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A necessidade de proteger a pele contra os raios ultravioleta (UV) é imprescindível, tendo em vista os efeitos deletérios gerados pelos mesmos. Compostos convencionais com atividade fotoprotetora sofrem interações, instabilidade e em sua maioria protegem contra os raios UVB. Poucos compostos apresentam proteção contra os raios UVA. No ambiente marinho, como nas algas marinhas, adversidades do ambiente relacionadas, principalmente, a exposição solar, aumentam as defesas naturais contra os raios UV por meio da produção de metabólitos secundários que podem absorver/refletir os raios solares, ou agir como antioxidantes. Tais moléculas podem ser produzidas pelas próprias algas ou pelos fungos endofíticos associados a elas. Assim, o presente estudo tem como objetivo investigar o potencial fotoprotetor de extratos, frações e substâncias isoladas dos fungos endofíticos Xylaria sp e Annulohypoxylon stygium, associados à alga vermelha Bostrychia radicans. Bem como de quatro espécies de algas da Antártica, Palmaria decipiens, Monostroma hariotii, Desmarestia anceps e a Gigartina skottsbergii. As algas da Antártica foram disponibilizadas pelo grupo do Prof. Dr. Pio Colepicolo Neto do IQ-USP e o cultivo dos fungos, a extração e o isolamento foram desenvolvidos em parceria com o Laboratório de Química Orgânica do Ambiente Marinho - NPPNS da FCFRP-USP. Os extratos e frações obtidos foram submetidos à análise dos espectros de absorção no UV e à fotodegradação. A seguir, os extratos e frações mais promissoras foram submetidos à avaliação da fototoxicidade em cultura de fibroblastos 3T3 para a determinação da viabilidade celular na presença e ausência da radiação, de acordo com o protocolo OECD TG 432. O fungo A. stygium apresentou frações com absorção no UVB, duas subfrações se mostraram não cito/fototóxicas e são provenientes de frações fotoestáveis. Destas subfrações, duas substâncias, ambas inéditas, foram isoladas e elucidadas por RMN H1, 1D, 2D, IV, ESI, e estas foram consideradas potenciais ativos para fotoproteção no UVB. O fungo Xylaria sp apresentou frações com absorção na região do UV, porém apenas uma, com absorção no UVB se mostrou fotoestável. Esta mesma fração foi considerada cito e fototóxica. Não foi possível identificar as duas substâncias isoladas pela baixa massa. Entretanto, um metabólito já isolado dessa linhagem anteriormente, o ácido gentísico, foi considerado não fototóxico, devendo ser melhor investigado quanto ao seu potencial como filtro biológico. Quanto às macroalgas antárticas, três espécies apresentaram absorção no UV. Apesar de a maioria dos extratos terem sido considerados fotoinstáveis, o extrato da alga D. anceps, de maior rendimento, apresentou frações com ampla absorção no UVA/VIS, no entanto elevada citotoxicidade. Desta fração foi isolada e identificada a fucoxantina (RMN H1 1D e 2D, ESI), um carotenoide marinho que apresentou potencial fototóxico no modelo monocamadas, mas não foi considerada citotóxica. Sendo assim, este pode ser considerado um promissor candidato a ativo cosmético, pelas suas propriedades antioxidantes e de filtro biológico, pois devido a sua alta massa molecular, 658.90 g/mol, pode-se inferir baixa permeação nas camadas viáveis da pele e ausência de fototoxicidade in vivo. Nesse sentido, novos estudos em modelo de pele 3D deverão ser realizados a fim de se comprovar a segurança de uso tópico da fucoxantina. As espécies também devem ser investigadas para outras atividades biológicas uma vez que este material pertence a uma região pouco estudada e estas podem apresentar potencial para os mais diversos empregos farmacológicos ou cosméticos. / The need to protect the skin against ultraviolet (UV) radiation is essential, due to the deleterious effects caused by them. Conventional UV-filters undergo chemical interactions, instability and mostly protect against UVB rays. Few compounds have protection against UVA rays. In the marine environment, especially in marine algae, adversities, mainly related to sun exposure, increases the natural defenses against UV radiation through the production of secondary metabolites that can absorb/reflect the UV radiation, or even act as antioxidants. Such molecules may be produced by algae themselves or by the endophytic fungi associated with them. The present study aims to investigate the potential of new sunscreen extracts, fractions and isolated compounds from endophytic fungi Xylaria sp and Annulohypoxylon stygium, associated with red algae Bostrychia radicans, and four species of algae from Antarctica: Palmaria decipiens, Monostroma hariotii, Desmarestia anceps and Gigartina skottsbergii. The algae material from Antarctica were given by the group of Prof. Dr. Pio Colepicolo Neto, IQ-USP, and the fungi cultivation, extractions and the compounds isolation were obtained in partnership with the Laboratory of Organic Chemistry of the Marine Environment - NPPNS of FCFRP-USP. The extracts and fractions obtained were submitted to analysis of the absorption spectra in the UV and to photodegradation. After that, the most promising extracts and fractions were submitted to the assessment of phototoxicity in 3T3 fibroblasts in culture for determination of cell viability in the presence and absence of radiation, in accordance with the OECD TG 432 protocol. The fungus A. stygium showed fractions with UVB absorption, from there were isolated two novel compounds and had the structure elucidated (RMN H1 1D/2D; IV; ESI). These compounds were considered potential ingredients for photoprotection on the UVB range. The fungus Xylaria sp. showed fractions with absorption on the UV, but only one of them, with UVB absorption, was considered photostable. The same fraction was also considered cyto/ phototoxic. It was not possible identify two of the compounds isolated due its low weight. Therefore, the gentisic acid, a metabolite already isolated from this fungus, was not considered phototoxic, what suggests that it should be better investigated as a potential biological UV filter. Regarding the seaweeds, three species have shown absorption in UV region. Most of the extracts were considered photounstable, but the D. anceps extract, of highest yield, showed fractions with large UVA/VIS absorption, but higher cytotoxic potential. Fucoxanthin, a marine carotenoid, was isolated from this fraction and identified by NMR H1 1D/2D, ESI. This compound showed phototoxic potential in a monolayer model, but it was not considered cytotoxic. Therefore, fucoxanthin is a promising candidate to a cosmetic ingredient, once it has antioxidant and biological UV filter properties and its high molecular weight, 658.90 g/mol, suggests low skin permeation into viable epidermis and absence of in vivo phototoxicity. Thus, more studies using 3D skin model should be performed in order to prove the safety of the fucoxanthin topical use. The species should be also investigated for other biological activities once this material belongs to a poorly studied region and can show potential for many of the pharmacological/cosmetics activities.

