Conselho Nacional de Desenvolvimento Científico e Tecnológico / A radiação ultravioleta (UV) induz diversos efeitos nocivos nos organismos e a quantidade desta radiação que atinge a biosfera é afetada pela concentração de ozônio, latitude, altitude, clima e reflexão especular. As respostas de briófitas em relação aos efeitos da radiação UV e a presença de compostos que absorvem esta radiação têm sido estudadas. Sanionia uncinata, Holomitriopsis laevifolia e Leucobryum laevifolium são espécies de musgos encontrados em locais expostos a alta incidência de radiação UV e com habitats distintos. Considerando que as respostas de musgos contra os efeitos da radiação UV e seus mecanismos de proteção ainda são pouco caracterizados, o objetivo deste estudo foi investigar o potencial fotoprotetor e possíveis riscos toxicológicos associados aos extratos dos musgos S. uncinata, proveniente da Antártica e H. laevifolia e L. laevifolium, proveniente do Amazonas. Seus extratos metanólico (EM), aquoso (EA), hidroalcoólico (EH) e etanólico (EE) foram estudados com a caracterização química por absorção ao UV e visível e pela cromatografia líquida de alta eficiência; quantificação do índice total de compostos fenólicos; determinação da capacidade captadora do radical 2,2-difenil-1-picril-hidrazila a fim de avaliar as atividades antioxidantes; avaliação do potencial de fotoproteção cutânea pela determinação do fator de proteção solar; avaliações do potencial mutagênico e citototóxico, através do ensaio de Salmonella/microssoma, utilizando as cepas TA97, TA98, TA100, TA102 e TA104; do potencial fotomutagênico através do ensaio de fotomutagenicidade, usando as cepas TA102 e TA104; e investigação dos efeitos genotóxicos e fotogenotóxicos, pelo ensaio de micronúcleo e fotomicronúcleo, respectivamente, usando diferentes linhagens celulares estabelecidas. Foram encontradas atividades fotoprotetoras e antioxidantes e observou-se que os extratos se apresentaram singulares devido a sua composição química. Os resultados fotoprotetores, além dos mutagênicos/fotomutagênicos, genotóxicos/fotogenotóxicos e suas respectivas avaliações citotóxicas também permitiram selecionar extratos e suas concentrações, como promissores candidatos em fotoproteção Assim, os EA e EH de H. laevifolia e L. laevifolium apresentam, no geral, os resultados mais significativos, tornando-se potenciais para avaliações refinadas em fotoproteção e na separação de componentes que possam levar a futuras aplicações como antioxidantes e protetores solares ou como adjuvantes. / The ultraviolet radiation (UV) induces many harmful effects in all living organisms and the amount of this radiation that reaching the ground is affected by many factors including ozone concentration, latitude, altitude, climate and specular reflection. The responses of bryophytes against the effects of UV radiation and the presence of compounds that absorb the UV region have been studied. Mosses Sanionia uncinata, Holomitriopsis laevifolia and Leucobryum laevifolium are found in locations exposed to UV at high levels of radiation and in different habitats. Whereas that the responses of mosses against the effects of UV radiation and their protection systems are poorly characterized yet, the aim of this study was to investigate photoprotective potential and possible toxicological risks associated with extracts of mosses S. uncinata (from Antarctica) and H. laevifolia and L. laevifolium (from Amazônia). Methanol (ME), aqueous (AE), hydroalcoholic (HE) and ethanolic (EE) were studied by: chemical characterization by UV/visible spectrophotometry and by High performance liquid chromatography; phenolic content estimation; 2,2-diphenyl-1-picrylhydrazyl scavenging activity; potential of skin photoprotection by in vitro determination of sun protection factor; the mutagenic potential, and cytotoxic by Salmonella/microsome assay, using the TA97, TA98, TA100, TA102 and TA104 strains; photomutagenic potential by photomutagenicity test, using TA102 and TA104 strains and; investigation of genotoxic and photogenotoxic effects by micronucleus test and photo-micronucleous assay, respectively, using different established cell lines. Photoprotective and antioxidant activities were found and it was observed that the extracts showed strong uniqueness due to its chemical composition. From the photoprotective, mutagenic/photomutagenic and genotoxic/photogenotoxic results and their cytotoxic evaluations it was possible to select extracts and their concentrations as promising candidates for photoprotection. Thus, the EA and EH of H. laevifolia and L. laevifolium demonstrated the most significant results, becoming potential for refined evaluations in photoprotection and separating components that can lead to future applications such as sunscreens and antioxidants or as adjuvants.