological reactions and the risk of graft rejection by pretreatment of the donor cornea, ultimately prolonging graft survival. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Die Arbeit umreißt das Krankheitsbild der Myopie (Kurzsichtigkeit) und deren unterschiedliche Ausprägungen, im Speziellen der progressiven und pathologischen Myopie. Hierbei wird ein Einblick in die Symptomatik, die anatomischen Ursachen und die heutigen medizinischen Interventionen gegeben. Hierdurch wird die Problematik einer zu „weichen“ Sklera (Lederhaut des Auges) und des damit einhergehenden fortschreitenden Augenwachstums deutlich. Im Zentrum der Arbeit steht ein neuer therapeutischer Ansatz zur vorbeugenden Behandlung der pathologischen Myopie; das Riboflavin/Blaulicht Cross-Linking der Sklera des Kaninchenauges. Dessen Wirkungsweise ermöglicht die biomechanische Versteifung von kollagenem Gewebe. Aus diesem Sachverhalt ergibt sich die Fragestellung der Arbeit: Ist das sklerale Riboflavin/Blaulicht Cross Linking geeignet das Augenwachstum im Tiermodell (junge Kaninchen) verträglich zu hemmen? Operationsbeeinflussende Parameter wie die Riboflavin-Durchdringungsdauer der Sklera und die sklerale Lichtdurchlässigkeit werden untersucht und für die Optimierung der Operationsmethode herangezogen und diskutiert. Zur Einschätzung des Versuchsansatzes werden die im Methodikteil dargelegten Anwendungen an adulten und jungen Kaninchen/Kaninchenaugen durchgeführt. In Tierversuchen wird die Schadensschwelle in Abhängigkeit der Blaulichtintensität, des Alters und der Pigmentierung untersucht, wobei histologische, immunhistochemische und elektronenmikroskopische Verfahren angewendet werden. Der inhibitorische Einfluss des Riboflavin/Blaulicht Cross-Linkings auf das Augenwachstum kann im Jungtiermodell durch verschiedene metrische Verfahren und MRT-Untersuchungen belegt werden.

Background: A mouse model of combined UVA/riboflavin irradiation to eliminate stromal cells and other antigen-presenting cells in the cornea provides the basis for a probably low risk of corneal transplantation. Methods: After abrasion of the epithelium, the central corneas of mouse eyes were treated with UVA/riboflavin in vitro. Histological studies of hematoxylin-eosin and immunohistochemical staining with caspase 3 were performed. Dissected mouse corneas were analyzed by Western blot. Results: Apoptotic cells were shown on the central corneal stroma; a cell-free zone was displayed in the cornea. Numbers of dead cells increased according to cultivation time. However, the endothelium survived due to the adjustment of the irradiation dose. Conclusions: A cell-free zone in the stroma of the mouse cornea was produced by UVA/riboflavin irradiation in vitro. The technique makes possible to prevent or reduce immunological reactions and the risk of graft rejection by pretreatment of the donor cornea, ultimately prolonging graft survival. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Background: Collagen cross-linking of the sclera is a promising approach to strengthen scleral rigidity and thus to inhibit eye growth in progressive myopia. Additionally, cross-linking might inhibit degrading processes in idiopathic melting or in ocular inflammatory diseases of the sclera. Different cross-linking treatments were tested to increase resistance to enzymatic degradation of the rabbit sclera. Methods: Scleral patches from rabbit eyes were cross-linked using paraformaldehyde, glutaraldehyde or riboflavin combined with UV-A-light or with blue light. The patches were incubated with collagenase I (MMP1) for various durations up to 24 h to elucidate differences in scleral resistance to enzymatic degradation. Degraded protein components in the supernatant were detected and quantified using measurements of Fluoraldehyde o-Phthaldialdehyde (OPA) fluorescence . Results: All cross-linking methods reduced the enzymatic degradation of rabbit scleral tissue by MMP1. Incubation with glutaraldehyde (1%) and paraformaldehyde (4%) caused nearly a complete inhibition of enzymatic degradation (down to 7% ± 2.8 of digested protein compared to control). Cross-linking with riboflavin/UV-A-light reduced the degradation by MMP1 to 62% ± 12.7 after 24 h. Cross-linking with riboflavin/blue light reduced the degradation by MMP1 to 77% ± 13.5 after 24 h. No significant differences could be detected comparing different light intensities, light exposure times or riboflavin concentrations. Conclusions: The application of all cross-linking methods increased the resistance of rabbit scleral tissue to MMP1-degradation. Especially, gentle cross-linking with riboflavin and UV-A or blue light might be a clinical approach in future.

