Phytochromes (Phys) are biliproteins that regulate light responses in plants, fungi, and microorganisms through photoconversion between a dark state and a photoproduct. Thermal reversion of the photoproduct is an intrinsic property of all Phys, typically occurring on a timescale of seconds to days. Despite methodological advances, the structural and spectroscopic determination of short-lived photoproducts has proven challenging. We herein present an innovative approach for photoproduct stabilisation by incorporating the protein into trehalose glasses (TGs). The resulting Phy–trehalose matrices were investigated by UV/Vis absorption and solid-state NMR spectroscopies. Our results demonstrate that the TGs strongly inhibit thermal reversion of the incorporated Phy proteins for periods as long as several weeks at room temperature (RT), during which the proteins fully sustain their native structures and spectral and biochemical properties. This sample preparation approach is beneficial for revealing bona fide structure/ function relationships of short-lived photoproducts that are otherwise not accessible, thus paving the way towards a deeper molecular understanding of the diversified spectral properties of Phys. Our results also provide new insights into the molecular mechanism of trehalose bioprotection.

A protoporfirina IX (PpIX) ocupa um lugar especial entre a família das porfirinas devido ao seu importante papel na natureza e diversas aplicações, inclusive em terapia fotodinâmica. Sob ação da luz visível, a PpIX transforma-se em fotoprodutos que podem ser citotóxicos ou fotocitotóxicos, e isto pode, de um lado, aumentar a eficiência do tratamento e, por outro lado, apresentar toxicidade no escuro, prejudicando o paciente. Como as características da sua fototransformação dependem do ambiente onde elas se encontram, tornam-se importantes os estudos dos efeitos do ambiente nas características da sua fototransformação. As propriedades espectroscópicas da PpIX sintética e endógena, extraída das glândulas harderianas de ratos da espécie Rattus novergicus albinus, e seus fotoprodutos e a atividade citotóxica foram estudadas no escuro e sob ação da luz visível em uma cultura de células malignas, em comparação com Photogem® e a porfirina TPPS4. A localização das protoporfirinas e seus fotoprodutos dentro da estrutura celular também foram observadas. Foi notado que existe uma diferença entre os espectros de absorção e fluorescência das PpIX endógena e sintética e também de seus fotoprodutos, e do grau de sua fototransformação. Associou-se estes efeitos à diferença do ambiente onde as porfirinas se encontravam, devido a interação da PpIX endógena e de seu fotoproduto com compostos celulares que poderiam estar presentes depois da purificação da PpIX obtida da glândula. A fotocitotoxicidade das PpIX sintética e endógena foram comparáveis. Os fotoprodutos, tanto da PpIX sintética quanto endógena não apresentaram fotocitotoxicidade ou mostraram fotocitotoxicidade menor do que a das porfirinas, resultado contrário ao encontrado na literatura. Em relação ao padrão Photogem®, as protoporfirinas apresentaram uma fotocitotoxicidade comparável a ele, mas uma citotoxicidade no escuro maior. A TPPS4 demonstrou tanto uma fototoxicidade, como uma citotoxicidade no escuro menor que o Photogem®. Da análise da distribuição intracelular das porfirinas PpIX e seus fotoprodutos com a de marcadores padrões de estruturas celulares, pode ser concluído que eles não penetraram no núcleo da célula, e sua distribuição nas demais partes do citoplasma foi difusa, sem nenhuma localização preferencial. / The protoporphyrin IX (PpIX) occupies a special place among the porphyrins family due their important role in nature and several applications, including Photodynamic Therapy. Under the action of visible light, PpIX becomes photoproducts that can be cytotoxic or photocytotoxic and these effects can, on one hand, increase the treatment efficiency and, on the other hand, be toxic in the dark, harming the patient. Since the characteristics of its phototransformation depend on the environment where they are finding, studies of the effects of environment on the characteristics of its phototransformation are important. The spectroscopic properties and cytotoxic activity were studied in the dark and under visible light action of synthetic and endogenous PpIX, extracted from mice harderian gland of the Rattus novergicus albinus species and its photoproducts, in a culture of malignant cells, compared with Photogem® and TPPS4 porphyrin. The cellular localization of protoporphyrin and its photoproducts inside cellular structure also were observed. It was observed that there is a difference between the fluorescence and absorption spectra of synthetic and endogenous PpIX and between its photoproducts and also the degree of its phototransformation. These effects can be linked to the difference of the environment where the porphyrins were, due the interaction of endogenous PpIX and its photoproducts with cellular components that could be present after purification of PpIX obtained from the gland. The photocytotoxic of synthetic and endogenous PpIX were comparable. The photoproducts of both synthetic and endogenous PpIX showed no photocytotoxic or were smaller than of porphyrins, a contrary result to that it was found in the literature. In relation to pattern composite Photogem®, the protoporphyrin showed a photocytotoxic comparable to it, but protoporphyrin a higher cytotoxicity in the dark. The TPPS4 demonstrated both a lower phototoxicity as a cytotoxicity in the dark than Photogem®. From analysis of intracellular distribution of the porphyrin PpIX and its photoproducts with markers patterns of cellular structures, may be concluded that they did not penetrate into the cell nucleus and its distribution in other parts of the cytoplasm was diffuse, without preferential location.