L’hygiène des mains (HDM) est déterminante dans la prévention du risque infectieux associé aux soins. Les pratiques actuelles privilégient l’utilisation de produits hydro-alcooliques (PHA), généralement formulés avec de l’éthanol, de l’isopropanol et/ou du n-propanol, en présence de co-formulants pour une meilleure acceptabilité cutanée. L’efficacité antimicrobienne sur un temps court, nécessaire en raison des situations fréquentes de pratique d’HDM, est atteinte par des teneurs élevées en alcools pouvant excéder 80 % p/p. Cela soulève la question de la toxicité cutanée et respiratoire associée à l’utilisation des PHA. L’évaluation de la toxicité cutanée basée sur les données publiées et des essais in vitro d’irritation cutanée (OCDE 439) et de phototoxicité (OCDE 432) conclut à l’absence d’irritation cutanée aiguë et de phototoxicité en relation avec l’exposition cutanée à ces alcools, y compris en présence de co-formulants, tels que fournis dans les PHA. Il est ressorti de nos essais le possible manque de spécificité, déjà décrit dans la littérature, des modèles d’épidermes humains reconstitués (RhE) vis-à-vis de certaines substances, qu’il convient de garder à l’esprit dans le cadre de l’évaluation de l’irritation cutanée in vitro. Des investigations complémentaires et une évaluation par l’approche Weight of Evidence peuvent être utiles avant de conclure aux propriétés irritantes d’un item d’essai. En termes de génotoxicité, une différence entre les alcools ressort de la revue bibliographique, avec des propriétés génotoxiques décrites uniquement pour l’éthanol. Dans une certaine mesure, nos essais ont confirmé une différence de profil. L’isopropanol et le n-propanol testés dans une batterie de tests in vitro permettant d’appréhender les différents mécanismes génotoxiques (test d’Ames et test du micronoyau (MN) sur cellules humaines p53 compétentes : cellules lymphoblastoïdes TK6 et cellules pulmonaires NCI H292) ont donné des résultats négatifs, y compris lorsqu’ils étaient formulés avec des co-formulants, ou administrés sous forme de vapeurs sur les cellules NCI H292 cultivées en interface air-liquide (IAL). Pour l’éthanol, la réalisation de la même batterie de tests a conduit à des résultats équivoques uniquement dans le test du MN sur cellules TK6 avec l’éthanol seul. Un test du MN supplémentaire sur cellules TK6 en co-culture avec un RhE mimant la barrière cutanée a donné des résultats négatifs. Par ailleurs, aucune exposition systémique significative aux alcools induite par les pratiques d’HDM ne ressort des études publiées chez l’homme, avec des taux indiscernables des valeurs endogènes existantes pour l’éthanol et l’isopropanol. L’ensemble de ces données est en faveur de l’absence de risque génotoxique systémique consécutif à l’utilisation des PHA, et de l’absence de génotoxicité pulmonaire locale liée à l’exposition aux vapeurs d’alcools. En conclusion, en situation d’utilisation des PHA pour l’HDM, aucun risque pour la santé humaine en termes d’irritation cutanée aiguë, de phototoxicité et de génotoxicité ne ressort de ce travail de recherche. / Hand hygiene (HH) is a key factor in preventing healthcare-associated infections. Current practices favor the use of alcohol-based hand rubs (AbHR), generally formulated with ethanol, propan-2-ol and/or propan-1-ol, in the presence of co-formulants for a better skin acceptability. The antimicrobial efficiency within a short time, essential because of the frequent situations of HH practice, is achieved by high levels of alcohols which can exceed 80% w/w. This raises the question of dermal and respiratory toxicity associated with the use of AbHR. The assessment of dermal toxicity based on published data and in vitro skin irritation (OECD 439) and phototoxicity tests (OECD 432) conclude to non acute dermal irritation and phototoxicity risk linked to dermal exposure to these alcohols, even in the presence of co-formulants, as provided in the AbHR. We encountered in our trials the possible lack of specificity, already described in the literature, of the reconstructed human epidermis (RhE) models for some substances, which should be kept in mind in the context of the evaluation of skin irritation in vitro. Additional investigations and an assessment using the Weight of Evidence approach may be useful before concluding the irritant properties of a test item. In terms of genotoxicity, a difference between the alcohols emerges from the bibliographic review, with genotoxic properties described only for ethanol. To a certain extent, our tests confirmed a difference in profile. Propan-2-ol and propan-1-ol tested in a battery of in vitro tests to explore the various genotoxic mechanisms (Ames test and micronucleus test (MN) on p53 competent human cells: lymphoblastoid cells TK6 and pulmonary cells NCI H292) gave negative results, even in the presence of co-formulants, or administered as vapors on air-liquid interface (ALI) NCI H292 cells. For ethanol, the same battery of tests gave equivocal results only in the MN test on TK6 cells with ethanol alone. An additional MN test on TK6 cells co-cultured with a RhE mimicking the existing skin barrier gave negative results. In addition, no significant systemic exposure to alcohols induced by HH practices is apparent from published studies in humans, with indiscernible levels of existing endogenous values for ethanol and isopropanol. All of these data support the absence of an increased systemic genotoxic risk resulting from the use of AbHR and the absence of local pulmonary genotoxicity due to exposure to alcohol vapors. In conclusion, during AbHR use for HH, no risk to human health in terms of acute skin irritation, phototoxicity and genotoxicity is apparent from this research.