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2017-03-08T18:16:41Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Katyane 2.pdf: 4899390 bytes, checksum: 591b77920f0440069d4c7a3d4ec83b37 (MD5) / Made available in DSpace on 2017-03-08T18:16:41Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Katyane 2.pdf: 4899390 bytes, checksum: 591b77920f0440069d4c7a3d4ec83b37 (MD5) Previous issue date: 2013-02-26 / Facepe e Capes / A radiação UV compreende uma região do espectro eletromagnético e é classificada conforme o comprimento de onda em: UVC (100-290 nm), UVB (290-320 nm) e UVA, UVA2 (320-340 nm) e UVA1 (340-400nm). Apesar de serem responsáveis pela maioria dos efeitos benéficos, também o são pelos efeitos prejudiciais e, mesmo tendo o organismo humano desenvolvido mecanismos de defesa, estes não são suficientes, por isso o uso de preparações fotoprotetoras tornou-se essencial. A associação de filtros solares, apesar de ser uma forma usual para aumentar os valores do fator de proteção solar (FPS) destas preparações, ocasiona uma série de problemas, como irritação, reações fototóxicas e alérgicas. Em vista disso, o objetivo desse estudo foi o uso de bentonitas como filtro físico para associação a filtros químicos UVA - avobenzona (AVO), UVB - metoxicinamato de etilexila (EHMC) ou de amplo espectro - oxibenzona (OXI). A escolha da bentonita justifica-se, além de ser um recurso natural, por sua estrutura lamelar, podendo gerar compostos de intercalação, o que pode proteger o filtro químico do sol, prolongando sua ação, e a pele do filtro solar. Foram produzidos, então, via solução, sistemas de cada filtro químico supracitado com bentonita sodica (SB) ou organofílica (OB), em diferentes proporções, obtidos por centrifugação [C] ou evaporação rotativa [R]. O rendimento dos produtos foi calculado indiretamente por espectrofotometria do sobrenadante dos centrifugados, de forma que foram escolhidas as proporções de maior rendimento de cada sistema, [C] e [R], para serem caracterizados. Os melhores sistemas com os respectivos rendimentos foram os seguintes: AVO-SB 3:1 (63 ± 1%); AVO-OB 2:1 (51 ± 1%); EHMC-SB 0,5:1 (79 ± 1%); EHMC-OB 0,5:1 (82 ± 1%); OXI-SB 1:1 (59 ± 2%); OXI-OB 1:1 (58 ± 1%). Os produtos foram caracterizados por difração de raios X (DRX), análise termogravimétrica (TG), calorimetria exploratória diferencial (DSC), espectroscopia de infravermelho com transformada de Fourier (FTIR) e microscopia eletrônica de transmissão (MET), os quais, de forma geral, confirmaram a formação dos compostos de intercalação. Para análise dos valores de FPS in vitro, todos os filtros foram incorporados em uma emulsão-base a 2,5%, em sua forma pura ou intercalada em bentonita. As preparações mostraram-se estáveis pelo teste de estabilidade preliminar e com valores de pH em conformidade com a via tópica. Os dados de transmitância revelaram um aumento significativo do FPS em todos os compostos de intercalação de AVO, se comparado ao filtro puro (0,7 ± 0,5), destacando-se o sistema AVO-OB [C] (17 ± 2); com o EHMC, mais uma vez o sistema com OB [C] se destacou (17 ± 1 versus 8 ± 1 para o EHMC puro), tendo sido o único, nesse caso, a ter um aumento significativo; os resultados dos compostos de intercalação contendo OXI não mostraram diferença nos valores de FPS, provavelmente porque o FPS da OXI pura já é bem alto (19 ± 3), pelo contrário, o sistema OXI-SB [C] reduziu o FPS (12 ± 0,5). Os resultados mostraram a viabilidade da formação de compostos de intercalação filtros solares – bentonita e da utilização, em especial, da OB em formulações fotoprotetoras. / UV radiation comprises a region of the electromagnetic spectrum and is classified as wavelength: UVC (100-290 nm), UVB (290-320 nm) and UVA UVA2 (320-340 nm) and UVA1 (340-400 nm). Despite being responsible for the most beneficial effects, so are the harmful effects and even having the human organism developed defense mechanisms, these are not enough, so the use of sunscreen preparations became essential. The combination of sunscreens, despite being a usual way to increase the values of the sun protection factor (SPF) of these preparations, causes a number of problems, such as irritation, allergic and phototoxic reactions. In view of this, the objective of this study was the use of bentonite as a physical filter for joining chemical filters UVA - avobenzone (AVO), UVB - methoxycinnamate etilexila (EHMC) or broad-spectrum - oxybenzone (OXI). The choice of bentonite is justified, besides being a natural resource for its lamellar structure, which can generate intercalation compounds, which can protect the chemical filter the sun, prolonging its action, and the skin of sunscreen. Produced were then via solution, each filter chemical systems above with sodium bentonite (SB) or organophilic (OB), in different proportions, obtained by centrifugation [C] or rotary evaporation [R]. The yield of the products was calculated indirectly by spectrophotometry of the supernatant centrifuged, were chosen so that the proportions of each system higher yield, [C] and [R], to be characterized. The best systems with their yields were as follows: AVO-SB 3:1 (63 ± 1%); AVO-OB 2:1 (51 ±2%); EHMCSB 0.5:1 (79 ± 1%); EHMC-OB 0.5:1 (82 ± 1%); OXI-SB 1:1 (59 ± 2%); OXI-OB 1:1 (58± 1%). The products were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TG), differential scanning calorimetry (DSC), infrared spectroscopy Fourier transform (FTIR) and transmission electron microscopy (TEM), which so Generally, confirmed the formation of intercalation compounds. For analysis of in vitro SPF values, all filters were incorporated into an emulsion base 2.5% in pure form or interleaved bentonite. The preparations were stable for preliminary stability test with pH values in accordance with topically. The transmittance data revealed a significant increase in FPS in all intercalation compounds of AVO, compared to pure filter (0.7 ± 0.5), highlighting the AVO-OB system [C] (17 ± 2); with EHMC, again with the system B [C] is highlighted (17 ± 1 versus 8 ± 1 for pure EHMC), being the only, in which case , to have a significant increase, the results of intercalation compounds containing OXI showed no difference in SPF values, probably because the SPF of pure OXI is already quite high (19 ± 3), in contrast, the system OXY -SB [C] reduced the FPS (12 ± 0.5). The results show the feasibility of the formation of intercalation compounds sunscreens - bentonite and use, especially in formulations of OB sunscreens.

