Este projeto teve como finalidade construir uma fonte de luz baseada em lâmpadas halógenas que apresentasse características técnicas adequadas para aplicações em fotobiologia e fotomedicina, versátil e comercialmente viável. Foram analisadas as características de diferentes lâmpadas halógenas e realizados os experimentos com duas lâmpadas escolhidas, das quais utilizamos a lâmpada de menor divergência luminosa. Foi analisado também o material do corpo da fonte, com relação a características de aquecimento e peso, sendo escolhido um corpo de alumínio produzido industrialmente. Foram avaliadas as características ópticas das lentes, sendo escolhidas as lentes de vidro utilizadas em retroprojetores. Foram analisados os materiais e a construção de filtros térmicos para diminuir o aquecimento do objeto sob irradiação, sendo que escolhemos construir um filtro térmico próprio, que utiliza a água como absorvedor de calor. Medimos e avaliamos as características: espectrais, térmicas, a potência radiante (irradiância) e sua distribuição espacial de três protótipos, onde cada um recebeu modificações de modo a torná-los mais adequados para aplicações na fotoquimeoterapia. Foram também realizados testes preliminares de fotocitotoxicidade do protótipo final com o composto fotossensibilizador de nome comercial Photogem® aplicado em células tumorais de adenocarcinoma de colo retal humano (HT29), cujo resultado foi a ativação do composto fotossensibilizador e indução da morte celular. / The aim of this work was to produce a light source based in halogen lamps. This source should have appropriated technical characteristic for application in photobiology and photomedicine, it has easy operation and be commercially viable. The characteristics of different halogen lamps were evaluated and the lamp of smaller luminous divergence was chosen. We also have evaluated the material of the source holder, especially heating characteristics and weight; finally we chose an aluminum holder. Then, the lenses optical characteristics were studied, and we decided that the glass lenses used in projectors were the best. The materials and the construction of thermal filters were evaluated in order to reduce the heating of the object under irradiation, and we preferred to construct a thermal filter, that uses water as an heat absorber. The spectral and thermal characteristics, the radiant power (irradiance) and its space distribution of three prototypes were measured and evaluated, each one received modifications to become more appropriated to applications in photochemotherapy. We also made preliminaries tests of photocytotoxicity and the final prototype was used with the photosensitizer (Photogem®) in human colon carcinoma cell lines HT-29, resulting in the photosensitizer activation, that induced cellular death.

A enzima peroxidase (HRP) , agindo como uma oxigenase em substratos apropriados, promove a formação de espécies excitadas no estado triplete. Estes produtos excitados podem ser formados em sistemas bioquímicos e promover processos fotoquímicos no escuro. Nesta linha de estudos, investigamos a oxidação aeróbica, na presença de HRP, de substratos contendo ligações de Schiff. Esses compostos são de grande importância biológica, uma vez que participam como intermediários em diversas reações enzimáticas (transaminação, biossíntese de aminoácidos, biossíntese de profirinas), nas ligações cruzadas do colágeno e da elastina, na hemoglobina AlC, na rodopsina e bacterionodopsina e como produtos finais da lipoperoxidação. A oxidação aeróbica de quatro iminas alifáticas (BPA, i-BMA, sec-BMA e BVA) , catalisada por HRP, é quimiluminescente. A natureza triplete da espécie excitada foi sugerida pelo espectro de quimiluminescência, por meio de estudos de transferência de energia para DBAS e clorofila e pela supressão de quimiluminescência por oxigênio, sorbato, indol e p-benzoquinona. Com base nos altos valores de kETo encontrados, concluimos que a transferência de energia esteja ocorrendo provavelmente por um mecanismo a longa distância. A análise dos produtos da reação e os dados cinéticos indicam que a reação ocorre provavelmente segundo a via: (VER Esquema no arquivo) Ressalta-se que esta reação constitui um modelo mais adequado para sistemas bioluminescentes do que o proposto por McCapra e Burford (1976) com bases de Schiff aromáticas em t-butóxido e DMSO. Os sistemas aqui descritos também apresentam a possibilidade de desenvolvimento de método analítico, empregando quimiluminescência, para detectar formação de bases de Schiff em sistemas biológicos. Os estudos com base de Schiff alifáticos (sistemas modelo) foram estendidos para adutos contendo ligação de Schiff entre glicolaldeído e aminoácidos (Lys, Arg, His e Phe) ou proteínas (lisozima, BSA e protaminas). Todos os sistemas estudados são quimiluminescentes na presença de HRP; a fluorescência característica da formação do aduto decai concomitantemente com a emissão de luz; e, durante a reação, a peroxidase encontra-se principalmente na forma de composto II. Observa-se também transferência de energia dos sistemas glicolaldeído-lisozima/HRP/O2 e glicolaldeídoprotamina/HRP/O2 para clorofila. Estes estudos têm grande interesse do ponto de vista da toxicidade associada à ingestão de álcool etílico, a qual, segundo trabalhos recentes da literatura, é atribuída à formação de bases de Schiff entre proteínas de membranas e acetaldeído. E notável também, a formação de iminas durante o processo de lipoperoxidação e a quimiluminescência que o acompanha. Nossos dados levantam a possibilidade de que a toxicologia do álcool pode envolver espécies eletronicamente excitadas formadas na oxidação aeróbica dos adutos aldeído-proteínas. / Horseradish peroxidase (HRP) , acting as an oxigenase on various substracts, promotes the generation of electronically excited triplet species. These species can also be formed in many biochemical reactions and drive photochemical processes in the absence of light. Enlighted by this hypothesis (photobiochemistry in the dark) we have investigated the HRP-catalyzed aerobic oxidation of substrates containing Schiff linkage. These compounds are of utmost biological importance as they participate as intermediates of several enzymatic reactions (transamination, amino-acid biosyntheses, porphyrin biosyntheses, a.s.o.), in the crosslinking of collagen and elastin, in hemoglobin AlC, in rhodopsin and bacteriorhodopsin, and as final products from lipid peroxidation. The aerobic oxidation of four aliphatic imines (BPA, i-BMA, sec-BMA and BVA) , catalyzed by HRP, has been shown to be chemiluminescent. The triplet nature of the products is suggested by the chemiluminescence spectrum, by energy transfer studies to DBAS and chlorophyll a and by quenching of the chemiluminescence by molecular oxygen, sorbate ion, indol and p-benzoquinone. Based on the high values of ETTo found in these experiments we have concluded that the energy transfer process occur through a long range mechanism. Analyses of the products found in the spent reaction mixtures and the kinetic data indicate that the reaction follows the route: (SEE scheme file) We stress that this reaction constitutes a more realistic model for bioluminescent systems than that reported by McCapra and Burford (1976), which uses aromatic Schiff bases in DMSO/t-butoxi. In addition, the reaction described here point out for the possible development of chemiluminescent analytical procedure to detect the formation of Schiff bases in biological systems. Our studies on aliphatic Schiff bases were extended to adducts of both amino acids (Lys, Arg, His and Phe) and proteins (lyzozyme, bovine serum albumin and fish protamins) with glycolaldehyde. All these adducts are chemiluminescent when exposed to HRP in an aerated buffered solution. The fluorescence typical of the adducts decays concomitantly with light emission and, during the reaction, the enzyme remains predominantely in the form of HRP Compound II. Energy transfer to chlorophyll a from the systems glycolaldenyde-lyzozyme/O2/HRP and glycolaldehyde-protamine/ 02/HRP occur. These studies are relevant with respect to the toxicity associated to ethyl alcohol intake which, according to recent reports, is attributed to the formation of Schiff linkages between membrane proteins and acetaldehyde. Also noteworthy is the formation of imines along lipid peroxidation and the accompanying chemiluminescence. Our data raise the hypothesis that the alcohol toxicology may involve generated electronically excited species formed during aerobic oxidation of protein-aldehyde adducts.