Cette étude s’inscrit dans le cadre de la dépollution de l’eau, elle a eu pour but d’examiner la dégradation photocatalytique d’herbicides phénylurées en solution aqueuse à pH acide, en présence d’un oxyde de fer naturel et d’acides carboxyliques. L’oxyde de fer naturel a été caractérisé par spectroscopie Raman, l’identification des produits de dégradation du fénuron et la proposition d’un mécanisme de dégradation ont été réalisées. Deux types d’irradiation ont été utilisés, i.e. l’irradiation solaire naturelle et l’irradiation par des UV générés par des lampes à vapeur de mercure. La photodégradation du fenuron en présence d’oxyde de fer naturel et l'acide oxalique obéit à une loi du premier ordre, les conditions expérimentales optimales pour ce système étaient [acide oxalique]0 = 10-3 M, [Hématite naturelle] = 0.1 g L-1 ; pH = 3. Le temps de demi-vie du fenuron est court (60 32 min) mais les résultats sont meilleurs sous irradiation solaire naturelle (30 15 min). L'acide oxalique est l’acide carboxylique le plus efficace en photodégradation du fénuron (disparition totale), l’ordre d’efficacité varie ainsi : acide oxalique > acide citrique > acide tartrique > acide malique. L’étude analytique de la dégradation du fénuron par ce procédé a permis l’identification de huit photoproduits aromatiques, de trois acides carboxyliques à courte chaîne de carbone et des ions inorganiques. Ce travail a permis de mettre en évidence le rôle de l’oxygène dissous dans le processus de dégradation du fenuron par le procédé de photo-Fenton-like, le processus ne peut pas avoir lieu en milieu désoxygéné car aucune production de H2O2 n’a été observée sous irradiation UV. L’oxygénation du milieu permet d’améliorer la capacité oxydative du procédé et d’augmenter le taux de minéralisation du carbone organique totale. En effet l’oxygénation du milieu augmente indirectement la production des radicaux hydroxyles par augmentation de la production de H2O2. Il a été mis en évidence pendant l’étude quantitative que l’oxygène était le réactif le plus consommé par ce procédé. Le traitement de la solution contenant 10-4 M de fenuron et 10-3 M d’oxalate a permis de réduire significativement la toxicité de la solution initiale après 360 minutes d’irradiation. / The photodegradation of the herbicide phenylurea by using a natural iron oxide (NIO) α-Fe2O3 in aqueous solution at acidic pH has been undertaken. The NIO was characterized by the Raman spectroscopy method. The degradation pathways and the formation of by-products were studied. A high-pressure mercury lamp and sunlight were employed as light source. Fenuron photodegradation using NIO with oxalic acid followed the pseudo-first order kinetics, the optimal experimental conditions were [oxalic acid]0 = 10−3 M and [NIO] = 0.1 g L−1 at pH 3. NIO/oxalic acid/UV system led to a low fenuron half-life (32 min). The results were even better when solar light is used (15 min). The effect of four carboxylic acids; oxalic, citric, tartaric and malic acids on the degradation efficiency was studied. Oxalic acid was the most effective carboxylic acid used on the fenuron photodegradation with NIO at pH 3. The analytical study showed many aromatic intermediates, short-chain carboxylic acids and inorganic ion.The role of dissolved oxygen (DO) in degradation and mineralization of fenuron in the photo-Fenton-like system by using the NIO was also studied; the NIO was used as an iron source. The DO effect has been elucidated by carrying out experiments with and without oxygen, it has been found that the degradation and mineralization rate were strongly affected by change in DO concentration. In absence of oxygen, there is no photo-Fenton-like because of the lack of hydrogen peroxide, the degradation was caused only by the photo-reduction of Fe(III) complex in this case. Oxygenation improved the hydrogen peroxide production under irradiation. The mass balance of the reactive species showed that dissolved oxygen was the main reactive consumed during the process. The toxicity decrease with irradiation time proves that the intermediates generated during photo-oxidation by the photo-Fenton-like system by using a NIO are less toxic than fenuron.