Foram estudadas trinta e uma cepas fúngicas não identificadas, as quais foram denominadasX1 a X31. O potencial fotoprotetor foi avaliado pela medida espectrofotométrica da absorçãodos extratos na região do UV (280-400 nm). Os extratos com os melhores perfis de absorção em cultura estacionária foram X1, X2, X6, X12, X13, X18, X19, X22, X24 e X31 e, em cultura agitada X4 e X17. A reprodutibilidade do processo foi avaliada e as cepas fúngicas que apresentaram coeficiente de variação menor que 15% foram selecionadas para o estudo de fotoestabilidade. A fotoestabilidade dos extratos foi avaliada pela medida da viabilidade celular de fibroblastos L929 tratados com extratos previamente irradiados sob radiação UVA (11,2 J/cm2) e UVB (3,43 J/cm2) e extratos não irradiados, bem como, pela comparação das áreas sob as curvas de absorção na região do UV dos extratos irradiados e não irradiados. Os extratos selecionados para o estudo de fotoestabilidade foram X4, X12, X19, X22, X24 e X31. Os extratos não irradiados apresentaram os seguintes valores deIC50 para viabilidade celular (citotoxidade): X4-130µg/ml, X19-20µg/ml, X22-10 µg/ml e X24-60µg/ml. Após a radiação UVA e UVB, os extratos apresentaram redução significativa da viabilidade celular em relação ao IC50 dos extratos não irradiados. Sob luz UVB, os extratos X12 (IC50 35µg/ml) e X31 (IC50 70µg/ml) mantiveram a mesma porcentagem de redução da viabilidade celular quando comparado ao IC50 dos extratos não irradiados. No entanto após exposição à luz UVA, o extrato X12 aumentou a viabilidade celular de 50% (quando não irradiado) para 75% (irradiado). Enquanto que o extrato X31, mesmo após a radiação UVA, manteve a mesma redução de 50% da viabilidade celular. Nessa etapa os extratos selecionados foram os X12 e X31. O espectro de absorção na região do UV obtido para o extrato X12 mostrou uma redução da absorbância de 28,3% sob radiação UVB e de 60% sob radiação UVA em relação ao extrato não irradiado. O extrato X31 apresentou uma redução da absorbância de 17,6% e30% sob radiação UVB e UVA respectivamente, em relação ao extrato não irradiado. Os fungos selecionados foram identificados por PCR, sugerindo que o fungo X12 seja o Aspergillus terreus e o X31 seja o Talaromyces pinophilus. Por fim, foi feita a identificação da substância ativa do extrato X12 empregando a técnica de desreplicação, a qual fez o uso da instrumentação analítica acoplada UHPLC-DAD-(ESI)-HRMS associada ao banco de dados Chapman& Hall\'s Dictionary of Natural Products (DNP). No extrato X12 o composto majoritário foi identificado como sendo a citreoviridina. Assim, os resultados do presente trabalho permitiu estabelecer um procedimento para a seleção de fungos produtores de compostos absorvedores de radiação UV, que poderia ser aplicado na obtenção de novos filtros orgânicos naturais para protetores solares. / It were studied thirty-one fungal strains not identified, which were named X1 to X31. The photoprotective potential was assessed spectrophotometrically by measuring absorption of the extract in the UV region (280-400 nm). The extracts that presented the best absorption profiles in stationary culture were X1, X2, X6, X12, X13, X18, X19, X22, X24 and X31, and X4 and X17 in stirred culture. The reproducibility of the process was evaluated and fungal strains that showed a coefficient of variation lower than 15% were selected for the study of photostability. The photostability of the extracts was assessed by measuring cell viability of L929 fibroblasts treated with extracts previously irradiated under UVA light (11,2 J/cm2) and UVB (3,43 J/cm2) and not irradiated extracts, as well as by comparison of the areas under the curves of absorption in the UV region of the irradiated and non-irradiated extracts. The extracts selected for the study of photostability were X4, X12, X19, X22, X24 and X31. The non-irradiated extracts showed the following IC50 values for cell viability (cytotoxicity): X4- 130?g/ml X19-20?g/ml, X22-10/ml and X24-60?g/ml. After UVA and UVB radiation, the extracts showed significant reduction in cell viability compared to the IC50 of the unirradiated extracts. Under UVB light, the X12 extracts (IC50 35?g/ml) and X31 (IC50 70mg/ml) maintained the same percentage of cell viability reduction when compared to the IC50 of the unirradiated extracts. However after exposure to UVA light, X12 extract increased the cell viability from 50% (when not irradiated) to 75% (irradiated). While X31 extract even after the UVA irradiation, remained the same 50% of reduction in cell viability. At this stage the selected extracts were X12 and X31. The absorption spectrum in the UV region obtained for X12 extract showed a decrease in absorbance of 28.3% under UVB and 60% under UVA radiation relative to non-irradiated extract. The X31extract showed a reduction in absorbance of 17.6% and 30% in UVA and UVB radiation, respectively, compared to non-irradiated extract. The selected fungi were identified by PCR, suggesting that X12 fungus is Aspergillus terreus and X31 is the Talaromyces pinophilus. Finally it was identified the active substance of X12 extract employing dereplication technique which makes use of coupled analytical instrumentation UHPLC-DAD- (ESI) HRMS associated to the Chapman and Hall\'s Dictionary of Natural Products (DNP) database. The majority compound of X12 extract was identified as the citreoviridin. Thus, the results of this study allowed us to establish a procedure for the selection of producers of UV absorbing compounds from fungi, which could be applied in obtaining new natural organic filters for sunscreens.

Photoprotection in Patients after Kidney Transplantation Author: Zuzana Hajšelová Tutor: doc. PharmDr. Josef Malý, PhD. Consultant: Mgr. Barbora Vaňková Department of Social and Clinical Pharmacy, Faculty of Pharmacy in Hradci Králové, Charles University Introduction and aims: Patients after kidney transplantation (KT) are in a higher risk of developing skin cancer as a result of lifelong immunosuppressive (IS) therapy. Thorough photoprotection is therefore recommended. The aim of this study was to determine patients' awareness and level of photoprotection and to analyse selected risk factors for skin cancer development in patients after KT. Methods: The cross-sectional study was undertaken from 15th April to 31st December 2019 at the Haemodialysis Centre in the Teaching Hospital Hradec Králové. Patients included in this study were above 18 years old, who visited Haemodialysis Centre in the defined period. The data was collected from the patients' medical documentation (e.g. occurrence of skin cancer and skin cancer type, current IS) and from the written version of questionnaires (e.g. phototype, sun exposure, photoprotection). The data was analysed using descriptive statistics. Results: There were 410 KT patients in regular care of the Haemodialysis Centre, 361 fulfilled the questionnaire (88.0...