Alpine zooplankton are exposed to a variety of stressors in their natural environment including ultraviolet radiation. Physiological coping mechanisms such as the accumulation of photoprotective compounds provide these zooplankton protection from many of these stressors. Elevated levels of carotenoid compounds such as astaxanthin have been shown to help zooplankton survive longer when exposed to ultraviolet radiation presumably due to the strong antioxidant properties of carotenoid compounds. This antioxidant capacity is important because it may ameliorate natural and anthropogenic stressor-induced oxidative stress. While previous researchers have shown carotenoid compounds impart increased resistance to ultraviolet radiation in populations of zooplankton, little work has focused on the toxicological implications of PAH induced phototoxicity on zooplankton containing high levels of carotenoid compounds. This thesis discusses research studying the role that carotenoid compounds play in reducing PAH induced phototoxicity. By sampling different lakes at elevations ranging from 9,500' to 12,700' in the front range of the Colorado Rocky Mountains, copepod populations containing different levels of carotenoid compounds were obtained. These populations were then challenged with fluoranthene and ultraviolet radiation. Results discussed include differences in survival and levels of lipid peroxidation among populations exhibiting different levels of carotenoid compounds, and the toxicological and ecological implications of these results.

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants potentiellement mutagènes et cancérigènes. Leur transfert de l’atmosphère vers les écosystèmes, notamment vers les plantes, conditionne leur entrée dans les chaines alimentaires mais les modalités de ce transfert restent encore mal connues. L’objectif de ce travail était donc de caractériser le transfert et d’identifier les effets biologiques des HAP atmosphériques sur un système sol-plante-microorganismes symbiotiques.Un dispositif expérimental a été conçu afin de recréer en laboratoire une pollution atmosphérique par les HAP avec comme HAP modèle le phénanthrène (PHE). Le dispositif a fait l’objet d’une validation et d’une calibration élaborée de manière originale par une double approche mêlant l’expérimental à la simulation mathématique. Les niveaux d’exposition en polluant (150 µg m-3), contrôlés par des échantillonneurs passifs, se sont avérés pertinents par rapport aux conditions in situ. Ce dispositif a donc été utilisé pour exposer durant un mois des microsystèmes sol-plante-microorganismes au PHE par voie atmosphérique.Les différentes études ont mis en évidence un transfert du PHE depuis l’atmosphère vers tous les compartiments du microsystème, avec une accumulation majeure vers les feuilles de trèfle ou de ray-grass (respectivement 170 et 70 µg g־ ¹MS) et un transfert phloémien vers les racines est suggéré. Chez le trèfle, la mycorhization n’a pas été affectée, tandis que le nombre de nodules actifs a diminué de manière significative. Contrairement aux racines, la biomasse aérienne du trèfle a été significativement affectée (environ – 25%) par l’exposition au PHE atmosphérique, suggérant un impact sur le métabolisme carboné de la plante. Une expérience de marquage des trèfles au ¹³C-CO2 a effectivement montré un impact négatif du PHE atmosphérique sur la croissance, l’allocation de biomasse et l’allocation carbonée. Pour conclure, ces études ont permis non seulement de caractériser les effets biologiques et physiologiques des HAP atmosphériques sur les végétaux mais également de proposer l’utilisation du potentiel mycorhizien comme indicateur de pollution atmosphérique par les HAP. / Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants potentially mutagenic and carcinogenic. Transfer from the atmosphere to ecosystems, especially to plants, conditioning their entry into food chains, but the terms of this transfer are still poorly understood. The aim of this study was to characterize the transfer and identify the biological effects of atmospheric PAHs on soil-plant-symbiotic microorganisms.An experimental device was designed to recreate in the laboratory air pollution with phenanthrene (PHE) as a model PAH. The device was been validated and a calibration developed in an original way by a dual approach combining the experimental mathematical simulation. The levels of exposure to pollutant (150 mg m־³), controlled by passive samplers, were relevant with field conditions. This device has been used to expose a month of the soil-plant micro-organisms in the PHE through the air.Various studies have demonstrated a transfer of PHE from the atmosphere to all compartments of the microsystem, with a major accumulation to leaves in clover or ryegrass (respectively 170 and 70 µg g ־¹ dry weight) and a phloemic transfer to the roots is suggested. In clover, mycorrhization was not affected, while the number of active nodules decreased significantly. Unlike roots, aboveground biomass of clover was significantly affected (approximately – 25%) by exposure to air PHE, suggesting an impact on the carbon metabolism of the plant. A labelling experiment with ¹³C- CO2 in clover has actually shown a negative impact of PHE air on growth, biomass and carbon allocation.In conclusion, these studies have not only characterized the biological and physiological effects of atmospheric PAHs on plants but also proposed the use of mycorrhizal potential as an indicator of air pollution by PAHs.