A protoporfirina IX (PpIX) ocupa um lugar especial entre a família das porfirinas devido ao seu importante papel na natureza e diversas aplicações, inclusive em terapia fotodinâmica. Sob ação da luz visível, a PpIX transforma-se em fotoprodutos que podem ser citotóxicos ou fotocitotóxicos, e isto pode, de um lado, aumentar a eficiência do tratamento e, por outro lado, apresentar toxicidade no escuro, prejudicando o paciente. Como as características da sua fototransformação dependem do ambiente onde elas se encontram, tornam-se importantes os estudos dos efeitos do ambiente nas características da sua fototransformação. As propriedades espectroscópicas da PpIX sintética e endógena, extraída das glândulas harderianas de ratos da espécie Rattus novergicus albinus, e seus fotoprodutos e a atividade citotóxica foram estudadas no escuro e sob ação da luz visível em uma cultura de células malignas, em comparação com Photogem® e a porfirina TPPS4. A localização das protoporfirinas e seus fotoprodutos dentro da estrutura celular também foram observadas. Foi notado que existe uma diferença entre os espectros de absorção e fluorescência das PpIX endógena e sintética e também de seus fotoprodutos, e do grau de sua fototransformação. Associou-se estes efeitos à diferença do ambiente onde as porfirinas se encontravam, devido a interação da PpIX endógena e de seu fotoproduto com compostos celulares que poderiam estar presentes depois da purificação da PpIX obtida da glândula. A fotocitotoxicidade das PpIX sintética e endógena foram comparáveis. Os fotoprodutos, tanto da PpIX sintética quanto endógena não apresentaram fotocitotoxicidade ou mostraram fotocitotoxicidade menor do que a das porfirinas, resultado contrário ao encontrado na literatura. Em relação ao padrão Photogem®, as protoporfirinas apresentaram uma fotocitotoxicidade comparável a ele, mas uma citotoxicidade no escuro maior. A TPPS4 demonstrou tanto uma fototoxicidade, como uma citotoxicidade no escuro menor que o Photogem®. Da análise da distribuição intracelular das porfirinas PpIX e seus fotoprodutos com a de marcadores padrões de estruturas celulares, pode ser concluído que eles não penetraram no núcleo da célula, e sua distribuição nas demais partes do citoplasma foi difusa, sem nenhuma localização preferencial. / The protoporphyrin IX (PpIX) occupies a special place among the porphyrins family due their important role in nature and several applications, including Photodynamic Therapy. Under the action of visible light, PpIX becomes photoproducts that can be cytotoxic or photocytotoxic and these effects can, on one hand, increase the treatment efficiency and, on the other hand, be toxic in the dark, harming the patient. Since the characteristics of its phototransformation depend on the environment where they are finding, studies of the effects of environment on the characteristics of its phototransformation are important. The spectroscopic properties and cytotoxic activity were studied in the dark and under visible light action of synthetic and endogenous PpIX, extracted from mice harderian gland of the Rattus novergicus albinus species and its photoproducts, in a culture of malignant cells, compared with Photogem® and TPPS4 porphyrin. The cellular localization of protoporphyrin and its photoproducts inside cellular structure also were observed. It was observed that there is a difference between the fluorescence and absorption spectra of synthetic and endogenous PpIX and between its photoproducts and also the degree of its phototransformation. These effects can be linked to the difference of the environment where the porphyrins were, due the interaction of endogenous PpIX and its photoproducts with cellular components that could be present after purification of PpIX obtained from the gland. The photocytotoxic of synthetic and endogenous PpIX were comparable. The photoproducts of both synthetic and endogenous PpIX showed no photocytotoxic or were smaller than of porphyrins, a contrary result to that it was found in the literature. In relation to pattern composite Photogem®, the protoporphyrin showed a photocytotoxic comparable to it, but protoporphyrin a higher cytotoxicity in the dark. The TPPS4 demonstrated both a lower phototoxicity as a cytotoxicity in the dark than Photogem®. From analysis of intracellular distribution of the porphyrin PpIX and its photoproducts with markers patterns of cellular structures, may be concluded that they did not penetrate into the cell nucleus and its distribution in other parts of the cytoplasm was diffuse, without preferential location.