If a beer is exposed to visible light, it will become light-damaged over time, which is characterised by an undesirable odour and flavour of the drink. The confirmed cause of the above-mentioned flavour in beer is 3-methyl-2-buten-1-thiol (MBT). This substance is formed by non-enzymatic reactions in which riboflavin (vitamin B2) plays the role of photocatalyst and breaks down after transferring its excitation energy, charged by absorption of visible light. The loss of riboflavin results in a change in optical properties of the sample. The aim of this work is to investigate the possibilities of optical detection of light damage, through the analysis of riboflavin content, based on fluorescence and absorption of the sample. Since riboflavin is degraded when light damage occurs, it is possible to determine whether or not a sample is light damaged from its content in beer. Optical detection of light damage is non- invasive and can therefore determine the quality of the beer directly in the commercial bottle without the need to open it.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

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.

Complex I (NADH:ubiquinone oxidoreductase) couples electron transfer to proton translocation across the inner mitochondrial membrane, to drive the synthesis of ATP. Its distinctive L-shaped structure comprises 45 subunits, encoded by both the mitochondrial and nuclear genomes, which are assembled by a complicated modular pathway. Complex I genetic defects are the most common cause of mitochondrial disorders and often present in early childhood, with high mortality rates. Recent high-resolution electron cryo-microscopy structures of mammalian complex I provide a foundation for both interpreting biochemical and biomedical data and understanding the catalytic mechanism. First, this thesis explores how the flavin cofactor is inserted into the NADH-binding (N-) domain of complex I. Genetic manipulation of cultured human cells, to starve them of flavin, revealed a hierarchal impact on the mitochondrial flavoproteome. High riboflavin content in the growth media ameliorated observed phenotypes, requiring cell conditioning in low riboflavin conditions. CRISPR knockout of the putative mitochondrial flavin transporter SLC25A32 demonstrated the severe impact of decreased flavin on complexes I and II, and mass spectrometry 'complexome' analyses suggest that the N-domain is still assembled onto complex I in the absence of the flavin. Second, the model organism Yarrowia lipolytica was used to assess the importance of residues in the quinone-binding site of complex I. Three residues with proposed roles in binding the quinone head-group were targeted. One variant was catalytically inactive, while two retained some activity. They showed decreased ability to reduce physiologically-relevant, long chain quinones, but their ability to reduce short-chain analogues was affected less severely. The results suggest a complicated picture in which interactions between the protein and both the hydrophilic quinone head-group and hydrophobic isoprenoid chain contribute to quinone-binding affinity and catalysis. Finally, a model for human complex I, generated from a recent high-resolution structure of mouse complex I, was used to investigate whether the pathogenicity of human variants could be predicted. Structural information on variant residues, including their secondary structure, proximity to key features and surface exposure, was collated and the power of each property to predict pathogenicity investigated. The analysis was then extended to the whole structure, to identify potential pathogenic hotspots in the enzyme, inform future studies of functionally important regions in complex I, and aid the diagnosis of clinically relevant pathogenic variants.

The aim of the study in this thesis was to assess the stability of the mycotoxin, cyclopiazonic acid (CPA) in milk and dairy products processed from contaminated milk. A method was developed to detect CPA in milk and milk products using micellar electrokinetic capillary chromatography (MEKC), a technique of capillary electrophoresis (CE), which was rapid and non-labour-intensive. The quantifying efficiency of CE in detecting CPA was compared to Reverse Phase Liquid Chromatography. Heat-stability of CPA in milk was assessed under different conditions. A longer heat treatment of 60 degrees centigrade for 30 minutes led to a 10% decrease in the level of CPA. The results from this thesis demonstrate that CPA in milk at concentrations found in naturally contaminated milk could not be eliminated by the heat-treatment during milk processing, storage, processing and manufacture of dairy products. Occurrence of CPA in cheese curd, butter or cream following manufacture with contaminated milk was demonstrated. CPA is left in milk despite UV-visible radiation treatment with or without hydrogen peroxide and/or riboflavin. Chemical treatment, which is capable of completely eliminating CPA, is prohibited and impractical to use for milk treatment. Stability of CPA in milk and milk products confirms the potential of the toxin to reach consumers of dairy products. / Doctor of Philosophy (PhD)