[ES] El cáncer es una de las principales causas de muerte a nivel mundial. Los tratamientos anticancerígenos generalmente usados tienen diversos efectos secundarios producidos por su baja especificidad. Esta es una de las razones por las que se sigue en continua búsqueda de nuevos tratamientos. Dentro de estas nuevas investigaciones se encuentra el extenso campo de la nanomedicina, es decir, el estudio de nuevos materiales a escala nanométrica. Esta permite reducir dichos efectos secundarios aumentando la selectividad y especificidad de los tratamientos. Dentro de los nanomateriales se encuentran las nanopartículas de upconversion que son capaces de absorber luz en el infrarrojo cercano y emitirla en la región ultravioleta-visible. Por otro lado, desde el principio de la historia de la medicina la luz se ha empleado como forma de tratamiento teniendo un rol muy importante. Un inconveniente para dichos tratamientos suele ser la necesidad de emplear luz de la región ultravioleta-visible, pues las biomoléculas son capaces de absorber y produce daño celular. En este contexto, la presente Tesis Doctoral se centra en el estudio de nuevas formas de tratamiento anticancerígeno combinando nanomedicina y luz. Para ello se han desarrollado nuevos fármacos fototóxicos y nuevos materiales capaces de ser activados mediante luz infrarroja cercana. En primer lugar, se sintetizaron nuevas fluoroquinolonas para explorar sus propiedades fototóxicas para su uso en fotoquimioterapia (Capítulo 3 de la Tesis). Se estudiaron las características fotofísicas y fotoquímicas de los nuevos compuestos, además de su capacidad para producir mayor fototoxicidad en las células en comparación con las fluoroquinolonas como la lomefloxacina mediante la aplicación de luz ultravioleta. En base a los resultados obtenidos se realizó un estudio para determinar las diferencias entre las interacciones de algunas fluoroquinolonas dihalogenadas, incluidas las comentadas anteriormente, y biomoléculas como ADN y proteínas. La reactividad de sus intermedios fotogenerados también se estudió en el Capítulo 4. Tras conocer en profundidad la capacidad fototóxica de los nuevos fármacos, en el Capítulo 5 se llevó a cabo el diseño de un nanosistema compuesto por fluoroquinolonas y nanopartículas de conversión ascendente. Se demostró la alta capacidad fototóxica de este nuevo nanosistema. De esta manera, se generó actividad fototóxica a partir de una fluoroquinolona sin el uso de luz ultravioleta Por otro lado, la formación de profármacos abre la puerta a la administración selectiva de fármacos contra el cáncer. Los profármacos consisten en la unión fotolábil de una molécula capaz de ser activada por la luz y el fármaco de interés. Sin embargo, un conocimiento profundo de las propiedades fotofísicas y fotoquímicas del fotodisparador y de los potenciales redox de ambos miembros de la diada puede ser crucial para obtener la fotoliberación deseada. Así, en el Capítulo 6, se destacó la relevancia de estos datos utilizando un profármaco formado por un derivado de cumarina como molécula fotoactivable y colchicina como fármaco. Finalmente, en el Capítulo 7 se exploró la síntesis de un nuevo nanosistema que contiene un profármaco formado por un derivado de cumarina unido al fármaco contra el cáncer clorambucilo y nanopartículas biocomatibles de conversión ascendente. La adición de albúmina de suero humano como recubrimiento de las nanopartículas cumple la doble función de obtener nanopartículas biocompatibles y ser el lugar de carga del profármaco. / [CA] El càncer és una de les principals causes de mort a nivell mundial. Els tractaments anticancerígens generalment usats tenen diversos efectes secundaris produïts per la seva baixa especificitat. Aquesta és una de les raons per les que se segueix en contínua recerca de nous tractaments. Dins d'aquestes noves investigacions es troba l'extens camp de la nanomedicina, és a dir, l'estudi de nous materials a escala nanomètrica. Aquesta permet reduir aquests efectes secundaris augmentant la selectivitat i especificitat dels tractaments. Dins dels nanomaterials es troben les nanopartícules de upconversion que són capaços d'absorbir llum en l'infraroig proper i emetre-la en la regió ultraviolada-visible. D'altra banda, des del principi de la història de la medicina la llum s'ha emprat com a forma de tractament tenint un paper molt important. Un inconvenient per aquests tractaments sol ser la necessitat d'emprar llum de la regió ultraviolada-visible, ja que les biomolècules són capaços d'absorbir-la i produïr dany cel·lular. En aquest context, la present Tesi Doctoral es centra en l'estudi de noves formes de tractament anticancerigen combinant nanomedicina i llum. Per això s'han desenvolupat nous fàrmacs fototòxics i nous materials capaços de ser activats mitjançant llum infraroja propera. En primer lloc, es van sintetitzar noves fluoroquinolones per explorar les seves propietats fototòxiques per al seu ús en fotoquimioteràpia (Capítol 3 de la Tesi). Es van estudiar les característiques fotofísiques i fotoquímiques dels nous compostos, a més de la seva capacitat per produir major fototoxicitat en les cèl·lules en comparació amb les fluoroquinolones com la lomefloxacina