L’objectif de ce travail est l’étude de la plante face aux molécules issues de la phototransformation de la sulcotrione. La recherche d’anomalies a été menée sur des extrémités racinaires d’Allium cepa L. var. aggregatum par un dénombrement au microscope des anomalies chromosomiques causées par l’ajout des différents xénobiotiques. L’étude a été réalisée en deux temps. Tout d’abord l’impact génotoxique d’un mélange constitué de la sulcotrione et de ses photoproduits a été évalué. Puis les caractéristiques de chaque produit de dégradation ont été analysées indépendamment. Il s’est avéré que la sulcotrione et ses produits de transformation CMBA et XDD ont des caractéristiques de toxicité qui sont spécifiques de chacun d’entre eux, avec une toxicité plus importante pour XDD. Il a été observé que XDD peut s’hydrolyser. Les études sur les produits de seconde génération ont permis d’identifier HMBA qui présente de nombreuses similitudes avec l’acide salicylique et provoque notamment une croissance racinaire accrue chez les espèces monocotylédones testées. / Cytotoxicity and genotoxicity of non irradiated and irradiated sulcotrione were investigated in Allium cepa test. Then, the cytotoxic effects of 2-chloro-4-mesylbenzoic acid (CMBA) and xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl (XDD), the two main photoproducts of sulcotrione were investigated at different concentrations. We analyze plant response treated with sulcotrione phototransformation by-products. Chromosomal aberrations were observed in Allium cepa root meristems. First, we investigated the genotoxic impact of a sulcotrione mixture and its photoproducts. Then each by-product was independently tested. Sulcotrione CMBA and XDD have specific toxicity, and XDD has a greater toxicity. In our latest analysis we observed that XDD can be hydrolyzed. HMBA was identified as a salicylic acid derivative and can increase root growth in monocotyledonous species.

Certains pesticides présentent une très bonne activité biologique en serre, lorsqu’ils sont protégés du rayonnement UV, mais pas en champs lorsqu’ils sont exposés à la totalité de la lumière solaire. Une dégradation photochimique par le rayonnement UV est alors suspectée. C’est le cas de la famille de fongicides contenant un groupement oxime dont nous avons étudié en détail la photodégradation d’un point de vue expérimental et théorique. La cinétique de phototransformation de ces fongicides a été mesurée en solution et en phase solide et les photoproduits de réaction ont été identifiés et quantifiés. L’ensemble des résultats nous a alors permis de proposer un mécanisme de photodégradation. Ces fongicides subissent à la fois une photoisomérisation et une photodégradation par rupture homolytique de la liaison oxime. Le rôle de certains éléments structuraux dans ces transformations a pu être établi grâce à plusieurs analogues. Une étude de modélisation moléculaire menée en parallèle a permis de déterminer la nature des voies réactionnelles mises en jeu après l’excitation de la molécule et ainsi d’expliquer les observations expérimentales. Ce travail avait pour objectif d’améliorer la photostabilité de cette famille de fongicides par des modifications structurales tout en respectant l’activité biologique par ailleurs tout à fait remarquable de ces composés. Le remplacement de l’oxime par un oxétane préparé par une réaction de Paternò-Büchi a été envisagé. / Some pesticides present a great biological activity in green house, while they are protected from UV radiations, but not in the field, while they are exposed to the whole spectrum of solar light. A photochemical degradation by UV light is then suspected. That is the case of the group of fungicides which contain an oxim moiety and that we studied the photodegradation in detail, both from experimental and theoretical points of view. The kinetics of phototransformation of these fungicides have been mesured in solution and in the solid phase and photoproducts have been identified and quantified. These results allowed us to propose a mecanism of photodegradation. These fungicides undergo simultanously a photoisomerisation and a photodegradation by a homolytic cleavage of the oxim bond. The role of some structural elements in these transformations has been set up thanks to several analogues. A parallel study of molecular modelisation allowed the determination of the nature of reactive paths followed after the excitation of the molecule and therefore the explanation of experimental observations. The objective of this work was to improve the photostability of this group of fungicides by structural modifications while keeping the remarquable biological activity of these compounds. The replacement of the oxim moiety by an oxetane prepared by a Paternò-Büchi reaction has been considered.