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Orientador: Prof. Dr. Luciano Soares da Cruz / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017. / O desenvolvimento das plantas depende fortemente das condições ambientais em que estes organismos se encontram. Assim, as plantas desenvolveram mecanismos de adaptação e defesa que permitem seu crescimento e proliferação, mesmo sem a habilidade de se deslocar. Devido a este altamente sofisticado sistema de defesa e adaptação das plantas, as suas características (fenotípicas e bioquímicas) são fortemente influenciadas pelo ambiente no qual a planta se desenvolve. Para garantir a reprodutibilidade dos experimentos em biologia de plantas é fundamental a mensuração e, se possível, o controle adequado dos parâmetros ambientais essenciais ao crescimento das plantas. A não observação disso pode incorrer na não reprodutibilidade do experimento e, consequentemente, obtenção de conclusões incorretas ou distorcidas dos resultados experimentais. Neste trabalho analisamos a manipulação do espectro luminoso a fim de controlar as características da luz incidente nas plantas. Para isto, foram estudados materiais poliméricos para uso como filtros da luz solar e isolamento dos espécimes do ambiente externo. Dessa forma, analisamos os efeitos destes filtros na qualidade e quantidade de luz e seu uso no redirecionamento das trajetórias metabólicas e propriedades biométricas no ciclo de crescimento das plantas. Além disso, para fins práticos, também verificamos as mudanças nas propriedades físicas destes materiais ao longo do tempo por meio de exposição a processos de degradação acelerada. Dessa forma, verificamos os possíveis efeitos de deterioração nos materiais poliméricos devido a sua exposição ao meio ambiente por longos períodos, o que nos permite garantir o uso das câmaras de crescimento desenvolvidas por um período de tempo adequado, no qual suas características essenciais não sofram danos que poderiam influir nos resultados experimentais esperados. / The development of the plans depends heavily on the environmental conditions in which these organisms are. Thus, plants are developed to adapt and defend maximizing their growth and proliferation, even without the ability to move. Due to this highly sophisticated system of defense and adaptation of the plants, their characteristics (phenotypic and biochemical) are strongly influenced by the environment where plant develops. In order to guarantee the reproducibility of the experiments in plant biology, it is fundamental the measurement and, if it is possible, adequate control of environmental parameters essential to plant growth. Failure to observe this may result in non-reproducibility of the experiment and consequently incorrect or distorted conclusions of the experimental results. In this work, we analyzed the manipulation of the light spectrum in order to control the characteristics of the incident light in the plants. For this, we studied polymeric materials for use as sunlight filters and isolation of the specimens from the external environment. Thus, we analyzed the effects of these filters on the quality and quantity of light and their effects in the redirection of the metabolic trajectories and biometric properties in the plant growth cycle. In addition, for practical purposes, we also monitoring at changes in the physical properties of these materials over time through exposure to accelerated degradation processes. In this way, we verify the possible effects of deterioration in the polymeric materials due to their exposure to the environment for long periods, which allows us to guarantee the use of developed growth chambers for an adequate period of time, in which their essential characteristics do not suffer degradation that could influence incorrectly the experimental results.