mitjançant l'aplicació de llum ultraviolada. En base als resultats obtinguts es va realitzar un estudi per determinar les diferències entre les interaccions d'algunes fluoroquinolones dihalogenades, incloses les comentades anteriorment, i biomolècules com ADN i proteïnes. La reactivitat de les seves intermedis fotogenerats també es va estudiar en el Capítol 4. Després de conèixer en profunditat la capacitat fototòxica dels nous fàrmacs, en el Capítol 5 es va dur a terme el disseny d'un nanosistema compost per fluoroquinolones i nanopartícules de upconversion. Es va demostrar l'alta capacitat fototòxica d'aquest nou nanosistema. D'aquesta manera, es va generar activitat fototòxica a partir d'una fluoroquinolona sense l'ús de llum ultraviolada D'altra banda, la formació de profàrmacs obre la porta a l'administració selectiva de fàrmacs contra el càncer. Els profàrmacs consisteixen en la unió fotolábil d'una molècula capaç de ser activada per la llum i el fàrmac d'interès. No obstant això, un coneixement profund de les propietats fotofísiques i fotoquímiques del fotodisparador i dels potencials redox de tots dos membres de la diada pot ser crucial per obtenir el fotoalliberament desitjada. Així, en el Capítol 6, es va destacar la rellevància d'aquestes dades utilitzant un profàrmac format per un derivat de cumarina com a molècula fotoactivable i colquicina com a fàrmac. Finalment, en el Capítol 7 es va explorar la síntesi d'un nou nanosistema que conté un profàrmac format per un derivat de cumarina unit a l'fàrmac contra el càncer clorambucilo i nanopartícules biocomatibles de upconversion. L'addició d'albúmina de sèrum humà com a recobriment de les nanopartícules compleix la doble funció d'obtenir nanopartícules biocompatibles i ser el lloc de càrrega del profàrmac. / [EN] Cancer is one of the leading causes of death worldwide. Generally used anticancer treatments have various side effects produced by their low specificity. This is one of the reasons why the search for new treatments continues. Within these new investigations is the extensive field of nanomedicine, which can be explained as the study of new materials on a nanometric scale. It can be translated in the reduction of these side effects by increasing the selectivity and specificity of the treatments. Among the nanomaterials are upconversion nanoparticles that are capable of absorbing light in the near infrared and emit it in the ultraviolet-visible region. On the other hand, since the beginning of the history of medicine, light has been used as a form of treatment, having a very important role. A drawback for such treatments is sometimes the need to use light from the ultraviolet-visible region since biomolecules are capable of absorbing and causing cell damage. In this context, this Doctoral Thesis focuses on the study of new forms of anticancer treatment combining nanomedicine and light. For this, new phototoxic drugs and new materials capable of being activated by near infrared light have been developed. First, new fluoroquinolones were synthesized to explore their phototoxic properties for using in photochemotherapy (Chapter 3 of the Thesis). The photophysical and photochemical characteristics of the new compounds were studied, in addition to their ability to produce greater phototoxicity in cells than fluoroquinolones such as lomefloxacin by applying ultraviolet light. Based on the results obtained, a study was carried out to determine the differences between the interactions of some dihalogenated fluoroquinolones including the above commented, and biomolecules such as DNA and proteins. The reactivity of their photo-generated intermediates was also studied in Chapter 4. After a deep knowledge of the phototoxic capacity of the new drugs, design of a nanosystem composed of fluoroquinolones and upconversion nanoparticles was carried out in Chapter 5. The high phototoxic capacity of this new nanosystem was demonstrated. In this way phototoxic activity was generated from a fluoroquinolone without the use of ultraviolet light. On the other hand, the formation of prodrugs opens a door to the selective administration of anticancer drugs. Prodrugs consist of the photolabile binding of a molecule capable of being activated by light and the drug of interest. However, a knowledge of the photophysical and photochemical properties of the phototrigger as well as the redox potentials of both members of the dyad can be crucial to obtain the desired photorelease. Thus, in Chapter 6, the relevance of these data was highlighted using a prodrug formed by a coumarin derivative as a photoactivatable molecule and colchicine as a drug. Finally, in Chapter 7 the synthesis of a new nanosystem containing a prodrug formed by a derivative of coumarin linked to the anticancer drug chlorambucil, and upconversion biocompatible nanoparticles was explored. The addition of human serum albumin as a coating for the nanoparticles fulfills the dual function of obtaining biocompatible nanoparticles and being the loading site for the prodrug. / Anaya González, C. (2021). Design of New Up-conversion Systems for Anticancer Therapies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172665 / TESIS

No description available.

