Indiana University-Purdue University Indianapolis (IUPUI) / Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(R-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.

<p>Bacterial endospores have been a topic of research interest over the last several decades given their high resistance to ultraviolet (UV) damage. Unlike vegetative bacterial cells, which form cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs) as the major product upon UV irradiation, endospore bacteria form a spore photoproduct (5-(<em>R</em>-thyminyl)-5,6-dihydrothymine or SP) as the major product. Vegetative bacteria cells are subject to regular cell activities and processes such as division and deoxyribonucleic acid (DNA) replication, which are prone to damage from UV exposure. However, in endospores, which have a largely anhydrous inner environment, the DNA remains dormant when bound to spore-specific small acid-soluble proteins (SASP) and dipicolinic acid, making spores highly resistant to radiation, heat, desiccation, and chemical harm. During spore germination, SP lesions in DNA are repaired by a distinctive repair enzyme, spore photoproduct lyase (SPL). In this thesis, molecular dynamics (MD) simulations were carried out to (i) examine how the formation of the SP lesion in DNA affects the global and local structural properties of duplex DNA and (ii) study how this lesion is recognized and repaired in endospore. The first part of this work was focused on designing and developing a structurally and dynamically stable model for dinucleotide SP molecule (TpT), which was subsequently used as an SP patch incorporated into duplex DNA. Computationally, this requires modifications of the bond and nonbonded force field parameters. The stability of the patch and developed parameters was tested via solution-phase MD simulations for the SP lesion incorporated within the B-DNA dodecamer duplex (PDB 463B). The second part involved applying the new SP patch to simulate the crystallographic structure of the DNA oligomer containing SP lesions. Solution-phase MD simulations were performed for the SP-containing DNA oligomers (modeled based on PDB 4M94) and compared to the simulations of the native structure (PDB 4M95). Our analysis of the MD trajectories revealed a range of SP-induced structural and dynamical changes, including the weakened hydrogen bonds at the SP sites, increased DNA bending, and distinct conformational stability and distribution. In the third part of this thesis project, we carried out MD simulations of SP-containing DNA bound with SASPs to examine how the DNA interacts differently with SASP in the presence and absence of the SP lesion. The simulation results suggested decreased electrostatic and hydrogen bonding interactions between SASP and the damaged DNA at the SP site compared to the undamaged DNA-protein complex. In addition, decreased helicity percentage was observed in the SASPs that directly interact with the SP lesion. The last part of this this thesis work focused on the SP-dimer flipping mechanism, as the lesion is likely flipped out to its extrahelical state to be recognized and repaired by SPL. Using steered molecular dynamic (SMD) simulations and a pseudo-dihedral angle reaction coordinate, we obtained possible SP flipping pathways both in the presence and absence of SASP. Collectively, these simulation results lend new perspectives toward understanding the unique behavior of the SP lesion within the DNA duplex and the nucleoprotein complex. They also provide new insights into how the SP lesion is efficiently recognized and repaired during spore germination.</p>

Vitamin D deficiency due to, e.g., nutritional and life style reasons is a health concern that is gaining increasing attention over the last two decades. Vitamin D3, the most common isoform of vitamin D, is only available in food derived from animal sources. However, mushrooms and yeast are rich in ergosterol. This compound can be converted into vitamin D2 by UV-light, and therefore act as a precursor for vitamin D. Vitamin D2 from UV-irradiated mushrooms has become an alternative source of vitamin D, especially for persons pursuing a vegan diet. UV-irradiated baker’s yeast (Saccharomyces cerevisiae) for the production of fortified yeast-leavened bread and baked goods was approved as a Novel Food Ingredient in the European Union, according to Regulation (EC) No. 258/97. The Scientific Opinion provided by the European Food Safety Authority Panel on Dietetic Products, Nutrition, and Allergies has assessed this Novel Food Ingredient as safe under the intended nutritional use. However, recent findings on the formation of side products during UV-irradiation, e.g., the photoproducts tachysterol and lumisterol which are compounds with no adequate risk assessment performed, have only been marginally considered for this EFSA opinion. Furthermore, proceedings in analytics can provide additional insights, which might open up new perspectives, also regarding the bioavailability and potential health benefits of vitamin D-fortified mushrooms and yeast. Therefore, this review is intended to give an overview on the current status of UV irradiation in mushrooms and yeast in general and provide a detailed assessment on the potential health effects of UV-irradiated baker’s yeast.