El objetivo principal de esta tesis es contrastar el papel de dichos compuestos 1,3-dicarbonilicos como agentes que dañan el ADN con respecto a su potencial fotoprotector. Primero, 5,6-dihidropirimidinas han sido derivatizadas utilizando el grupo fotolábil t-Bu cetona con el fin de estudiar la generación de radicales en C5 en un medio no acuoso. Después, el estudio por fotólisis de destello láser en ACN de los derivados 1,3-dicarbonilicos diseñados da lugar a la detección de los supuestos radicales 5,6-dihidropirimidin-5-ilo. Su caracterización muestra especies transitorias de vida larga y están centrados a 400-420 nm o 350-400 nm para los derivados 5,6-dihidrouridina o 5,6-dihidrotimidina, respectivamente. Además, la generación de radicales también se ha evidenciado mediante experimentos de fluorescencia en estado estacionario mediante el uso de una sonda profluorescente (AAA-TEMPO) que atrapa el radical. Por lo tanto, la irradiación de los derivados fotolábiles del ácido nucleico en presencia de AAA-TEMPO produce un aumento de la emisión, de acuerdo con la captura del radical C5 por la sonda paramagnética. La formación del aducto se ha confirmado mediante UPLC-HRMS. Los datos experimentales se han corroborado con cálculos teóricos ab initio CASPT2 // CASSCF. Segundo, otro derivado 1,3-dicarbonílico de la pirimidina se ha investigado. De hecho, el daño 5-formiluracilo (ForU) presenta características interesantes como potencial agente fotosensibilizador intrínseco del ADN. Por lo tanto, los estudios espectroscópicos revelan que ForU tiene una absorción en el rango UVA/UVB y también presenta un estado triplete excitado (3ForU *) con un tiempo de vida algunos micros y con una ET suficientemente alta como para fotosensibilizar la formación de los conocidos dímeros de pirimidina de tipo ciclobutano (CPDs) a través de una transferencia de energía triplete-triplete. Este proceso ha sido confirmado por medio de la síntesis de díadas modelo Thy-Thy y Cyt-Cyt, ya que su irradiación en presencia de ForU ha demostrado que producen CPDs. Asimismo, el estudio en ADN plasmídico permitió establecer la capacidad de ForU para inducir roturas de cadena simple y CPDs. A continuación, se ha desarrollo una nueva estrategia para la fotoprotección de moléculas bioactivas aprovechando la reactividad fotoquímica del tautómero 1,3-dicetona de la avobenzona (AB), un filtro del UVA. Los compuestos seleccionados son dos fármacos antiinflamatorios no esteroideos de uso tópico con propiedades fotosensibilizantes, (S)-ketoprofeno (KP) y diclofenaco (DF). El tautómero dicetona de la AB contiene dos restos fenacilo, que es un grupo protector fotolábil muy establecido. Por lo tanto, un diseño juicioso de una díada profármaco/profiltro permite la fotoliberación del fármaco y de su protector, la AB. La viabilidad de esta liberación controlada de los ingredientes se verificó en diferentes disolventes con carácter dador de H y viscosidad para simular la formulación tópica. Además, los estudios de fotólisis de destello láser en EtOH permiten la caracterización de una especie transitoria a 400-420 nm, la cual ha sido asignada al estado excitado triplete de AB-KP. Finalmente, se ha evaluado la fotoseguridad de la díada fotoactivable AB-KP. Los espectros de absorción transitoria de la díada AB-KP en ciclohexano muestra que la especie observada es el estado excitado triplete del KP y no el de la AB en su forma dicetona. El impacto de la díada sobre la membrana celular se ha abordado mediante irradiación UVA de soluciones de ácido linoleico en presencia de AB-KP y su potencial fototóxico se ha evidenciado mediante espectrofotometría UV-Vis revelando la formación de derivados hidroperóxidos diénicos conjugados del ácido linoleico. Sin embargo, la diada AB-KP no exhibe un potencial fotogenotóxico como lo demuestran los experimentos del ensayo comet, donde a diferencia del KP, la forma redonda no / The main objective of this thesis is to contrast the role of these 1,3-dicarbonyl compounds as DNA damaging agents to their photoprotective potential. Firstly, 5,6-dihydropyrimidines have been derivatized using a tert-butyl ketone photolabile group in order to study the generation of C5-centered radicals in non aqueous media. Then, laser flash photolysis study in acetonitrile of the designed 1,3-dicarbonyl derivatives yields the formation of the purported 5,6-dihydropyrimidin-5-yl radicals. Their characterization shows long lived transient species, which do not decay in the µs range and are centered at 400-420 nm or 350-400 nm for the 5,6-dihydrouridine or 5,6-dihydrothymidine derivatives, respectively. Moreover, radical generation has also been evidenced by steady state fluorescence experiments by using a profluorescent radical trap (AAA-TEMPO). Thus, irradiation of the photolabile nucleic acid derivatives in the presence of AAA-TEMPO results in an increased emission, in agreement with the trapping of C5 radical by the paramagnetic probe. Formation of the resulting adduct has been confirmed by UPLC-HRMS. Experimental data have been corroborated with ab initio CASPT2//CASSCF theoretical calculations. In a second chapter, another 1,3-dicarbonyl derivative of pyrimidine has been investigated. Indeed, 5-formyluracil (ForU) presents interesting features as a potential intrinsic DNA photosensitizing agent. Thus, spectroscopic studies reveal that ForU has not only an absorption in the UVA/UVB range, but also a triplet excited state (3ForU*) with a lifetime of some micros and with an energy high enough to photosensitize the well-known cyclobutane pyrimidine dimers (CPDs) through triplet-triplet energy transfer. This process has been confirmed by means of the synthesis of model Thy-Thy and Cyt-Cyt dyads, which after irradiation in the presence of ForU have been demonstrated to produce CPDs. Finally, the study extended to plasmid DNA allows establishing the ability of ForU to produce single strand breaks and CPDs. Next, the attention has been focused on the development of a new strategy for photoprotection of bioactive molecules taking advantage of the photochemical reactivity of the 1,3-diketo tautomer of the UVA filter avobenzone (AB). The selected bioactive compounds are two photosensitive topical non steroidal anti-inflammatory drugs, (S)-ketoprofen (KP) and diclofenac (DF). In this context, the diketo tautomer of avobenzone contains two phenacyl moieties, which are well-known photoremovable protecting groups. Thus, a judicious design of a pro-drug/pro-filter dyad allows the photorelease of the drug and its protecting shield, avobenzone. The viability of this controlled release of the active ingredients was checked in different solvents of different H donating properties and viscosity to simulate topical formulation.Plus, laser flash photolysis studies in ethanol allow characterization of a transient absorption band at 400-420 nm assigned to the triplet excited state of the dyad by comparison with that of the diketo form of AB. Finally, the photosafety of the photoactivatable AB-KP dyad has been assessed. The transient absorption spectra obtained for AB-KP dyad in cyclohexane showed the triplet excited state of KP and not that of the AB in its diketo form. The impact on the cellular membrane has been addressed by UVA irradiation of linoleic acid solutions in the presence of the dyad. Phototoxic potential of the dyad has been evidenced by UV-Vis spectrophotometry through the formation of the conjugated dienic hydroperoxides derived from linoleic acid. However, AB-KP does not exhibit a photogenotoxic potential as demonstrated by comet assay experiments, where by contrast with KP, the non damaged round shape of the cell is still observed after UVA irradiation. / L'objectiu principal d'aquesta tesi és contrastar el paper d'aquests compostos 1,3-dicarbonil com a agents que danyen l'ADN respecte al seu potencial fotoprotector. En primer lloc, 5,6-dihidropirimidines han sigut derivatitzades utilitzant el grup fotolàbil t-Bu cetona amb la finalitat d'estudiar la generació de radicals centrats en C5 en un mitjà no aquós. Després, l'estudi de fotòlisi de flaix làser en acetonitril dels derivats 1,3-dicarbonil dissenyats produeix la formació dels suposats radicals 5,6-dihidropirimidin-5-il. La seua caracterització mostra espècies transitòries de vida llarga i estan centrats a 400-420 nm o 350-400 nm per als derivats 5,6-dihidrouridina o 5,6-dihidrotimidina, respectivament. Per tant, la irradiació dels derivats fotolàbils d'àcid nucleic en presència de AAA-TEMPO dóna com resultat un augment de l'emissió, d'acord amb la captura del radical C5 per la sonda paramagnètica. La formació del adducte resultant s'ha confirmat mitjançant UPLC-HRMS. Així mateix, les dades experimentals s'han corroborat amb càlculs teòrics ab initio CASPT2 // CASSCF. En un segon capítol, un altre derivat 1,3-dicarbonil de la pirimidina ha sigut investigat. De fet, el dany 5-formiluracil (ForU), presenta característiques interessants com a potencial fotosensibilitzador intrínsec de l'ADN. Per tant, els estudis espectroscòpics revelen que ForU té una absorció en el rang UVA/UVB i també presenta un estat triplet excitat (3ForU*) amb un temps de vida d'alguns micros i amb una ET prou alta com per a fotosensibilitzar la formació dels coneguts dímers de pirimidina de tipus ciclobutà (CPDs) a través d'una transferència d'energia triplet-triplet. Aquest procés ha sigut confirmat per mitjà de la síntesi de diades model Thy-Thy i Cyt-Cyt, que després de la irradiació en presència de ForU s'ha demostrat que produeixen CPDs. Finalment, l'estudi en ADN plasmídic ha permès establir la capacitat de ForU per a produir trencaments de cadena simple i CPDs. A continuació, s'ha desenvolupat una nova estratègia per a la fotoprotecció de molècules bioactives aprofitant la reactivitat fotoquímica del tautòmer 1,3-dicetona del filtre de l'UVA Avobenzone (AB). Els compostos seleccionats són dos fàrmacs antiinflamatoris no esteroïdals d'ús tòpic amb propietats fotosensibilizants, (S)-ketoprofè (KP) i diclofenac (DF). En aquest context, el tautòmer dicetona de l'AB conté dues fraccions fenacil, que es un grup protector fotolàbil ben conegut. Per tant, un disseny judiciós d'una diada profàrmac / profiltre permet el fotoalliberament del fàrmac i del seu escut protector, l'AB. La viabilitat d'aquest alliberament controlat dels ingredients actius s'ha verificat en diferents dissolvents de diferent caràcter dador d'hidrogen i viscositat per a simular la formulació tòpica. A més, els estudis de fotòlisi de flaix làser en EtOH permeten la caracterització d'una banda d'absorció transitòria a 400-420 nm, la qual ha sigut assignada a l'estat excitat triplet de AB-KP. Finalment, s'ha avaluat la fotoseguretat de la diada fotoactivable AB-KP. Els espectres d'absorció transitòria de la diada AB-KP en ciclohexà mostres que l'espècie observada és l'estat excitat triplet del KP i no el de la AB en la seua forma dicetònica. L'impacte sobre la membrana cel·lular s'ha abordat mitjançant la irradiació UVA de solucions d'àcid linoleic en presència de AB-KP. El potencial fototòxic de la diada s'ha evidenciat mitjançant espectrofotometria UV-Vis revelant la formació de derivats hidroperòxids diènics conjugats de l'àcid linoleic. No obstant açò, la diada AB-KP no exhibeix un potencial fotogenotòxic com ho demostren els experiments de l'assaig comet, on a diferència del KP, la forma redona no danyada de la cèl·lula encara s'observa després de la irradiació UVA. / Aparici Espert, MI. (2018). Photochemistry of 1,3-Dicarbonyl Compounds: DNA Photodamage vs. Photoprotection [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/105782

No / Ultraviolet radiation (UVR) in sunlight has deleterious effects on skin, while behavioural changes have resulted in people gaining more sun exposure. The clinical impact includes a year-on-year increase in skin cancer incidence, and topical sunscreens alone provide an inadequate measure to combat overexposure to UVR. Novel methods of photoprotection are being targeted as additional measures, with growing interest in the potential for systemic photoprotection through naturally sourced nutrients. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are promising candidates, showing potential to protect the skin from UVR injury through a range of mechanisms. In this review, we discuss the biological actions of n-3 PUFA in the context of skin protection from acute and chronic UVR overexposure and describe how emerging new technologies such as nutrigenomics and lipidomics assist our understanding of the contribution of such nutrients to skin health.