Photosensitizing effects of xenobiotics are of increasing concern in public health since modern lifestyle often associates sunlight exposure with the presence of chemical substances in the skin. An important number of chemicals like perfumes, sunscreen components, or therapeutic agents have been reported as photosensitizers. In this context, a considerable effort has been made to design a model system for photosafety assessment. Indeed, screening for phototoxicity is necessary at the early phase of drug discovery process, even before introducing drugs and chemicals into clinical therapy, to prevent undesired photoreactions in humans. In the case of new pharmaceuticals, their phototoxic potential has to be tested when they absorb in the regions corresponding to the solar spectrum, that is, for wavelengths >290 nm. So, there is an obvious need for a screening strategy based on in vitro experiments. The goal of the present thesis was the photochemical study of different photoactive drugs to investigate the key molecular aspects responsible for their photosensitivity side effects. In a first stage, rosuvastatin was considered in chapter 3 as representative compound of the statin family. This lipid-lowering drug, also known as “superstatin”, contains a 2-vinylbiphenyl-like moiety and has been previously described to decompose under solar irradiation, yielding stable dihydrophenanthrene analogues. During photophysical characterization of rosuvastatin, only a long-lived transient at ca. 550 nm was observed and assigned to the primary photocyclization intermediate. Thus, the absence of detectable triplet-triplet absorption and the low yield of fluorescence ruled out the role of the parent drug as an efficient sensitizer. In this context, the attention was placed on the rosuvastatin main photoproduct (ppRSV). Indeed, the photobehavior of this dihydrophenanthrene-like compound presented the essential components needed for an efficient biomolecule photosensitizer i.e. (i) a high intersystem crossing quantum yield (ΦISC =0.8), (ii) a triplet excited state energy of ca. 67 kcal mol−1 , and (iii) a quantum yield of singlet oxygen formation (Φ∆) of 0.3. Furthermore, laser flash photolysis studies revealed a triplet-triplet energy transfer from the triplet excited state of ppRSV to thymidine, leading to the formation of cyclobutane thymidine dimers, an important type of DNA lesion. Finally, tryptophan was used as a probe to investigate the Type I and/or Type II character of ppRSV-mediated oxidation. In this way, both an electron transfer process giving rise to the tryptophanyl radical and a singlet oxygen mediated oxidation were observed. On the basis of the obtained results, rosuvastatin, through its major photoproduct ppRSV, should be considered as a potential sensitizer. Then, itraconazole (ITZ), a broad-spectrum antifungal agent, was chosen as main character of chapter 4. Its photochemical properties were investigated in connection with its reported skin photosensitivity disorders. Steady state photolysis, fluorescence and phosphorescence experiments were performed to understand ITZ photoreactivity in biological media. The drug is unstable under UVB irradiation, suffering a primary dehalogenation of the 2,4-dichlorophenyl moiety that occurs mainly at the ortho-position. In poorly H-donating solvents, as acetonitrile, the major photoproduct arises from intramolecular attack of the initially generated aryl radical to the triazole ring. In addition, reduced compounds resulting from homolytic cleavage of the C-Cl bond in ortho or para positions and subsequent Habstraction from the medium are obtained to a lesser extent. In good H-donating solvents, such as ethanol, the main photoproducts are formed by reductive dehalogenation. Furthermore, irradiation of a model dyad containing a tryptophan unit and the reactive 2,4-dichlorophenyl moiety of itraconazole leads to formation of a new covalent link between these two substructures revealing that homolysis of the C-Cl bond of ITZ can result in alkylation of reactive amino acid residues of proteins, leading to formation of covalent photoadducts. Therefore, it has been established that the key process in the photosensitization by itraconazole is cleavage of the carbon-halogen bond, which leads to aryl radicals and chlorine atoms. These highly reactive species might be responsible for extensive free radical-mediated biological damage, including lipid peroxidation or photobinding to proteins. In chapter 5, photobehavior of imatinib (IMT) was addressed. This is a promising tyrosine kinase inhibitor used in the treatment of some types of human cancer, which constitutes a successful example of rational drug design based on the optimization of the chemical structure to reach an improved pharmacological activity. Cutaneous reactions, such as increased photosensitivity or pseudoporphyria, are among the most common nonhematological IMT side effects; however, the molecular bases of these clinical observations have not been unveiled yet. Thus, to gain insight into the IMT photosensitizing properties, its photobehavior was studied together with that of its potentially photoactive anilino-pyrimidine and pyridyl-pyrimidine fragments. In this context, steady-state and time resolved fluorescence, as well as laser flash photolysis experiments were run, and the DNA photosensitization potential was investigated by means of single strand breaks detection using agarose gel electrophoresis. The obtained results revealed that the drug itself and its anilino-pyrimidine fragment are not DNA-photosensitizers. By contrast, the pyridyl-pyrimidine substructure displayed a marked photogenotoxic potential, which was associated with the generation of a long-lived triplet excited state. Interestingly, this reactive species was efficiently quenched by benzanilide, another molecular fragment of IMT. Clearly, integration of the photoactive pyridyl-pyrimidine moiety in a more complex structure strongly modifies its photobehavior, which in this case is fortunate as it leads to an improved toxicological profile. Thus, on the bases of the experimental results, direct in vivo photosensitization by IMT seems unlikely. Instead, the reported photosensitivity disorders could be related to indirect processes, such as the previously suggested impairment of melanogenesis or the accumulation of endogenous porphyrins. Finally, a possible