A enzima peroxidase (HRP) , agindo como uma oxigenase em substratos apropriados, promove a formação de espécies excitadas no estado triplete. Estes produtos excitados podem ser formados em sistemas bioquímicos e promover processos fotoquímicos no escuro. Nesta linha de estudos, investigamos a oxidação aeróbica, na presença de HRP, de substratos contendo ligações de Schiff. Esses compostos são de grande importância biológica, uma vez que participam como intermediários em diversas reações enzimáticas (transaminação, biossíntese de aminoácidos, biossíntese de profirinas), nas ligações cruzadas do colágeno e da elastina, na hemoglobina AlC, na rodopsina e bacterionodopsina e como produtos finais da lipoperoxidação. A oxidação aeróbica de quatro iminas alifáticas (BPA, i-BMA, sec-BMA e BVA) , catalisada por HRP, é quimiluminescente. A natureza triplete da espécie excitada foi sugerida pelo espectro de quimiluminescência, por meio de estudos de transferência de energia para DBAS e clorofila e pela supressão de quimiluminescência por oxigênio, sorbato, indol e p-benzoquinona. Com base nos altos valores de kETo encontrados, concluimos que a transferência de energia esteja ocorrendo provavelmente por um mecanismo a longa distância. A análise dos produtos da reação e os dados cinéticos indicam que a reação ocorre provavelmente segundo a via: (VER Esquema no arquivo) Ressalta-se que esta reação constitui um modelo mais adequado para sistemas bioluminescentes do que o proposto por McCapra e Burford (1976) com bases de Schiff aromáticas em t-butóxido e DMSO. Os sistemas aqui descritos também apresentam a possibilidade de desenvolvimento de método analítico, empregando quimiluminescência, para detectar formação de bases de Schiff em sistemas biológicos. Os estudos com base de Schiff alifáticos (sistemas modelo) foram estendidos para adutos contendo ligação de Schiff entre glicolaldeído e aminoácidos (Lys, Arg, His e Phe) ou proteínas (lisozima, BSA e protaminas). Todos os sistemas estudados são quimiluminescentes na presença de HRP; a fluorescência característica da formação do aduto decai concomitantemente com a emissão de luz; e, durante a reação, a peroxidase encontra-se principalmente na forma de composto II. Observa-se também transferência de energia dos sistemas glicolaldeído-lisozima/HRP/O2 e glicolaldeídoprotamina/HRP/O2 para clorofila. Estes estudos têm grande interesse do ponto de vista da toxicidade associada à ingestão de álcool etílico, a qual, segundo trabalhos recentes da literatura, é atribuída à formação de bases de Schiff entre proteínas de membranas e acetaldeído. E notável também, a formação de iminas durante o processo de lipoperoxidação e a quimiluminescência que o acompanha. Nossos dados levantam a possibilidade de que a toxicologia do álcool pode envolver espécies eletronicamente excitadas formadas na oxidação aeróbica dos adutos aldeído-proteínas. / Horseradish peroxidase (HRP) , acting as an oxigenase on various substracts, promotes the generation of electronically excited triplet species. These species can also be formed in many biochemical reactions and drive photochemical processes in the absence of light. Enlighted by this hypothesis (photobiochemistry in the dark) we have investigated the HRP-catalyzed aerobic oxidation of substrates containing Schiff linkage. These compounds are of utmost biological importance as they participate as intermediates of several enzymatic reactions (transamination, amino-acid biosyntheses, porphyrin biosyntheses, a.s.o.), in the crosslinking of collagen and elastin, in hemoglobin AlC, in rhodopsin and bacteriorhodopsin, and as final products from lipid peroxidation. The aerobic oxidation of four aliphatic imines (BPA, i-BMA, sec-BMA and BVA) , catalyzed by HRP, has been shown to be chemiluminescent. The triplet nature of the products is suggested by the chemiluminescence spectrum, by energy transfer studies to DBAS and chlorophyll a and by quenching of the chemiluminescence by molecular oxygen, sorbate ion, indol and p-benzoquinone. Based on the high values of ETTo found in these experiments we have concluded that the energy transfer process occur through a long range mechanism. Analyses of the products found in the spent reaction mixtures and the kinetic data indicate that the reaction follows the route: (SEE scheme file) We stress that this reaction constitutes a more realistic model for bioluminescent systems than that reported by McCapra and Burford (1976), which uses aromatic Schiff bases in DMSO/t-butoxi. In addition, the reaction described here point out for the possible development of chemiluminescent analytical procedure to detect the formation of Schiff bases in biological systems. Our studies on aliphatic Schiff bases were extended to adducts of both amino acids (Lys, Arg, His and Phe) and proteins (lyzozyme, bovine serum albumin and fish protamins) with glycolaldehyde. All these adducts are chemiluminescent when exposed to HRP in an aerated buffered solution. The fluorescence typical of the adducts decays concomitantly with light emission and, during the reaction, the enzyme remains predominantely in the form of HRP Compound II. Energy transfer to chlorophyll a from the systems glycolaldenyde-lyzozyme/O2/HRP and glycolaldehyde-protamine/ 02/HRP occur. These studies are relevant with respect to the toxicity associated to ethyl alcohol intake which, according to recent reports, is attributed to the formation of Schiff linkages between membrane proteins and acetaldehyde. Also noteworthy is the formation of imines along lipid peroxidation and the accompanying chemiluminescence. Our data raise the hypothesis that the alcohol toxicology may involve generated electronically excited species formed during aerobic oxidation of protein-aldehyde adducts.

Este projeto teve como finalidade construir uma fonte de luz baseada em lâmpadas halógenas que apresentasse características técnicas adequadas para aplicações em fotobiologia e fotomedicina, versátil e comercialmente viável. Foram analisadas as características de diferentes lâmpadas halógenas e realizados os experimentos com duas lâmpadas escolhidas, das quais utilizamos a lâmpada de menor divergência luminosa. Foi analisado também o material do corpo da fonte, com relação a características de aquecimento e peso, sendo escolhido um corpo de alumínio produzido industrialmente. Foram avaliadas as características ópticas das lentes, sendo escolhidas as lentes de vidro utilizadas em retroprojetores. Foram analisados os materiais e a construção de filtros térmicos para diminuir o aquecimento do objeto sob irradiação, sendo que escolhemos construir um filtro térmico próprio, que utiliza a água como absorvedor de calor. Medimos e avaliamos as características: espectrais, térmicas, a potência radiante (irradiância) e sua distribuição espacial de três protótipos, onde cada um recebeu modificações de modo a torná-los mais adequados para aplicações na fotoquimeoterapia. Foram também realizados testes preliminares de fotocitotoxicidade do protótipo final com o composto fotossensibilizador de nome comercial Photogem® aplicado em células tumorais de adenocarcinoma de colo retal humano (HT29), cujo resultado foi a ativação do composto fotossensibilizador e indução da morte celular. / The aim of this work was to produce a light source based in halogen lamps. This source should have appropriated technical characteristic for application in photobiology and photomedicine, it has easy operation and be commercially viable. The characteristics of different halogen lamps were evaluated and the lamp of smaller luminous divergence was chosen. We also have evaluated the material of the source holder, especially heating characteristics and weight; finally we chose an aluminum holder. Then, the lenses optical characteristics were studied, and we decided that the glass lenses used in projectors were the best. The materials and the construction of thermal filters were evaluated in order to reduce the heating of the object under irradiation, and we preferred to construct a thermal filter, that uses water as an heat absorber. The spectral and thermal characteristics, the radiant power (irradiance) and its space distribution of three prototypes were measured and evaluated, each one received modifications to become more appropriated to applications in photochemotherapy. We also made preliminaries tests of photocytotoxicity and the final prototype was used with the photosensitizer (Photogem®) in human colon carcinoma cell lines HT-29, resulting in the photosensitizer activation, that induced cellular death.