Vitamin D deficiency due to, e.g., nutritional and life style reasons is a health concern that is gaining increasing attention over the last two decades. Vitamin D3, the most common isoform of vitamin D, is only available in food derived from animal sources. However, mushrooms and yeast are rich in ergosterol. This compound can be converted into vitamin D2 by UV-light, and therefore act as a precursor for vitamin D. Vitamin D2 from UV-irradiated mushrooms has become an alternative source of vitamin D, especially for persons pursuing a vegan diet. UV-irradiated baker’s yeast (Saccharomyces cerevisiae) for the production of fortified yeast-leavened bread and baked goods was approved as a Novel Food Ingredient in the European Union, according to Regulation (EC) No. 258/97. The Scientific Opinion provided by the European Food Safety Authority Panel on Dietetic Products, Nutrition, and Allergies has assessed this Novel Food Ingredient as safe under the intended nutritional use. However, recent findings on the formation of side products during UV-irradiation, e.g., the photoproducts tachysterol and lumisterol which are compounds with no adequate risk assessment performed, have only been marginally considered for this EFSA opinion. Furthermore, proceedings in analytics can provide additional insights, which might open up new perspectives, also regarding the bioavailability and potential health benefits of vitamin D-fortified mushrooms and yeast. Therefore, this review is intended to give an overview on the current status of UV irradiation in mushrooms and yeast in general and provide a detailed assessment on the potential health effects of UV-irradiated baker’s yeast.

Indiana University-Purdue University Indianapolis (IUPUI) / Spore photoproduct lyase (SPL) is a radical SAM (S-adenosylmethionine) enzyme that is responsible for the repair of the DNA UV damage product 5-thyminyl-5,6-dihydrothymine (also called spore photoproduct, SP) in the early germination phase of bacterial endospores. SPL initiates the SP repair process using 5'-dA• (5'-deoxyadenosyl radical) generated by SAM cleavage to abstract the H6proR atom which results in a thymine allylic radical. These studies provide strong evidence that the TpT radical likely receives an H atom from an intrinsic H atom donor, C141 in B. subtilis SPL. I have shown that C141 can be alkylated in native SPL by iodoacetamide treatment indicating that it is accessible to the TpT radical. Activity studies demonstrate a 3-fold slower repair rate of SP by C141A which produces TpTSO2 - and TpT simultaneously with no lag phase observed for TpTSO2- formation. Additionally, formation of both products shows a Dvmax kinetic isotope effect (KIE) of 1.7 ± 0.2 which is smaller than the DVmax KIE of 2.8 ± 0.3 for the WT SPL reaction. Removal of the intrinsic H atom donor by this single mutation disrupts the rate-limiting process in the enzyme catalysis. Moreover, C141A exhibits ~0.4 turnover compared to the > 5 turnovers in the WT SPL reaction. In Y97 and Y99 studies, structural and biochemical data suggest that these two tyrosine residues are also crucial in enzyme catalysis. It is suggested that Y99 in B. subtilis SPL uses a novel hydrogen atom transfer pathway utilizing a pair of cysteinetyrosine residues to regenerate SAM. The second tyrosine, Y97, structurally assists in SAM binding and may also contribute to SAM regeneration by interacting with radical intermediates to lower the energy barrier for the second H-abstraction step.