Rhododendron maximum L. is a dominant subcanopy species in the southern Appalachian Mountains. R. maximum undergo distinct thermonastic leaf movements (TLM). The purpose of these movements has not yet been determined. Previous studies have suggested TLM are a photoprotective mechanism for the dynamic light environment of the subcanopy in a deciduous forest during winter. The present study aimed to determine the effects of restricting TLM on photoinhibition, net photosynthesis, and other gas exchange parameters, particularly during the early spring. After restricting TLM on certain leaves, we observed the above parameters from autumn 2005 to late spring 2006. Our results indicated that photoinhibition increased (lower Fv/Fm) in treatment leaves over reference leaves throughout the winter. The difference became greater during the early spring, when reference leaves began to return to normal levels of photochemical efficiency and treatment leaves sustained low Fv/Fm. Net photosynthesis was lower for treatment leaves than reference leaves. This became most significant during the early spring, when maximum carbon gain is possible. Finally, gas exchange parameters as measured by light and CO2 response curves did not indicate any significant difference between treatment and reference leaves post canopy closure. Out results suggest that TLM are an important mechanism for photoprotection, allowing leaves of R. maximum to recover quickly during the early spring and maximize their early spring carbon gain. / Master of Science

Skin is daily exposed to sun radiation. Among them, UVA reach the basal layer of epidermis composed of keratinocyte stem cells (KSC) and progenitors commonly called transitory amplifying cells (TA). KSC and TA, responsible of the epidermal renewal, are sensitive to genotoxic agents and more particularly to UV. Indeed, KSC, usually quiescent but composing the stem cell pool all lifelong, as well as TA, are specific targets for photocarcinogenesis and photoageing. In this context, the aim of the project was to develop a method able to isolate KSC and TA for characterizing their response to UVA, and in a more industrial objective, to value a photoprotective and/or genoprotective ingredient by investigating their mechanisms of action. A parallel aim was to define culture conditions suitable for maintaining the stemness of KSC in culture, which is quickly lost from their enter into a proliferative state. Thus we showed that adjunction of adipose derived stem cells (ASC) to fibroblasts in dermis of a skin equivalent model (SE) in order to reproduce the physiological environment of KSC, significantly increases the thickness of the epidermis and preserves the keratinocytes from their senescence. ASC act partially via the increase of fibroblasts proliferation in dermis and potentially via a synergic effect of factors secreted by the combination of ASC and fibroblasts. To compare the behavior of KSC to the one of TA, we firstly optimized the rapid adhesion method ; then compared it to the cell sorting by flow cytometry following the alpha6high/CD71low phenotype, which appeared more efficient. Thus, KSC (alpha6high/CD71low) and TA (alpha6high/CD71high) were then irradiated to UVA. KSC showed a photoresistance compared to TA with a better cell viability and a clonogenic potential superior as well as a better ability to reconstruct a pluristratified epidermis in vitro. We also investigated mechanisms of resistance. Our results demonstrate that the induction of the three types of DNA damage immediately induced by UVA is similar in both populations, but that the repair of single strand breaks (SSB) and of thymin dimers (CPDs) is faster for KSC. Finally, PE1, ingredient preselected by Gattefossé was characterized for its photoprotective and genoprotective effect. We showed that PE1 is able to i) preserve capacity of keratinocytes to form holoclones after UVA radiation, ii) decrease DNA damage, notably 8oxoGuanin and CPDs, and iii) improve several repair genes expression and activities. To conclude, this thesis project showed for the first time that KSC are more resistant to UVA radiation than their direct progeny, TA notably via improved DNA repair systems. Moreover, it allowed to identify a plant extract (PE1) able to protect genome of proliferative keratinocytes to UVA radiation / La peau est quotidiennement exposée aux rayons UV du soleil dont les UVA qui atteignent la couche basale de l'épiderme, composée de cellules souches kératinocytaires (KSC) et de cellules progénitrices appelées communément les cellules d'amplification transitoire (TA). Les KSC et TA, responsables du renouvellement de l'épiderme, sont vulnérables à l'action des agents génotoxiques et plus particulièrement aux rayonnements UV. En effet, Les KSC normalement quiescentes mais constituant la réserve de cellules souches tout au long de la vie de l'organisme, comme les TA, sont des cibles préférentielles pour la photocarcingénèse et le photovieillissement cutanés. Dans ce contexte, le but du projet était de développer une méthode capable d'isoler les KSC et les TA afin de caractériser leur réponse biologique vis-à-vis des UVA et dans un objectif plus industriel, de