source of errors in the TEMPO/EPR method for singlet oxygen detection was analyzed. For many biological and biomedical studies, it is essential to detect the production of 1O2 and to quantify its production yield. Among the available methods, detection of the characteristic 1270 nm phosphorescence of singlet oxygen by time-resolved near infrared (TRNIR) emission constitutes the most direct and unambiguous approach. An alternative indirect method is electron paramagnetic resonance (EPR) in combination with trapping. This is based on the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6- tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has been largely employed, it can produce misleading data. This was demonstrated by the present study, where the quantum yields of singlet oxygen formation obtained by TRNIR emission and by the TEMPO/EPR method were compared for a set of well-known photosensitizers. The results revealed that the TEMPO/EPR method leads to significant overestimation of singlet oxygen yield when the singlet or triplet excited state of the photosensitizers were efficiently quenched by TEMP, acting as electron donor. In such case, generation of the TEMP+• radical cation, followed by deprotonation and reaction with molecular oxygen gives rise to a EPR detectable TEMPO signal that is not associated with singlet oxygen production. This knowledge is essential for an appropriate and error-free application of the TEMPO/EPR method in chemical, biological and medical studies. / Nardi, G. (2014). MECHANISTIC STUDIES ON THE PHOTOTOXICITY OF ROSUVASTATIN, ITRACONAZOLE AND IMATINIB [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535 / TESIS

[ES] En la presente tesis doctoral se ha desarrollado una metodología multidisciplinar para la evaluación del potencial fotoquímico y fotobiológico in vitro de los fármacos inhibidores de la poli (ADP-ribosa) polimerasa (PARP), combinando tanto estudios fotofísicos y de caracterización como estudios fotobiológicos en biomoléculas modelo y en cultivos celulares, concretamente en queratinocitos humanos inmortalizados (HaCaT). Así, el objetivo general es investigar si estos fármacos, en combinación con la radiación solar, son capaces de inducir reacciones de fotosensibilidad y, por tanto, poder alertar a los oncólogos de estos hallazgos para que puedan indicar pautas de fotoprotección adecuadas a sus pacientes, y así prevenir estos efectos indeseados. Esta tesis se divide en un total de 7 capítulos. En el primero (introducción), se recogen los hitos más importantes relacionados con los inhibidores de la PARP, así como un resumen acerca de las reacciones de fotosensibilización y de los mecanismos químicos y biológicos involucrados en las mismas. Tras una exhaustiva búsqueda bibliográfica acerca de estos fármacos, se hizo un primer cribado de los cinco de la tercera y última generación (rucaparib, talazoparib, niraparib, olaparib y veliparib), identificando así tres de ellos como posibles agentes fotosensibilizantes: rucaparib, talazoparib y niraparib, cuyo estudio se describe en profundidad en los capítulos 4, 5 y 6 de la tesis. En el capítulo 4 se evalúa la fototoxicidad del rucaparib (RCP), un fármaco que, a pesar de tener un cromóforo indol en su estructura química, posee un desplazamiento batocrómico hacia la región UVA de la luz solar, siendo así capaz de inducir reacciones de fotosensibilidad. Se observó mediante experimentos de fluorescencia y fotólisis de destello láser que, tras absorción de luz en esta región del espectro, tiene lugar la generación de especies reactivas de oxígeno (ROS), que podrían ser las responsables de generar un daño fotooxidativo hacia el ADN celular y a las proteínas transmembranales, originando como resultado una foto(geno)toxicidad. Además, se estableció que el mecanismo de la muerte celular fotoinducida por RCP es por apoptosis. El capítulo 5 se centra en la evaluación del perfil de fotoseguridad de talazoparib (TLZ), el cual, tras su irradiación con luz UVA da lugar a un fotoproducto que, tras su aislamiento y caracterización, resultó no presentar fotorreactividad. Este fotoproducto resultó además ser el responsable de reducir significativamente el potencial fototóxico del fármaco original, ya que no se detectaron especies transitorias tras su irradiación con luz UVA. Además, para el TLZ se reveló la producción in vitro de ROS, especies que podrían ser las responsables de generar un daño fotooxidativo tanto hacia el ADN celular como a las proteínas de membrana, originando como resultado una foto(geno)toxicidad. En el capítulo 6 se evalúa la fototoxicidad del niraparib (NRP) y su principal metabolito (N-M1). A pesar de que ambos compuestos resultaron ser fototóxicos, dicha fototoxicidad no afectó igual a las principales dianas celulares, ya que el daño fotooxidativo de NRP se observó en lípidos, en proteínas transmembranales y en el ADN celular, mientras que en N-M1 se aprecia fundamentalmente en las proteínas de membrana. Además, la toxicidad observada del NRP en células de cáncer de ovario (A2780 y A2780cis) indicó que este fármaco podría contemplarse como candidato para su uso en un futuro en la terapia fotodinámica para el tratamiento del cáncer de ovario. Finalmente, con todo ello se concluye que estos fármacos de la familia de los inhibidores de la PARP (RCP, TLZ y NRP) son capaces de inducir reacciones de fotosensibilidad, con valores de factor de fotoirritación (PIF) de 41, 7 y 46, respectivamente. / [CAT] En la present tesi doctoral s'ha desenvolupat una metodologia multidisciplinària per a l'avaluació del potencial fotoquímic i fotobiològic in vitro dels fàrmacs inhibidors de la poli(ADP-ribosa) polimerasa (PARP), combinant tant estudis fotofísics i de caracterització com estudis fotobiològics en biomolècules model i en cultius cel·lulars, concretament en queratinòcits humans immortalitzats (HaCaT). Així, l'objectiu general és investigar si aquests fàrmacs, en combinació amb la radiació solar, són capaços d'induir reaccions de fotosensibilitat i, per tant, poder alertar als oncòlegs d'aquestes fites perquè puguen indicar pautes de fotoprotecció adequades als seus pacients, i així previndre aquests efectes no desitjats. Aquesta tesi es divideix en un total de 7 capítols. En el primer (introducció), es recullen les fites més importants relacionades amb els inhibidors de la PARP, així com un resum sobre les reaccions de fotosensibilització