Le rayonnement UV solaire induit des modifications de la structure chimique des bases nucléiques de l'ADN essentiellement au niveau de site dipyrimidinique et conduit principalement à la formation des adduits cyclobutanique et (6-4). Ces adduits sont impliqués dans le photovieillisement et le cancer cutané. Les adduits (6-4) sont potentiellement les plus dangereux en raison de leurs propriétés mutagènes intrinsèques.La conformation de l'ADN représente l'un des facteurs gouvernant la formation de l'adduit (6-4). Afin, d'identifier les paramètres structuraux favorisant sa formation, des analogues du dinucléotide de thymine, de conformation choisie, ont été synthétisés. Les modifications chimiques apportées au sein des dinucléotodes ont permis de déplacer l'équilibre conformationnel Nord/Sud du désoxyribose de la thymidine, vers une conformation majoritaire souhaitée. Une analyse structurale par dichroïsme circulaire et une étude préliminaire de photoréactivité ont été réalisées sur ces dinucléotides modifiés. Les résultats obtenus révèlent l'implication de formes minoritaires empilées de ces dinucléotides dans la formation de l'adduit (6-4). De plus, l'étude photochimique montre que l'augmentation de conformères Nord et Sud du dinucléotide aux extrémités 5' et 3', respectivement, favorise la formation de l'adduit (6-4). Cependant, une conformation totalement contrainte Nord et Sud aux extrémités 5' et 3', respectivement, défavorise sa formation.Ces résultats ont permis d'identifier une conformation augmentant le rendement de formation de l'adduit (6-4) et permettront de concevoir de nouveaux modèles conduisant à la formation exclusive de ces adduits. Ces modèles pourront être utilisés en biologie en vue d'applications thérapeutiques ou comme outil en biophysique afin d'élucider le mécanisme de formation de cet adduit. / Exposure of DNA to solar UV light induces chemical modifications on its nucleic bases that are responsible for photoaging and skin cancer. Two major DNA photoproducts class exist: cyclobutane and (6-4) adducts. The latter is intrinsically more mutagenic than the cyclobutane adduct and therefore potentially more dangerous.DNA conformational parameters influence (6-4) adducts formation. To identify structural factors governing (6-4) adduct formation; we synthesized dinucleotides endowed with a specific conformation. Nucleoside moieties exist in a dynamic equilibrium between North and South conformation. Nucleoside conformational changes have been introduced by chemical modifications to obtain desired conformations. Then a structural study by circular dichroism correlated with photochemical study was done. It showed that minor stacked conformers promote (6-4) adduct formation. Moreover, the photochemical study reveals that the increase of North and South conformers at the 5' and 3' end, respectively, increased (6-4) adduct formation but a locked dinucleotide almost precluded (6-4) adduct formation. This result helps to design new models favoring (6-4) adduct formation. Understanding the conformational parameters that govern (6-4) adduct formation will facilitate the development of new therapeutic strategies and the elucidation of the (6-4) adducts formation mechanism.

Ultraviolet light is damaging but fish have evolved protective mechanisms, which allows them to live in shallow water reefs, high in UV radiation. This thesis details my investigation into the physiological ecology of solar ultraviolet (UV) absorbing compounds, known as mycosporine-like amino acids found in the external epithelial mucus, and examines the supporting role potentially played by a UV-induced DNA repair mechanism in coral reef fish of the Indo-Pacific. Using reverse phase chromatography and UV spectrophotometry, I examined whether the distribution of MAA compounds across different areas of the body is correlated with differential UV exposure. Comparisons were made between the MAA content and the absorbance spectra of mucus from the dorsal, ventral, caudal and head body surface areas in five species of Scaridae (Chlorurus sordidus, Scarus schlegeli, S. niger, S. psittacus and S. globiceps) from Ningaloo Reef, Coral Bay, Western Australia. All fish analysed had at least five MAAs present, and results showed that fish had increased UV absorbance in mucus over the dorsal area, which receives the brunt of UV radiation. Little UV protection was found in mucus from the ventral area, which receives the lower level of UV radiation mostly via reflection of the sand and reef surfaces. Furthermore, UV absorbance per mg dry mucus versus standard fish length showed that there is a positive relationship in C. sordidus with increasing size. I examined whether there is a difference in the quantity of UV screening compounds found in the mucus of fish along a longitudinal geographical gradient from inshore reefs (Lizard Island, Great Barrier Reef) to the outer edge reefs to oceanic reefs (Osprey Reef). MAA absorbance increased with longitudinal distance from the mainland landmass of Australia to more oligotrophic outer reefs, where UV attenuation is reduced and the ocean is more transparent to UV wavelength. I determined that fish living on inshore, more turbid reefs where UV attenuation in shallow waters is high have lower levels of MAA protection than fish from clear oceanic reefs. Furthermore, there seems to be a direct relationship between light attenuation and exposure with the quantity of protective sunscreening found in the mucus of reef fish. It is know that UV irradiation decreases with water depth and that mucus from fish with deep habitats absorbs less UV than that of fish from shallow habitats. It is unknown however, whether this UV protection is variable within the same individuals and if so, how fast changes 11 occur. To test this, I relocated 9 ambon damselfish from a deep reef (18 m) to a shallow reef (1.5 m) to expose fish to increased levels of UV and relocated another 7 fish from a shallow to a deep reef to expose fish to decreased levels of UV. One week after relocation, all fish were returned to their original reef site to determine whether MAA levels would return to their initial levels. Fish relocated to a shallower depth were recovered and had a 60% (SD+/-2%) increase in mucus UV absorbance. Conversely, the fish relocated to a deeper depth were recovered and had a 