Einleitung: Fibroblasten bilden essentielle Komponenten des Bindegewebes und reagieren auf Bestrahlung mit langwelligem Licht. In der Zellkultur lässt sich durch Lichtsignale eine gerichtete Migration (= Phototaxis) induzieren. Da die Migration von Fibroblasten eine zentrale Rolle für die Wundheilung und Regeneration des Bindegewebes spielt, hat die Phototherapie mit langwelligem, nicht-thermischem Licht besondere klinische Bedeutung erlangt. Eindeutige Vorgaben für phototherapeutische Anwendungsparameter existieren bisher jedoch nicht. Fragestellung: Frühere Versuchsanordnungen zur vitalmikroskopischen Untersuchung der Phototaxis von 3T3-Fibroblasten arbeiteten in geschlossenen Kulturflaschen mit Latex- Kügelchen als Lichtquellen, die von externen Strahlungsquellen aktiviert wurden. Die vorliegende Arbeit beschreibt die Entwicklung einer faseroptischen Anordnung mit freier Positionierbarkeit in der offenen Kulturschale. Die primäre Fragestellung war die Eignung der Methode zur Erstellung eines Aktionsspektrums für Phototaxis photoresponsiver, adhärent wachsender Zellen. Material und Methoden: Die Versuche wurden mit NIH-3T3-Zellen der Schweizer Maus durchgeführt. Um die Ergebnisse nicht durch Fremd-Chromophore zu verfälschen, wurden native Zellen ohne Färbemethoden vitalmikroskopisch untersucht. Eine speziell entwickelte Faseroptik und LED-Lichtquellen mit verschiedenen Wellenlängen ermöglichten die direkte Einbringung und Positionierung der Lichtsignale auf dem Boden der Kulturschale. Ein wissenschaftliches Bildbearbeitungsprogramm wurde durch ein Plug-In so modifiziert, dass die Bewegungs- und Zeitparameter halbautomatisch erfasst werden konnten. Ergebnisse: Bei 18 von 44 Langzeitversuchen konnte eindeutiges phototaktisches Verhalten mit Zellkontakt zur Lichtquelle induziert werden. In 16 Fällen kam es zur migratorischen Annäherung und in 10 Fällen zu keiner Reaktion bzw. einer diskret negativen Phototaxis. Das positive phototaktische Verhalten der beiden reagierenden Gruppen war hochsignifikant, der zweiseitige p-Wert des Binomialtests beträgt 0,000388. Bei 77,3 % der Versuche konnte positive Phototaxis demonstriert werden. Schlussfolgerungen: Das neu entwickelte Verfahren eignet sich zur Untersuchung einer Phototaxis und kann damit prinzipiell zur Erstellung von phototaktischen Aktionsspektren eingesetzt werden. Im Vergleich zu bisherigen Methoden können die Bestrahlungsstärken direkt am Lichtwellenleiter gemessen und unerwünschte Fremdkörper-induzierte Wechselwirkungen eliminiert werden. Durch die freie Positionierbarkeit der Lichtquelle in der Kulturschale können die zu beobachtenden Zellen beliebig ausgewählt werden. / Background: Fibroblasts are essential constituents of connective tissue and react upon irradiation with long-wavelength light. In cell culture, directed migration (= phototaxis) can be induced by light stimuli. Fibroblast migration and activity play a central role for wound healing and connective tissue regeneration. In consequence, phototherapy with non-thermal, long wavelength light obtained increasing clinical importance. However, accepted guidelines for phototherapeutical treatment parameters are still lacking. Objective: Existing assays for microscopic observation of phototactic reaction of 3T3- fibroblasts in a live cell chamber use closed cell culture flasks with small light-scattering latex particles attached to the surface of the flask bottom prior to cell seeding, which can be illuminated by an external light source. The present work describes the development of a fiberoptic assembly providing free positioning of the light source in an open culture dish. The primary objective is the eligibility of this method for determination of a phototactic action spectrum for photoresponsive adherent cell species. Materials and Methods: Experiments were conducted using Swiss mouse NIH-3T3-cells. Native cells without staining were used for elimination of potential dye-induced cell-light interferences. A light microscope with field illumination shutter simulation and live cell chamber was used in combination with a specially devised LED-light-fed fiberoptic assembly providing different wavelengths, which could be directly positioned on the inner bottom of the culture dish. A scientific image processing application was modified by a special plug-in for semi-automatic acquisition of cellular locomotion parameters and temporal data token. Results: A total of 44 experiments were conducted. 18 essays resulted in full phototactic reaction characterized by pseudopodium contact with the fiberoptic aperture, another 16 essays showed migratory approximation without direct contact with the light source. 10 essays displayed no reaction or discrete negative phototaxis. The positive phototaxis of the two responding groups was highly significant in the two-sided binomial test (p = 0.000388). For 77.3 % of the experiments positive phototaxis could be demonstrated. Conclusions: The newly developed assay is appropriate for the induction of phototactic behaviour and can be utilized for the determination of phototactic action spectra. In contrast to existing methods the irradiances can be measured directly at the aperture of the optical fiber and biasing foreign object-induced effects can be eliminated. Due to arbitrary positioning of the light source the cells can be chosen freely for examination.