valoriser un actif photoprotecteur et/ou génoprotecteur par l'investigation de ses mécanismes d'action. Un objectif parallèle était de définir des conditions de culture optimales pour garder le phénotype souche des KSC en culture, qui est rapidement perdu dès leur entrée en prolifération. Ainsi, nous avons montré que l'ajout des cellules souches adipeuses (ASC) aux fibroblastes d'une peau reconstruite (PR), pour reproduire l'environnement des KSC, augmente significativement l'épaisseur de l'épiderme et surtout préserve les kératinocytes de leur entrée en sénescence, en partie par l'augmentation de la prolifération des fibroblastes au niveau du derme et potentiellement par un effet synergique des facteurs solubles sécrétés par la combinaison des ASC/fibroblastes. Afin de comparer le comportement des KSC à celui des TA nous avons donc tout d'abord optimisé la méthode d'adhésion rapide, puis l'avons comparée au tri par cytométrie de flux selon le phénotype alpha6high/CD71low, qui s'est avérée plus efficace. Les KSC (alpha6high/CD71low) et les TA (alpha6high/CD71high) ont ensuite été irradiés aux UVA. Les KSC ont montré une photorésistante plus importante que les TA avec une viabilité cellulaire et un potentiel clonogénique supérieurs ainsi qu'une meilleure capacité à reconstruire un épiderme pluristratifié in vitro. Nous avons aussi recherché les mécanismes de résistance. Nos résultats démontrent que l'induction des 3 types de dommages à l'ADN immédiatement après irradiation est identique pour les deux populations mais que la réparation des cassures simple brin (SSB) et des dimères de pyrimidine (CPDs) est plus rapide pour les KSC. Enfin, PE1, actif préselectionné par Gattefossé, a été caractérisé pour son effet photoprotecteur et génoprotecteur. Nous avons montré que PE1 est capable de (i) préserver la capacité des kératinocytes à former des holoclones après irradiation, (ii) diminuer les lésions à l'ADN, notamment la 8oxoGuanine et les CPDs (iii) améliorer l'expression de plusieurs gènes et les activités de réparation de l'ADN. Pour conclure, ce travail de thèse a montré pour la première fois que les KSC (alpha6high/CD71low), sont plus résistantes vis-à-vis des UVA que les TA (alpha6high/CD71high) notamment grâce à des systèmes de réparation de l'ADN plus actifs dans les KSC, et a permis d'identifier un extrait végétal (PE1) capable de protéger le génome des kératinocytes proliférants contre les UVA

Un criblage phytochimique à partir de quatre lichens saxicoles hydrophiles du genre Dermatocarpon a mis en évidence la présence inattendue de composés de type mycosporines dans ces chlorolichens. Leur présence a été confirmée dans des échantillons historiques d'herbiers couvrant une période de plus de 170 ans. Leur teneur était comprise entre 0,1 et 1,4 mg/g de lichen sec. Trois mycosporines ont été isolées de Dermatocarpon luridum collecté en Bretagne sur des rochers d'eau vive: deux mycosporines natives, la mycosporine glutaminol et la mycosporine glutamicol ainsi qu'un artéfact d'isolement, l'ester éthylique de la mycosporine glutamicol. Leurs propriétés physico-chimiques incluant le calcul de leur pKa ont été déterminées. L'étude phytochimique a également conduit à l'isolement de sept autres composés dont un acide aminé non protéique, un dipeptide et un céramide identifiés pour la première fois dans un lichen et le cérévistérol jusqu'ici non décrit dans D. luridum. Les activités cytotoxiques et photoprotectrives des composés disponibles en quantité suffisante ont été évaluées. Le cérévistérol a montré une cytotoxicité modérée sur huit lignées cellulaires contrairement aux cinq autres composés. Les propriétés photoprotectrices et activités antioxydantes de l'ester éthylique de la mycosporine glutamicol et d'un extrait aqueux semi-purifié de D. miniatum présentent un intérêt pour un possible développement comme actif solaire. / A screening of mycosporine-like compounds in four hydrophilic Dermatocarpon lichen species has highlighted the unexpected occurrence of such metabolites in chlorolichens. These compounds were confirmed and quantified in historical herbarium samples (15-50 mg) even more than 170 years old. Total mycosporine contents ranged from 0.1 to 1.4 mg/g of dry lichens. Two genuine mycosporines (mycosporine glutaminol and the more stable mycosporine glutamicol) with an artifact mycosporine glutamicol ethyl ester were isolated from Dermatocarpon luridum collected in a breton river. Their physico-chemical properties were also determined including pKa. The phytochemical studies led to the isolation of seven additional compounds in which a non-protein amino acid, a dipeptide and a ceramide were identified for the first time in lichens and cerevisterol for the first time in D. luridum. Compounds obtained in suitable quantity were evaluated for their cytotoxic and photoprotective properties. Except a moderate cytotoxicity of cerevisterol, the five tested compounds were not found toxic on the eight cell lines. Photoprotective properties of the mycosporine glutamicol ethyl ester and a semi-purified aqueous extract of Dermatocarpon miniatum along with some antioxidant activities are promising to develop new sunscreens.