i dels mecanismes químics i biològics involucrats en aquestes. Després d'una exhaustiva cerca bibliogràfica sobre aquests fàrmacs, es va fer un primer estudi dels cinc de la tercera i última generació (rucaparib, talazoparib, niraparib, olaparib i veliparib), identificant així tres d'ells com a possibles agents fotosensibilizants: rucaparib, talazoparib i niraparib, l'estudi del qual es descriu en profunditat en els capítols 4, 5 i 6 de la tesi. En el capítol 4 s'avalua la fototoxicitat del rucaparib (RCP), un fàrmac que, malgrat tindre un cromòfor indole en la seua estructura química, posseeix un desplaçament batocròmic cap a la regió UVA de la llum solar, sent així capaç d'induir reaccions de fotosensibilitat. Mitjançant experiments de fluorescència i fotòlisi de flaix làser es va observar que, després de l¿absorció de llum en aquesta regió de l'espectre, té lloc la generació d'espècies reactives d'oxigen (ROS), que podrien ser les responsables de generar un dany fotooxidatiu cap a l'ADN cel·lular i a les proteïnes transmembranals, originant com a resultat una foto(geno)toxicitat. A més, es va establir que el mecanisme de la mort cel·lular fotoinduïda per RCP és per apoptosi. El capítol 5 se centra en l'avaluació del perfil de fotoseguretat de talazoparib (TLZ), el qual, després de la seua irradiació amb llum UVA dona lloc a un fotoproducte que, després del seu aïllament i caracterització, va resultar no presentar fotorreactivitat ninguna. Aquest fotoproducte va resultar a més ser el responsable de reduir significativament el potencial fototòxic del fàrmac original, ja que no es van detectar espècies transitòries després de la seua irradiació amb llum UVA. A més, per al TLZ es va revelar la producció in vitro de ROS, espècies que podrien ser les responsables de generar un dany fotooxidatiu tant cap a l'ADN cel·lular com a les proteïnes de membrana, originant com a resultat una foto(geno)toxicitat. En el capítol 6 s'avalua la fototoxicitat del niraparib (*NRP) i el seu principal metabòlit (N-M1). A pesar que tots dos compostos van resultar ser fototòxics, aquesta fototoxicitat no va afectar per igual a les principals dianes cel·lulars, ja que el dany fotooxidatiu de NRP es va observar en lípids, en proteïnes i en l'ADN cel·lular, mentre que en N-M1 s'aprecia fonamentalment en les proteïnes de membrana. A més, la toxicitat observada del NRP en cèl·lules de càncer d'ovari (A2780 i A2780cis) va indicar que aquest fàrmac podria contemplar-se com a candidat per al seu ús en un futur en la teràpia fotodinàmica per al tractament del càncer d'ovari. Finalment, amb tot això es conclou que aquests fàrmacs de la família dels inhibidors de la PARP (RCP, TLZ i NRP) són capaços d'induir reaccions de fotosensibilitat, amb valors de factor de fotoirritació (PIF) de 41, 7 i 46, respectivament. / [EN] In this doctoral thesis, a multidisciplinary methodology has been performed for the evaluation of the in vitro photochemical and photobiological potential of the poly (ADP-ribose) polymerase (PARP) inhibitors, combining photophysical and photobiological studies in biomolecules and cell cultures, specifically in human immortalized keratinocytes (HaCaT). Thus, the general objective is to investigate if these drugs, in combination with solar radiation, can trigger photosensitivity reactions; this will allow oncologists to indicate appropriate photoprotective guidelines to their patients in order to prevent these undesirable effects. This thesis is divided into 7 chapters. The first one (introduction) contains the essential issues concerning PARP inhibitors, a background of the photosensitization reactions and a description of the involved chemical and biological mechanisms. After in-depth bibliographical research about these drugs, a screening of the five PARP inhibitors (rucaparib, talazoparib, niraparib, olaparib and veliparib) of the third and last generation was carried out, identifying three of them as possible photosensitizing drugs: rucaparib, talazoparib and niraparib. Their behaviour is thoroughly described in chapters 4, 5 and 6. Chapter 4 evaluates the phototoxicity of rucaparib (RCP), a drug that possess an indole chromophore in its chemical structure but displays a bathochromic shift towards the UVA region of sunlight, which makes it able to induce photosensitivity reactions. Generation of reactive oxygen species (ROS) after UVA light absorption was detected by fluorescence and laser flash photolysis experiments. These species could generate photooxidative damage to cellular DNA and transmembrane proteins, resulting in photo(geno)toxicity. In addition, it was established that the mechanism of RCP photoinduced cell death is by apoptosis. Chapter 5 focuses on the photosafety profile of talazoparib (TLZ), a compound that, after UVA irradiation, gives rise to a photooxidized product; after isolation and characterization, the photoproduct did not display any photoreactivity, and no transient species were detected after UVA light irradiation. Therefore, it was responsible for the significantly reduced phototoxic potential of the parent drug. Additionally, for TLZ, in vitro ROS production was detected. These species could lead to photooxidative damage to both cellular DNA and membrane proteins, resulting in photo(geno)toxicity. Chapter 6 deals with the phototoxicity of niraparib (NRP) and its main metabolite (N-M1). Although both compounds are phototoxic, the phototoxicity was found to be different for the main cellular targets: thus, the photooxidative damage of NRP was noticed in lipids, transmembrane proteins and cellular DNA, whereas in N-M1 it was mainly observed in membrane proteins. In addition, NRP was cytotoxic in ovarian cancer cells (A2780 and A2780cis), indicating that this drug could be considered as a future candidate for its use in photodynamic therapy to treat ovarian cancer. Finally, it can be concluded that these PARP inhibitors (RCP, TLZ and NRP) are able to induce photosensitivity disorders, with PIF values of 41, 7 and 46, respectively. / We thank the Agencia Estatal de Investigación (PID2020-115010RB-I00/AEI/10.13039/501100011033 and the Generalitat Valenciana (CIAICO/2021/061 and ACIF/2018/153 fellowship for A. M.-P. / Mateos Pujante, A. (2023). Evaluación del potencial fotoquímico y fotobiológico de los inhibidores de la poli (ADP-ribosa) polimerasa [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196092