Polymorphic light eruption (PLE) is the most common photodermatosis, affecting ~17% of the population. PLE is a delayed-type hypersensitivity response to an antigen induced by solar ultra-violet radiation (UVR). Its effects vary between patients, but the main symptom is a non-scarring, red papular rash in areas exposed to UVR. An effective therapy is low dose ultra-violet B (NBUVB) phototherapy. It is thought that NBUVB phototherapy desensitises the skin to further UVR exposure, but the mechanism by which this happens is unknown. Current immune based studies have been unable to clarify a mechanism as to how PLE arises. However, research in other skin diseases, such as psoriasis and atopic dermatitis, has shown that the barrier function of the skin is compromised by these disorders. Furthermore, research in lesional PLE skin showed an increase in barrier permeability of the skin. Recent research has specifically linked claudin proteins of tight junctions to the barrier dysfunction. Therefore, this study used quantitative immunofluorescent staining to measure tight junction (TJ) proteins and other barrier proteins of interest. Barrier function was also measured by transepidermal water loss (TEWL); a tracer dye penetration assay was used to measure TJ barrier function specifically. All measurements were made in non-lesional PLE skin, as compared to skin from healthy human volunteers. In photoprotected PLE skin the TJ protein claudin-1 was significantly reduced compared to healthy skin. The use of a tracer dye highlighted there was a reduction in TJ barrier function in PLE skin compared to healthy individuals. PLE and healthy skin were then exposed to ultra-violet B (UVB) and 24h later TJ proteins and TJ barrier function were measured. There was no change to claudin-1 after UVB exposure in PLE skin, but claudin-7 was reduced and claudin-12 increased. In contrast, in UVB-irradiated skin in normal controls after UVB exposure claudin-7 and claudin-12 were both increased, whilst claudin-1 was reduced. In PLE patients there was no further change to TJ barrier function, however, in normal controls, skin TJ barrier function was reduced post UVB. Both in healthy and PLE skin TEWL was unchanged before and after UVB exposure. Lastly TJ proteins were investigated after NBUVB in PLE patients. There was a further reduction in claudin-1 in PLE patients as well as a reduction in the TJ protein occludin, however the stratum corneum was significantly thickened. It could be suggested that this is a compensatory measure for the reduction seen in TJ barrier proteins, however further studies are needed to understand this. These data show significant differences in the TJ skin barrier in patients with PLE as compared to healthy human volunteers before and after UVB exposure. Furthermore, in PLE skin there is a significant change to the epidermis after NBUVB phototherapy. These data demonstrate that TJ protein expression and function is altered in PLE skin and may contribute to aetiology of the disorder, however the role of TJ barrier in aetiology is yet to be firmly established.

Orientador: Helio Amante Miot / Resumo: FUNDAMENTOS: Melasma é hipermelanose crônica, focal, adquirida decorrente de patogênese não totalmente compreendida, resultado da alteração funcional e arquitetural dos melanócitos. A predisposição genética, aspectos hormonais e exposição à radiação solar são os elementos mais associados ao desenvolvimento da doença e essenciais para entendimento da fisiopatologia. É bem estabelecida a relação entre a exposição à radiação solar e a piora do quadro, entretanto, o efeito independente do UVB, UVA, assim como a atuação da luz visível (LV) são pouco estudados. OBJETIVOS: Padronização de um modelo de cultura organoide cutâneo primário para estudo da melanogênese da pele com melasma e pele normal adjacente, à irradiação com diferentes comprimentos de onda (UVB, UVA, LV). MÉTODOS: Etapa 1: Amostras de pele da região retroauricular (punch 3mm), de 10 voluntários foram seccionados longitudinalmente, e cultivados em meio DMEM segundo protocolo estabelecido por Ayres, para padronização de viabilidade e dosimetria das radiações induzindo melanogênese. Um dos fragmentos foi irradiado e o outro mantido ao abrigo da luz por 72h. Foram avaliados aspectos morfológicos e arquiteturais da histologia (H&E e Fontana-Masson) e por rt-PCR para comparação da expressão quantitativa de gene constitucional (GAPDH) entre as peles recém-coletadas e as cultivadas. Foram padronizadas as doses de radiação e tempo de cultura que promovessem viabilidade da amostra e aumento de 10% na intensidade de pigmentação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: FUNDAMENTALS: Melasma is chronic hypermelanosis, focal, acquired due to pathogenesis not fully understood, resulting from the functional and architectural alteration of melanocytes. Genetic predisposition, hormonal aspects and exposure to solar radiation are the elements most associated with the development of the disease and essential for understanding the pathophysiology. The relationship between exposure to solar radiation and