41% (SD+/-1%) decrease mucus UV absorbance. No difference was found between UV absorbance of relocated and original fish at both depth. Six days after fish were returned to their original reef, mucus UV absorbance levels had returned to 67% +/- 4% of the original level. These results show that mucus UV absorbance is variable in individual ambon damselfish and that the sunscreen protection typical for a certain depth is reached in relocated fish within just a few days of relocation. The rate of MAA loss is higher than the accumulation of MAAs suggesting that diet is not the sole determining factor involved in the sequestration of MAAs to mucus. The cleaner fish Labroides dimidiatus performs a mutualistic service by removing ectoparasites such as gnathiid isopods as well other dead infected tissue from its clients. Cleaner fish however are also known to feed on client mucus. The benefits of eating mucus until recently were unclear. In this study, we analysed the mucus of several cleaner fish clients to determine whether mucus feeding has a nutritional advantage over gnathiids and whether cleaner fish obtain their own MAA protection through this dietary mucus ingestion. Results show that host fish that are infected with gnathiids of poor nutritional value, in contrast to those that harbour gnathiids with higher nutritional value, continuously exude mucus that has both high nutritional value and high MAA content. These findings support the conclusion that in a competitive market for cleaners some host fish are forced to offer more than parasites to cleaners. Ultraviolet light that is not filtered by UV absorbing compounds such as MAA may still lead to DNA damage such as the formation of cyclobutane pyrimidine dimers (CPDs) or 6-4 photoproducts (6-4 PPs). However, coral reef fish have alternative mechanisms to overcome UV induced damage via the photolyase DNA repair mechanisms. We experimentally demonstrated for the first time that a coral reef fish species, the moon wrasse Thalassoma lunare has the ability to repair DNA damage via photoreactivation. Fish both with and without MAA protection were irradiated with UVB wavelength to induce DNA lesions. Half of the experimental fish were then exposed to photoreactivating wavelength to induce DNA repair 12 while the other fish were blocked from the repair mechanisms. Fish which had undergone DNA repair had the lowest number of lesions regardless of mucus MAA protection. When fish were blocked from photoreactivation wavelengths MAA sunscreens clearly served a photoprotective role. The amount of damage was greatest in fish which both lacked MAAs and which were also blocked from photoreactivating wavelengths. Thus for the overall UV protection of fish both the MAA sunscreens as well as the DNA repair system play a significant role in counteracting UV damage. Ultraviolet protection by MAA sunscreens is ubiquitous in marine fish. To date the same 5 MAA compounds (palythine (λmax 320 nm), asterina (λmax 330 nm), palythinol (λmax 332 nm), usujirene (λmax 357 nm) and palythene (λmax 360nm) have been identified in the mucus of several different species of reef fish from Australia. Here we report the first evidence of the presence of additional UV absorbing compounds found in the mucus of fish from Indonesia. Using UV spectroscopy the mucus of four species of fish was compared between both geographical regions. The presence of an additional peak between 294-296 nm wavelengths suggests the presence of gadusol and/or deoxygadusol, which are photoprotective compounds, thought to be the precursors of MAAs. Thus, UV protecting compounds in the mucus of fish may not be as conserved between different regions as previously assumed. Our knowledge concerning the effect of UV radiation has advanced considerably in the past decade and my research findings contribute to the better understanding of protective mechanisms of marine fish. The correlations I have found between UV attenuation/exposure, depth, and longitude of sampled individuals lead me to believe that mucus UV absorbing MAA compounds are a highly efficient adaptive defence.

The kinetics of drugs photodegradation have traditionally been treated using thermal kinetic analysis methods consisting most commonly in zero and first order kinetics. These treatment strategies were shown to lack specificity and present a number of limitations when applied to photoreactions kinetics. Nevertheless, these methods have widely been used due to a lack of integrated rate-laws for the majority of photoreactions types, in turn, due to the presence of a variable time-dependent factor in most photoreactions rate-laws that prevents their mathematical integration. To address these limitations, a new methodology for the development and validation of semi-empirical integrated rate-laws that faithfully describe photoreactions kinetics and photoreactions simulated cases generated by numerical integration methods (NIMs), is hereby presented. Using this methodology, a new kinetic order was ascribed to photoreactions namely the Φ-order kinetics. Semi-empirical integrated rate-laws were, thus, developed for three photoreaction types namely, unimolecular, AB(1Φ), photoreversible ,AB(2Φ), and consecutive, AB4(4Φ), photoreactions. The proposed models were further tested experimentally on drugs following these photodegradation mechanisms using; nifedipine and nisoldipine for unimolecular photoreactions; montelukast and fluvoxamine for photoreversible reactions; and riboflavin for consecutive photoreactions. The developed models not only accurately described the photoreaction kinetics of these drugs but also allowed the determination of all the kinetic parameters that characterise them. Furthermore, the above studied drugs were shown to act as precise and simple actinometers when analytically treated with the Φ-order kinetic methods, hereby presented. A universal standard method for the precise and worldwide reproducible study of drugs stability and compounds photoreactions, based on monochromatic irradiation and Φ-kinetics data analysis, is also detailed and adopted throughout the thesis. Finally, two new kinetic parameters namely, the pseudo-rate-constant and pseudo-initial velocity have been identified and shown to be more reliable and accurate in the description and universal comparison of photoreactions kinetics.