A exposição excessiva à radiação Ultravioleta (UV) resulta em manifestações clínicas à pele humana como queimaduras, fotoenvelhecimento e câncer. A radiação UVA, preferencialmente, induz à formação de espécies reativas de oxigênio, enquanto que a radiação UVB é absorvida diretamente pelo DNA. Apesar de mecanismos endógenos auxiliarem na prevenção/reparação dos danos causados pela radiação UV, quando o dano excede a capacidade de reparação celular, diversos efeitos lesivos ocorrem na pele como alterações da matriz dérmica, resposta inflamatória e desidratação do estrato córneo. O uso de compostos fenólicos com atividade antioxidante pode auxiliar na prevenção das consequências patológicas da exposição à radiação UV. O presente trabalho teve como objetivo estudar em cultura de células da pele (HaCaT -queratinócito humano imortalizado e FHPD - fibroblasto humano primário dermal) exposta às radiações UVA e UVB a atividade fotoprotetora de 3 compostos fenólicos, ácido cafeico (AC), clorogênico (ACG) e rosmarínico (AR). Inicialmente, células HaCaT e FHPD cultivadas em monocamada foram expostas às doses crescentes de radiação UVA ou UVB e, após 24 horas, foram analisadas quanto a viabilidade, marcadores de morte celular, mediadores inflamatórios, presença de aquaporina e lesões de DNA. HaCaT quando exposta às radiações UVA e UVB são conduzidas à morte por apoptose, com aumento de Caspases 3 e 9, p53 e redução de PARP. Após a exposição à radiação UVA, HaCaT responde com aumento na liberação de IL-6, TNF-α e COX-2, internalização/redução de AQP3 da membrana, redução na liberação de MMP-2 e 9, aumento na liberação de MMP-1 e na produção de ERO. Quando expostos à radiação UVB, HaCaT aumenta a liberação de IL-6 e COX-2, promove internalização/redução de AQP3 na membrana e redução na liberação de MMP-2 e 9. FHPD são menos sensíveis à exposição a ambas as radiações, mostrando redução de viabilidade com parada de ciclo apenas frente à radiação UVA. Além disto, FHPD exposto a radiação UVA responde com aumento na liberação de IL-6 e danos no DNA do tipo 8-oxo-dG. Dentre os compostos, o ACG apresentou melhor atividade fotoquimioprotetora perante ambas as radiações UVA e UVB, pois foi capaz de reverter em HaCaT a morte celular induzida por ambas as radiações e de reverter a parada de ciclo em FHPD expostos à radiação UVA. HaCaT tratado com ACG e exposto à radiação UVA responde com aumento na expressão de AQP3 e PARP, aumento na expressão gênica de AQP3, redução na expressão gênica de CDKN1A e na liberação de MMP-1, 2 e 9. Após a radiação UVB, o tratamento com ACG aumenta a expressão gênica de AQP3, reduz a expressão gênica de CDKN1A, reduz a produção de COX-2 e aumenta a liberação de MMP-2 e 9. O tratamento com o AR apresentou atividade fotoquimioprotetora frente à radiação UVA, com HaCaT respondendo a radiação com aumento na população de células viáveis, aumento na expressão de AQP3 e PARP e na expressão gênica de AQP3, redução na liberação de MMP-1 e 9 e redução na produção de COX-2. FHPD tratados com AR apresentaram aumento na população em fase G1, na expressão de p21, e redução de danos de DNA tipo 8-oxo-dG. O tratamento de HaCaT com AC foi capaz de reverter a morte celular, aumentar a expressão de p53 e aumentar a liberação de MMP-2 e 9 frente à radiação UVB e de reduzir a produção de ERO, a expressão de p21 e a liberação de MMP-1, 2 e 9 frente à radiação UVA. Para FHPD, o tratamento com AC foi capaz apenas de reduzir a formação de danos de DNA tipo 8-oxo-dG. Os resultados indicam que o modelo proposto foi capaz de discriminar a atividade fotoprotetora dos compostos frente à radiação UVA e UVB. Além disto, foi possível demonstrar que os compostos antioxidantes se comportam de maneira distinta enquanto fotoprotetores no modelo empregado. / Excessive exposure to Ultraviolet radiation (UV) results in clinical manifestations in human skin such as burns, photo-aging and cancer. UVA radiation preferentially induces formation of reactive oxygen species, while UVB radiation is absorbed directly by the DNA. Although endogenous mechanisms are able to prevent/repair cellular damages caused by UV radiation, excess cellular damage retains cells repair capacity and also results on diverse harmful effects on skin, such as, changes in the dermal matrix, inflammatory response and dehydration of the stratum corneum. The use of phenolic compounds with antioxidant activity may help preventing pathological conditions caused by UV radiation. This work aimed to study the photoprotective activity of three phenolic compounds, caffeic (CA), chlorogenic (CGA) and rosmarinic acid (RA) in human skin cells (HaCaT - immortalized human keratinocytes and HDSF - human dermal skin fibroblast) exposed to UVA and UVB radiation. Initially, HDSF and HaCaT cells were exposed to increasing doses of UVA and UVB radiation. After 24 hours of exposure, we evaluated cell viability, cell death, inflammatory mediators, aquaporin and DNA damage. Exposure to UVA and UVB radiation in HaCaT cells results on apoptotic cell death, with an increase of caspases 3 and 9, p53 and reduction of PARP. HaCaT cells when exposed to UVA radiation resulted on increased levels of IL-6, TNF-α and COX-2, internalization of the membrane AQP3, reduction of MMP-2 and MMP-9 release, increase of MMP-1 and ROS production. After UVB radiation, HaCaT cells resulted on an increase of IL-6 and COX-2 production, it also promoted internalization of membrane AQP3 and reduced release of MMP-2 and 9. HDSF were less sensitive to both radiations. Moreover, HDSF resulted in cell viability decrease and cell cycle arrest only after UVA radiation. Furthermore, HDSF when exposed to UVA radiation resulted on an increase of IL-6 production and in DNA damage (8-oxo-dG). Among the studied compounds, CGA presented better photochemiprotective activity towards UVA and UVB radiation. Also, this compound was able to reverse cell death in HaCaT after exposure to both radiations and inhibited cell cycle arrest in HDSF after UVA radiation exposure. HaCaT cells treated with CGA and exposed to UVA radiation resulted on an increase in AQP3 and PARP expression, increased in AQP3 gene expression, reduction in CDKN1A gene expression and reduction in MMP-1, 2 and 9 release. After UVB radiation, GCA treatment increases AQP3 gene expression, reduces CDKN1A gene expression, reduces COX-2 production and increase MMP-2 and 9 releases. The AR treatment showed photochemiprotective activity towards the effects of UVA radiation, with HaCaT responding with an increase on cells viability, increased in PARP and AQP3 expression and in AQP3 gene expression, decreased MMP-1 and 9 releases and reduced COX-2c production. HDSF when treated with AR showed an increase in G1 phase population, in p21 expression and reduced DNA damage-type 8-oxo-dG. HaCaT cells treated with AC reversed cell death, increased p53 expression and increased MMP-2 and 9 releases after UVB radiation and reduced ROS production, p21 expression and MMP -1, 2, 9 release after UVA radiation. HDSF treated with AC was only able to reduce the formation of 8-oxodG DNA damage. These results indicated that the proposed model was able to discriminate the photochemiprotective activity of the studied compounds against the UVA and UVB radiation. In addition, it was demonstrated that the each studied antioxidant have different photoprotective mode of action.