worsening of the condition is well established, however, the independent effect of UVB, UVA, as well as the action of visible light (VL) are poorly studied. OBJECTIVES: Standardization of a primary cutaneous organoid culture model to study melanogenesis of skin with melasma and adjacent normal skin to irradiation with different wavelengths (UVB, UVA, VL). METHODS: Step 1: Skin samples from the retroauricular region (punch 3mm) from 10 volunteers were longitudinally sectioned and cultured in DMEM medium according to the protocol established by Ayres, for viability standardization and radiation dosimetry inducing melanogenesis. One of the fragments was irradiated and the other kept in the dark for 72h. Morphological and architectural aspects of histology (H&E and Fontana-Masson) and rt-PCR were evaluated for comparison of quantitative constitutional gene expression (GAPDH) between newly collected and cultured skins. Radiation doses and culture time that promoted sample viability and a 10% increase in basal layer pigmentation intensity were standardized... (Complete abstract click electronic access below) / Mestre

Einleitung: Fibroblasten bilden essentielle Komponenten des Bindegewebes und reagieren auf Bestrahlung mit langwelligem Licht. In der Zellkultur lässt sich durch Lichtsignale eine gerichtete Migration (= Phototaxis) induzieren. Da die Migration von Fibroblasten eine zentrale Rolle für die Wundheilung und Regeneration des Bindegewebes spielt, hat die Phototherapie mit langwelligem, nicht-thermischem Licht besondere klinische Bedeutung erlangt. Eindeutige Vorgaben für phototherapeutische Anwendungsparameter existieren bisher jedoch nicht. Fragestellung: Frühere Versuchsanordnungen zur vitalmikroskopischen Untersuchung der Phototaxis von 3T3-Fibroblasten arbeiteten in geschlossenen Kulturflaschen mit Latex- Kügelchen als Lichtquellen, die von externen Strahlungsquellen aktiviert wurden. Die vorliegende Arbeit beschreibt die Entwicklung einer faseroptischen Anordnung mit freier Positionierbarkeit in der offenen Kulturschale. Die primäre Fragestellung war die Eignung der Methode zur Erstellung eines Aktionsspektrums für Phototaxis photoresponsiver, adhärent wachsender Zellen. Material und Methoden: Die Versuche wurden mit NIH-3T3-Zellen der Schweizer Maus durchgeführt. Um die Ergebnisse nicht durch Fremd-Chromophore zu verfälschen, wurden native Zellen ohne Färbemethoden vitalmikroskopisch untersucht. Eine speziell entwickelte Faseroptik und LED-Lichtquellen mit verschiedenen Wellenlängen ermöglichten die direkte Einbringung und Positionierung der Lichtsignale auf dem Boden der Kulturschale. Ein wissenschaftliches Bildbearbeitungsprogramm wurde durch ein Plug-In so modifiziert, dass die Bewegungs- und Zeitparameter halbautomatisch erfasst werden konnten. Ergebnisse: Bei 18 von 44 Langzeitversuchen konnte eindeutiges phototaktisches Verhalten mit Zellkontakt zur Lichtquelle induziert werden. In 16 Fällen kam es zur migratorischen Annäherung und in 10 Fällen zu keiner Reaktion bzw. einer diskret negativen Phototaxis. Das positive phototaktische Verhalten der beiden reagierenden Gruppen war hochsignifikant, der zweiseitige p-Wert des Binomialtests beträgt 0,000388. Bei 77,3 % der Versuche konnte positive Phototaxis demonstriert werden. Schlussfolgerungen: Das neu entwickelte Verfahren eignet sich zur Untersuchung einer Phototaxis und kann damit prinzipiell zur Erstellung von phototaktischen Aktionsspektren eingesetzt werden. Im Vergleich zu bisherigen Methoden können die Bestrahlungsstärken direkt am Lichtwellenleiter gemessen und unerwünschte Fremdkörper-induzierte Wechselwirkungen eliminiert werden. Durch die freie Positionierbarkeit der Lichtquelle in der Kulturschale können die zu beobachtenden Zellen beliebig ausgewählt werden.:INHALTSVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VII TABELLENVERZEICHNIS IX ABKÜRZUNGSVERZEICHNIS X 1. EINLEITUNG 1 2. DEFINITIONEN UND STAND DER FORSCHUNG 5 2.1 LICHT 5 2.1.1 Sonnenlicht 5 2.1.2 Licht in der Physik 5 2.1.3 Licht in der Biologie 6 2.1.3.1 Taxis 7 2.1.4 Licht in der Medizin 8 2.1.4.1 Historischer Überblick 8 2.2 PHOTOBIOLOGIE VON ROT UND NAHINFRAROT 10 2.2.1 Photochemische Grundlagen 10 2.2.2 Haut 11 2.2.2.1 Aufbau und Funktion 11 2.2.2.2 Optische Eigenschaften 12 2.2.3 Fibroblasten 13 2.2.4 Zellmigration 15 2.2.5 Mitochondrien 16 2.3 PHOTOTAXIS BEI 3T3-FIBROBLASTEN 17 3. FRAGESTELLUNG 23 4. MATERIAL UND METHODEN 25 4.1 GERÄTE, LABORMATERIALIEN UND SOFTWARE 25 4.2 ZELLMATERIAL UND KULTURBEDINGUNGEN 25 4.3 ZELLPRÄPARATION 27 4.4 VERSUCHSAUFBAU 27 4.5 SHUTTER-SIMULATION DER OBJEKTBELEUCHTUNG 32 4.6 ZUFUHR VON DESTILLIERTEM WASSER 33 4.7 VERSUCHSABLAUF 33 4.8 VERMESSUNG DES BEWEGUNGSVERHALTENS 35 4.9 STATISTISCHE BERECHNUNG 36 5. ERGEBNISSE 37 5.1 AUSWAHLKRITERIEN FÜR GÜLTIGE VERSUCHE 37 5.2 GRUPPENEINTEILUNG DER GÜLTIGEN VERSUCHE 38 5.2.1 Die Gruppe "Touchdown" (TD) 38 5.2.2 Die Gruppe "Annäherung/Closer" (CL) 39 5.2.3 Die Gruppe "still/entf./no reaction" (NO) 39 5.2.4 Zusammenfassung des Gruppenverhaltens 40 5.3 GRAFISCHE DARSTELLUNGEN DER AUSGEWERTETEN VERSUCHE 41 5.4 PRIMÄRE FRAGESTELLUNG: PHOTOTAKTISCHE REAKTION 49 5.5 FRAGESTELLUNG 2: EINFLUSS DER MIGRATIONSMODALITÄT 49 5.6 EINFLUSS VON WELLENLÄNGE UND LICHTMODULATION 51 5.6.1 Fragestellung 3: Einfluss der Wellenlänge 51 5.6.2 Fragestellung 4: Einfluss der Pulsationsfrequenz 52 5.7 FRAGESTELLUNG 5: ROLLE DER AUSGANGSPOSITION 53 5.8 LOKOMOTORISCHE PARAMETER 54 5.8.1 Fragestellung 6: Zurückgelegte Wegstrecke 54 5.8.2 Fragestellung 7: Migrationsgeschwindigkeit 56 5.9 FRAGESTELLUNG 8: EINFLUSS DER VITALITÄT 57 5.9.1 Definition der mittleren Vitalität vitmean 57 5.9.2 Definition der relativen Vitalität vitrel 57 5.9.3 Die relative Vitalität vitrel der Zellen FBc, FB1 und FB2 58 5.9.4 Die mittlere Vitalität vitmean in den Versuchsgruppen 59 5.9.5 Die relative Vitalität vitrel des zentralen Fibroblasten 60 5.10 ATYPISCHES ZELLVERHALTEN 61 5.10.1 Atypisches Verhalten in Versuchen mit normalen 3T3-Zellen 61 5.10.1.1 Hohe Geschwindigkeit und große Wegstrecke 61 5.10.1.3 "Überfall" eines Fibroblasten 63 5.10.1.2 Zentraler Fibroblast migriert unter den Lichtwellenleiter 64 5.10.2 Atypisches Verhalten bei frischen humanen Monozyten 66 6. DISKUSSION 68 6.1 ERGEBNISSE 68 6.1.1 Primäre Fragestellung: Phototaktische Reaktion 68 6.1.2 Fragestellung 2: Einfluss der Migrationsmodalität 70 6.1.3 Fragestellung 3: Einfluss der Wellenlänge 71 6.1.4 Fragestellung 4: Einfluss der Pulsationsfrequenz 74 6.1.5 Fragestellung 5: Rolle der Ausgangsposition 78 6.1.6 Fragestellung 6: Zurückgelegte Wegstrecke 79 6.1.6.1 Differenz Startposition - Minimalposition zum LWL 79 6.1.6.2 Insgesamt zurückgelegte Wegstrecke 80 6.1.7 Fragestellung 7: Migrationsgeschwindigkeit 80 6.1.8 Fragestellung 8: Einfluss der Vitalität 81 6.1.9 Atypisches Zellverhalten 83 6.1.9.1 Versuche mit 3T3-Zellen 83 6.1.9.1.1 Hohe Geschwindigkeit und große Wegstrecke 83 6.1.9.1.2 "Überfall" eines Fibroblasten 84 6.1.9.1.3 Phototaxis bei hyperosmotischem Stress 85 6.1.9.2 Atypisches Verhalten bei frischen humanen Monozyten 86 6.1.10 Zusätzliche Fragestellungen 87 6.1.10.1 Wie lange ist die maximale Versuchsdauer im offenen System 88 6.1.10.2 Spielt der Aspekt der potentiellen Verkeimung eine relevante Rolle? 88 6.1.10.3 Spielt die Dimension der Lichtquelle eine objektivierbare Rolle 88 6.1.10.4 Kann die Bestrahlungsstärke gemessen anstatt geschätzt werden 89 6.1.10.5 Können Rückschlüsse auf das "Auge der Zelle" gezogen werden? 90 6.2 SCHLUSSFOLGERUNG 93 6.3 AUSBLICK 94 7. ZUSAMMENFASSUNG 96 7.1 SUMMARY 97 8. LITERATURVERZEICHNIS 98 9. ANLAGEN 117 9.1 LABORGERÄTE UND MATERIALIEN 117 9.1.1 Mikroskope 117 9.1.2 Software: 117 9.1.3 Geräte: 118 9.1.4 Verbrauchsmaterialien 118 9.1.5 Medien, Puffer, Lösungen 119 9.2 EIGENE GERÄTSCHAFTEN 120 9.2.1 Frequenzgenerator 120 9.2.2 Halbleiter-Lichtquellen 121 9.2.2.1 Modulierbare Lichtquelle 121 9.2.2.2 Kohärente Lichtquelle 122 9.2.2.3 Photon Micro-Light LED-Lichtquellen mit Pulsmodus f = 1Hz 124 9.2.2.4 Bestrahlungsstärken der verwendeten Lichtquellen 124 9.2.3 Systemträger 125 9.2.4 LWL und Optokoppler 126 9.2.5 Modifizierte Microsoft-Maus 126 9.2.6 Eigene Software 127 10. DANKSAGUNG 128 11. EIDESSTATTLICHE ERKLÄRUNG 130 / Background: Fibroblasts are essential constituents of connective tissue and react upon irradiation with long-wavelength light. In cell culture, directed migration (= phototaxis) can be induced by light stimuli. Fibroblast migration and activity play a central role for wound healing and connective tissue regeneration. In consequence, phototherapy with non-thermal, long wavelength light obtained increasing clinical importance. However, accepted guidelines for phototherapeutical treatment parameters are still lacking. Objective: Existing assays for microscopic observation of phototactic reaction of 3T3- fibroblasts in a live cell chamber use closed cell culture flasks with small light-scattering latex particles attached to the surface of the flask bottom prior to cell seeding, which can be illuminated by an external light source. The present work describes the development of a fiberoptic assembly providing free positioning of the light source in an open culture dish. The primary objective is the eligibility of this method for determination of a phototactic action spectrum for photoresponsive adherent cell species. Materials and Methods: Experiments were conducted using Swiss mouse NIH-3T3-cells. Native cells without staining were used for elimination of potential dye-induced cell-light interferences. A light microscope with field illumination shutter simulation and live cell chamber was used in combination with a specially devised LED-light-fed fiberoptic assembly providing different wavelengths, which could be directly positioned on the inner bottom of the culture dish. A scientific image processing application was modified by a special plug-in for semi-automatic acquisition of cellular locomotion parameters and temporal data token. Results: A total of 44 experiments were conducted. 18 essays resulted in full phototactic reaction characterized by pseudopodium contact with the fiberoptic aperture, another 16 essays showed migratory approximation without direct contact with the light source. 