[ES] Los estudios sobre los daños en el ADN se han incrementado en las últimas décadas con el fin de profundizar en su implicación en la aparición del cáncer. Entre el gran número de lesiones del ADN, los aductos de tipo eteno han sido objeto de interés debido a su presencia en los tejidos humanos crónicamente inflamados. Asimismo, su cuantificación es útil para su uso como potencial biomarcador del cáncer de colon, próstata, pulmón, etc. Además, estas lesiones presentan propiedades altamente mutagénicas e inducen transiciones de bases o transversiones en las células de mamíferos. Los aductos de tipo eteno se generan principalmente de forma endógena como resultado de la peroxidación lipídica. Este proceso bioquímico produce aldehídos reactivos como el malondialdehído, que pueden combinarse con las bases del ADN creando un anillo exocíclico.Este anillo exocíclico proporciona a la nucleobase un sistema extendido p-conjugado que puede conferirles propiedades ópticas diferentes a las de las bases canónicas, lo que puede suponer una amenaza para la fotoestabilidad del ADN. Las bases canónicas tienen la capacidad de disipar la mayor parte de la energía recibida a través de canales no radiativos eficientes que conducen de nuevo al estado fundamental. Por lo tanto, los estudios sobre las propiedades ópticas de estos aductos etenos son clave para establecer si estas lesiones pueden poner en peligro la relajación eficiente de los estados excitados de las bases y desencadenar una fotorreactividad indeseada del ADN. La primera parte de la tesis trata de evaluar la fotoactividad potencial de estas lesiones del ADN mediante un estudio espectroscópico. En el Capítulo 3 se combinan experimentos de upconversión de fluorescencia en la escala de femtosegundos y cálculos teóricos (en los niveles PCM-TD-DFT y CASPT2/CASSCF) para proporcionar una imagen completa de la relajación de los estados excitados del aducto mutagénico 3,N4-eteno-2'-desoxicitidina (edC). El Capítulo 4 aborda las propiedades fotofísicas de los aductos junto con su fotorreactividad en presencia de fotosensibilizadores comunes como la Rosa de Bengala (RB) y la 4-carboxibenzofenona (CBP), prestando especial atención a la interacción con el 1O2. En condiciones aeróbicas, se observa una interacción con 1O2 para los tres aductos estudiados. Curiosamente, se observan los mismos fotoproductos, las nucleobases originales, para la irradiación en condiciones anaeróbicas, abriendo la posibilidad de un mecanismo mixto de Tipo I y Tipo II cuando se utiliza Rosa de Bengala como fotosensibilizador, y de Tipo I para la 4-carboxibenzofenona. Finalmente, el último capítulo trata de unir todos los conocimientos adquiridos sobre la fotorreactividad de los e-aductos para elegir el mejor cromóforo tratando de optimizar el proceso de reparación observado en el Capítulo 4. Para ello se utilizan sistemas híbridos de nanopartículas metálicas de Ag como matriz de soporte para la Rosa de Bengala. Las NPs metálicas, como las Ag NPs, poseen una resonancia plasmónica superficial localizada (LSPR), este efecto amplifica una gran variedad de fenómenos ópticos que pueden mejorar las propiedades ópticas de la Rosa de Bengala. / [CA] Els estudis sobre els danys en l'ADN s'han incrementat en les últimes dècades amb la finalitat d'aprofundir en la seua implicació en l'aparició del càncer. Entre el gran nombre de lesions de l'ADN, els adductes de tipus eteno han sigut objecte d'interés degut a la seua presència en els teixits humans crònicament inflamats, la qual cosa fa que la seua quantificació siga útil com a potencials biomarcadors del càncer de còlon, pròstata, pulmó, etc. A més, aquestes lesions presenten propietats altament mutagèniques i indueixen transicions de bases o transversions en les cèl·lules dels mamífers. Els adductes de tipus eteno es formen principalment de manera endògena com a resultat de la peroxidació dels lípids. Aquest procés bioquímic produeix aldehids reactius com el malondialdehid, que poden combinar-se amb les bases de l'ADN creant un anell exocíclic. Aquest anell exocíclic proporciona a les nucleobases un sistema estés p-conjugat que pot conferir-les propietats òptiques diferents a les de les bases canòniques, i que poden suposar una amenaça per a la fotoestabilitat de l'ADN. Les bases canòniques tenen la capacitat de dissipar la major part de l'energia d'excitació a través de canals no radiatius eficients que condueixen de nou a l'estat bàsic. No obstant això, els estudis sobre les propietats òptiques d'aquests adductes de tipus eteno són fonamentals per a deixar clar si aquestes lesions poden posar en perill