Made available in DSpace on 2015-07-22T20:49:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-28 / Objetivo: verificar a relação entre queixa, aspectos funcionais da deglutição e impacto na qualidade de vida referente à voz e à deglutição, em pacientes portadores da doença de Parkinson, submetidos à administração de riboflavina, no período de um ano. Métodos: participaram do estudo 16 pacientes portadores da Doença de Parkinson, com média de idade de 67,25 anos, média do nível de severidade da doença de II para III e média de tempo de diagnóstico da doença de Parkinson de 3,5 anos. As avaliações videofluoroscópicas da deglutição foram realizadas antes e após um ano da administração de riboflavina e restrição de carne vermelha e de aves. Foram realizadas a análise qualitativa da deglutição, a verificação da presença de queixa com relação à deglutição e aplicação de dois protocolos de qualidade de vida sendo relacionados um com o impacto da alteração vocal, e outro com o impacto da alteração na deglutição. A análise quantitativa compreendeu a realização de medidas de deslocamento do osso hióide, de abertura do esfíncter esofágico superior e de constrição da faringe. Resultados: foram observadas redução da queixa e discreta piora na qualidade de vida relacionada à voz e à deglutição, maior freqüência de ocorrência de deglutição normal, no momento pós-riboflavina. Não foram observadas diferenças significativas entre as medidas quantitativas. Conclusões: Conclui-se neste estudo que: houve melhora, apesar de não significativa, da queixa relacionada à deglutição; não ocorreram pioras significativas na dinâmica da deglutição, com exceção do deslocamento da cartilagem cricóidea na consistência de líquido fino após um ano de estudo; não houve piora com relação ao impacto na qualidade de vida, tanto relacionado à deglutição, quanto ao aspecto vocal; os pacientes que não apresentavam queixas de deglutição no momento pré apresentaram melhora na dinâmica da deglutição, com exceção da constrição da faringe; os pacientes com queixa de deglutição no momento pré apresentaram melhora nos índices de qualidade de vida e na auto-avaliação vocal segundo o QVV, no momento pós. / Objective: to verify the relation between clinical complaint, swallowing functional aspects and the impact on quality of life related to voice and swallowing in patients with Parkinson´s disease submitted to treatment with riboflavin during one year period. Method: sixteen patients with Parkinson´s disease participated in the study; mean age was 67.25 years old, mean degree of disease severity was II to III and mean time of disease diagnosis was 3.5 years. Videofluoroscopic evaluations were performed before and after one year of treatment with riboflavin and restriction diet of read meat and chicken. It were analyzed: qualitative analysis of swallowing, presence of complaints related to swallowing, application of questionnaires related to the impact of voice and swallowing alterations and quantitative analyses of swallowing, which included computerized measurements of hyoid bone and cricoid cartilage displacement, opening of the superior esophageal sphincter and pharyngeal constriction. Results: it were observed: decrease of complaints and slight worsening in quality of life impact regarding voice and swallowing and more frequent normal swallowing post riboflavin; there were no significant changes regarding quantitative measurements. Conclusions: there was a non significant improvement regarding swallowing complaints; it were not observed significant impairments of the swallowing dynamics, except for cricoid cartilage displacement during thin liquid ingestion at the end of the treatment; there was no impairment of quality of life regarding swallowing or voice; patients without swallowing complaints at the beginning of the treatment showed improvement in swallowing dynamics, except for pharyngeal constriction; patients with swallowing complaints at the beginning of the treatment showed improvement regarding quality of life and vocal selfevaluation after treatment. / TEDE / BV UNIFESP: Teses e dissertações