10 essays displayed no reaction or discrete negative phototaxis. The positive phototaxis of the two responding groups was highly significant in the two-sided binomial test (p = 0.000388). For 77.3 % of the experiments positive phototaxis could be demonstrated. Conclusions: The newly developed assay is appropriate for the induction of phototactic behaviour and can be utilized for the determination of phototactic action spectra. In contrast to existing methods the irradiances can be measured directly at the aperture of the optical fiber and biasing foreign object-induced effects can be eliminated. Due to arbitrary positioning of the light source the cells can be chosen freely for examination.:INHALTSVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VII TABELLENVERZEICHNIS IX ABKÜRZUNGSVERZEICHNIS X 1. EINLEITUNG 1 2. DEFINITIONEN UND STAND DER FORSCHUNG 5 2.1 LICHT 5 2.1.1 Sonnenlicht 5 2.1.2 Licht in der Physik 5 2.1.3 Licht in der Biologie 6 2.1.3.1 Taxis 7 2.1.4 Licht in der Medizin 8 2.1.4.1 Historischer Überblick 8 2.2 PHOTOBIOLOGIE VON ROT UND NAHINFRAROT 10 2.2.1 Photochemische Grundlagen 10 2.2.2 Haut 11 2.2.2.1 Aufbau und Funktion 11 2.2.2.2 Optische Eigenschaften 12 2.2.3 Fibroblasten 13 2.2.4 Zellmigration 15 2.2.5 Mitochondrien 16 2.3 PHOTOTAXIS BEI 3T3-FIBROBLASTEN 17 3. FRAGESTELLUNG 23 4. MATERIAL UND METHODEN 25 4.1 GERÄTE, LABORMATERIALIEN UND SOFTWARE 25 4.2 ZELLMATERIAL UND KULTURBEDINGUNGEN 25 4.3 ZELLPRÄPARATION 27 4.4 VERSUCHSAUFBAU 27 4.5 SHUTTER-SIMULATION DER OBJEKTBELEUCHTUNG 32 4.6 ZUFUHR VON DESTILLIERTEM WASSER 33 4.7 VERSUCHSABLAUF 33 4.8 VERMESSUNG DES BEWEGUNGSVERHALTENS 35 4.9 STATISTISCHE BERECHNUNG 36 5. ERGEBNISSE 37 5.1 AUSWAHLKRITERIEN FÜR GÜLTIGE VERSUCHE 37 5.2 GRUPPENEINTEILUNG DER GÜLTIGEN VERSUCHE 38 5.2.1 Die Gruppe "Touchdown" (TD) 38 5.2.2 Die Gruppe "Annäherung/Closer" (CL) 39 5.2.3 Die Gruppe "still/entf./no reaction" (NO) 39 5.2.4 Zusammenfassung des Gruppenverhaltens 40 5.3 GRAFISCHE DARSTELLUNGEN DER AUSGEWERTETEN VERSUCHE 41 5.4 PRIMÄRE FRAGESTELLUNG: PHOTOTAKTISCHE REAKTION 49 5.5 FRAGESTELLUNG 2: EINFLUSS DER MIGRATIONSMODALITÄT 49 5.6 EINFLUSS VON WELLENLÄNGE UND LICHTMODULATION 51 5.6.1 Fragestellung 3: Einfluss der Wellenlänge 51 5.6.2 Fragestellung 4: Einfluss der Pulsationsfrequenz 52 5.7 FRAGESTELLUNG 5: ROLLE DER AUSGANGSPOSITION 53 5.8 LOKOMOTORISCHE PARAMETER 54 5.8.1 Fragestellung 6: Zurückgelegte Wegstrecke 54 5.8.2 Fragestellung 7: Migrationsgeschwindigkeit 56 5.9 FRAGESTELLUNG 8: EINFLUSS DER VITALITÄT 57 5.9.1 Definition der mittleren Vitalität vitmean 57 5.9.2 Definition der relativen Vitalität vitrel 57 5.9.3 Die relative Vitalität vitrel der Zellen FBc, FB1 und FB2 58 5.9.4 Die mittlere Vitalität vitmean in den Versuchsgruppen 59 5.9.5 Die relative Vitalität vitrel des zentralen Fibroblasten 60 5.10 ATYPISCHES ZELLVERHALTEN 61 5.10.1 Atypisches Verhalten in Versuchen mit normalen 3T3-Zellen 61 5.10.1.1 Hohe Geschwindigkeit und große Wegstrecke 61 5.10.1.3 "Überfall" eines Fibroblasten 63 5.10.1.2 Zentraler Fibroblast migriert unter den Lichtwellenleiter 64 5.10.2 Atypisches Verhalten bei frischen humanen Monozyten 66 6. DISKUSSION 68 6.1 ERGEBNISSE 68 6.1.1 Primäre Fragestellung: Phototaktische Reaktion 68 6.1.2 Fragestellung 2: Einfluss der Migrationsmodalität 70 6.1.3 Fragestellung 3: Einfluss der Wellenlänge 71 6.1.4 Fragestellung 4: Einfluss der Pulsationsfrequenz 74 6.1.5 Fragestellung 5: Rolle der Ausgangsposition 78 6.1.6 Fragestellung 6: Zurückgelegte Wegstrecke 79 6.1.6.1 Differenz Startposition - Minimalposition zum LWL 79 6.1.6.2 Insgesamt zurückgelegte Wegstrecke 80 6.1.7 Fragestellung 7: Migrationsgeschwindigkeit 80 6.1.8 Fragestellung 8: Einfluss der Vitalität 81 6.1.9 Atypisches Zellverhalten 83 6.1.9.1 Versuche mit 3T3-Zellen 83 6.1.9.1.1 Hohe Geschwindigkeit und große Wegstrecke 83 6.1.9.1.2 "Überfall" eines Fibroblasten 84 6.1.9.1.3 Phototaxis bei hyperosmotischem Stress 85 6.1.9.2 Atypisches Verhalten bei frischen humanen Monozyten 86 6.1.10 Zusätzliche Fragestellungen 87 6.1.10.1 Wie lange ist die maximale Versuchsdauer im offenen System 88 6.1.10.2 Spielt der Aspekt der potentiellen Verkeimung eine relevante Rolle? 88 6.1.10.3 Spielt die Dimension der Lichtquelle eine objektivierbare Rolle 88 6.1.10.4 Kann die Bestrahlungsstärke gemessen anstatt geschätzt werden 89 6.1.10.5 Können Rückschlüsse auf das "Auge der Zelle" gezogen werden? 90 6.2 SCHLUSSFOLGERUNG 93 6.3 AUSBLICK 94 7. ZUSAMMENFASSUNG 96 7.1 SUMMARY 97 8. LITERATURVERZEICHNIS 98 9. ANLAGEN 117 9.1 LABORGERÄTE UND MATERIALIEN 117 9.1.1 Mikroskope 117 9.1.2 Software: 117 9.1.3 Geräte: 118 9.1.4 Verbrauchsmaterialien 118 9.1.5 Medien, Puffer, Lösungen 119 9.2 EIGENE GERÄTSCHAFTEN 120 9.2.1 Frequenzgenerator 120 9.2.2 Halbleiter-Lichtquellen 121 9.2.2.1 Modulierbare Lichtquelle 121 9.2.2.2 Kohärente Lichtquelle 122 9.2.2.3 Photon Micro-Light LED-Lichtquellen mit Pulsmodus f = 1Hz 124 9.2.2.4 Bestrahlungsstärken der verwendeten Lichtquellen 124 9.2.3 Systemträger 125 9.2.4 LWL und Optokoppler 126 9.2.5 Modifizierte Microsoft-Maus 126 9.2.6 Eigene Software 127 10. DANKSAGUNG 128 11. EIDESSTATTLICHE ERKLÄRUNG 130