aquesta relaxació eficient i desencadenar una fotoreactivitat indesitjada de l'ADN. La primera part de la tesi tracta d'estimar el potencial fotoactiu d'aquestes lesions de l'ADN mitjançant un estudi espectroscòpic. En el Capítol 3 es combinen experiments de "upconversió" de fluorescència en una escala de femtosegons i càlculs teòrics (en els nivells PCM-TD-DFT i CASPT2/CASSCF) per a proporcionar una imatge completa de la relaxació dels estats excitats del adducte mutagènic 3,N4-eteno-2'-desoxicitidina (edC). El Capítol 4 aborda les propietats fotofísiques dels adductes restants juntament amb el seua fotoreactivitat en presència d'alguns fotosensibilitzadors comuns com la Rosa de Bengala (RB) i la 4-carboxibenzofenona (CBP), prestant especial atenció a la interacció amb el 1O2. S'observa la interacció amb 1O2 per als tres adductes estudiats. Curiosament, s'observa la mateixa formació de nucleobases per a la irradiació en condicions anaeròbiques, obrint la possibilitat d'un mecanisme mixt de Tipus I i Tipus II quan s'utilitza Rosa de Bengala com fotosensibilitzador i de Tipus I per a la 4-carboxibenzofenona. Finalment, l'últim capítol tracta d'unir tots els coneixements adquirits sobre la fotoreactivitat dels adductes de tipus eteno per a triar el millor cromòfor tractant d'optimitzar el procés de reparació observat en el Capítol 4. Per a això s'utilitzen sistemes híbrids de nanopartícules metàl·liques de Ag com a matriu de suport per a la Rosa de Bengala. Les NPs metàl·liques, com les de Ag, posseeixen ressonància plasmónica superficial localitzada (LSPR), aquest efecte amplifica una gran varietat de fenòmens òptics que poden millorar les propietats òptiques de la Rosa de Bengala. / [EN] Studies dealing with DNA damages have increased during the last decades in order to get more insight into their involvement in the appearance of cancer. Among the large number of DNA lesions, etheno adducts have been the matter of interest because of their presence in chronically inflamed human tissues, making their quantification useful as potential biomarkers for cancer of colon, prostate, lung, etc. Moreover, these lesions exhibit highly mutagenic properties and induce base transitions or transversion in mammal cells. Etheno adducts are mainly formed endogenously as a result of lipid peroxidation. This biochemical process produces reactive aldehydes such as malondialdehyde, which can combine with DNA bases creating the exocyclic ring. This exocyclic ring provides to the nucleobases an extended p- conjugated system that might confer them optical properties different from those of the canonical bases, and can pose a threat to the DNA photostability. Canonical bases have the ability to dissipate most of the excitation energy through efficient nonradiative channels leading back to the ground state. However, the studies about the optical properties of this etheno adducts are basics to make it clear whether these lesions can jeopardize this efficient relaxation and trigger undesired DNA photoreactivity. The first part of the thesis establishes the potential photoactivity of these DNA lesions through a spectroscopic study. Chapter 3 joints femtosecond fluorescence upconversion experiments and theoretical calculations (at the PCM-TD-DFT and CASPT2/CASSCF levels) to provide a comprehensive picture of the mutagenic etheno adduct 3,N4-etheno-2'-deoxycytidine (edC) excited states relaxation. Chapter 4 addresses the photophysical properties of the adducts together with its photoreactivity in the presence of some common photosensitizers as Rose Bengal and 4-carboxybenzophenone, paying a special attention to interaction with 1O2. Interaction with 1O2 is observed for the three studied e-adducts. Interestingly, the same nucleobase formation is detected for irradiation under anaerobic conditions, opening the possibility of a mixed Type I and Type II mechanism when Rose Bengal is used as photosensitizer, and Type I for 4-carboxybenzophenone. Finally, the last chapter takes advantage of all the gained knowledge about the photoreactivity of e-adducts to choose the best chromophore and optimize the repair process observed in Chapter 4. To achieve this, hybrid systems of Ag metal nanoparticles are used as a support matrix for the Rose Bengal. Metal NPs, such as Ag NP, possess localized surface plasmon resonance (LSPR). This effect amplifies a wide variety of optical phenomena that can enhance the Rose Bengal optical properties. / Lizondo Aranda, P. (2022). Photoreactivity of DNA Etheno Adducts: Spectroscopic and Mechanistic Study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189688