Wird DNA mit UV-Licht bestrahlt, kommt es zur Bildung von Photoschäden, die zu Zelltod oder Krebs führen können. In dieser Arbeit wird die primäre Elektronentransferreaktion des lichtaktivierten Reparaturprozesses des (6-4)-Schadens in Drosophila melanogaster charakterisiert. Der katalytische Reparaturzyklus wird durch das Flavoprotein (6-4)-Photolyase (PL) realisiert. Der Elektronentransfer (ET) vom Flavin-Adenin-Dinukleotid (FADH⁻) Kofaktor zum Schaden initiiert die molekularen Umlagerungen. Diese Arbeit charakterisiert die primäre ET Reaktion mithilfe von molekulardynamischen Langzeitsimulationen (µs) in Kombination mit Quantenmechanik/Molekularmechanik-Simulationen. Ab initio lokale Coupled-Cluster- und Dichtefunktionaltheorierechnungen wurden angewendet, um die relative Energetik von lokal angeregten und Ladungstransferzuständen des (6-4)-Reparaturkomplexes zu charakterisieren. Es zeigt sich, dass die Reduktion des (6-4)-Schadens durch einen Ladungstransferzustand ermöglicht wird an dem die Adeninstruktur des FADH⁻ -Kofaktors beteiligt ist. Über die Simulationen wird ein mikroskopisches Bild der Reaktionskoordinate der Elektronentransferreaktion im Marcusbild entwickelt. Diese ist nicht vollständig durch parabolische freie Energiekurven beschrieben sondern wird, durch Wechselwirkungen in der aktiven Tasche, ein Multiminima-Reaktionspfad ausgebildet. Hierbei hat die Rotation der Seitenkette der benachbarten, geladenen Aminosäure Lys246 dominanten Einfluss. Dies legt nahe, dass die primäre ET Reaktion der (6-4) Schadensreparatur, einen vom Adenin unterstützten ET Weg von der PL zur 5’ Seite des Schadens nimmt. Dieser Prozess wird durch benachbarte Aminosäuren und einer Stärkung der Wasserstoffbrücken mit Wassermolekülen stabilisiert. Die Ergebnisse dieser Arbeit zeigen, dass ET-Reaktionen in komplexen enzymatischen Systemen nicht im Kontinuumsbild von ET beschrieben werden können, da lokale Wechselwirkungen drastischen Einfluss auf die ET Reaktionen haben. / UV-light irradiation of DNA leads to the formation of photolesions that can cause cell death and cancer. This thesis aims at the characterization of the primary electron transfer (ET) reaction in the photoactivated repair process of the (6-4) lesion in Drosophila melanogaster. The catalytic repair cycle is realized by a flavoprotein called photolyase (PL). The ET from the fully reduced flavin-adenine-dinucleotide (FADH⁻) cofactor of the PL to the lesion initiates molecular rearrangements. In this thesis fluctuation properties of the enzyme environment on the excited states are considered by conducting long-time (µs) molecular dynamics simulations combined with extensive quantum mechanical/molecular mechanical simulations. Ab initio local coupled cluster simulations and density functional theory are applied to characterize the relative energetics of locally excited and charge transfer (CT) states in the (6-4) lesion repair complex. Reduction of the (6-4) lesion is found to be enabled by a CT state involving the adenine moiety of the FADH⁻ cofactor. Microscopic characterization of a Marcus-type free energy reaction coordinate reveals that it cannot be fully described by parabolic free energy curves. Specifically, rotation of the side chain of nearby charged amino acid Lys246 imposes a double-well character on the potential energy surface along the reaction coordinate of the ET. For the ET reaction triggering the catalytic (6-4) lesion repair, the findings of this thesis suggest an ET pathway to the 5’ side of the (6-4) lesion mediated by the adenine moiety. The process is stabilized by neighboring amino acids and a strengthening of hydrogen bonds with water molecules. The presented results demonstrate that ET reactions in complex enzymatic systems cannot be described within the continuum ET picture, as local interactions drastically tune the ET reaction.

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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535

[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