(A) Photoregulation of DNA Functions by Cyclic Azobenzene-tethered Oligonucleotides (B) Site-specific Fluorescent Labeling of DNA using Inverse Electron Demand Diels-Alder Reaction between trans-Cyclooctene Derivatives and BODIPY-Tetrazine Adducts

(A) Most azobenzene-based photoswitches require UV light for photoisomerization, which limit their applications in biological systems due to possible photodamage. Cyclic azobenzene derivatives, on the other hand, can undergo cis-trans isomerization when exposed to visible light. A shortened synthetic scheme was developed for the preparation of a building block containing cyclic azobenzene and D-threoninol (cAB-Thr). trans-Cyclic azobenzene was found to thermally isomerize back to the cis-form in a temperature-dependent manner. cAB-Thr was transformed into the corresponding phosphoramidite and subsequently incorporated into oligonucleotides by solid phase synthesis. Melting temperature measurement suggested that incorporation of cis-cAB into oligonucleotides destabilizes DNA duplexes, these findings corroborate with circular dichroism measurement. Finally, Fluorescent Energy Resonance Transfer experiments indicated that trans-cAB can be accommodated in DNA duplexes. (B) Inverse Electron Demand Diels-Alder reactions (IEDDA) between trans-olefins and tetrazines provide a powerful alternative to existing ligation chemistries due to its fast reaction rate, bioorthogonality and mutual orthogonality with other click reactions. In this project, an attempt was pursued to synthesize trans-cyclooctene building blocks for oligonucleotide labeling by reacting with BODIPY-tetrazine. Rel-(1R-4E-pR)-cyclooct-4-enol and rel-(1R,8S,9S,4E)-Bicyclo[6.1.0]non-4-ene-9-ylmethanol were synthesized and then transformed into the corresponding propargyl ether. Subsequent Sonogashira reactions between these propargylated compounds with DMT-protected 5-iododeoxyuridine failed to give the desired products. Finally a methodology was pursued for the synthesis of BODIPY-tetrazine conjugates that will be used in future IEDDA reactions with trans-cyclooctene modified oligonucleotides.

Escala para valoración cliníca de fotoenvejecimiento cutáneo en la cara

Introducción: el fotoenvejecimiento cutáneo es causado principalmente por la exposición crónica y acumulativa a los rayos ultravioleta. Las clasificaciones y escalas existentes evalúan el envejecimiento cutáneo de forma global y no existe un instrumento que valore exclusivamente el fotoenvejecimiento cutáneo. Objetivo: Construir y realizar la validación de contenido, de una escala para valoración clínica del fotoenvejecimiento cutáneo en la cara. Materiales y métodos: Estudio de validez de contenido, de una escala para valoración clínica del fotoenvejecimiento cutáneo en cara, construida a partir del marco conceptual, y evaluada por 15 expertos con especialidades médicas que manejan el fotoenvejecimiento. Los datos obtenidos se analizaron así: porcentaje de la pertinencia; la media; coeficiente de variación (CV) e índice de validez de contenido por ítem (CVIi) de cada ítem incluido en la escala. Resultados: Para el grupo de 15 expertos evaluadores, la pertinencia de los 6 ítems incluidos en los criterios clínicos, arrojo una media de 4,2/5 con CVIi entre 73,3% y 100% y con CV por debajo del 20%; el ítem de surcos tuvo un CV de 26,93%; la pertinencia de los 5 ítems incluidos en los antecedentes, arrojo una media de 3,9/5, un CVIi entre 73,3% y 80% y un CV entre 33,67% y 39,84%. Discusión y conclusiones: La validez de contenido de los criterios clínicos de la escala cumplió en un nivel alto con la pertinencia, lo que indica que miden adecuada y exclusivamente las dimensiones del grado clínico de fotoenvejecimiento cutáneo en cara evaluado por los expertos.

Tryptophan photooxidation promoted by new hybrid materials prepared by condensation of naphthalene imides with silicate by the sol-gel process

Three novel hybrid organic/inorganic materials were synthesized from 4-substituted (NO(2), Br, H) 1,8-naphthalene imide-N-propyltriethoxysilane by the sol-gel process. These materials were obtained as a xerogel and partially characterized. The ability to photosensitize the oxidation and degradation of tryptophan indole ring by these materials was studied through photophysical and photochemical techniques. Although the derivatives containing Br and NO(2) as substituent do not cause efficient tryptophan photodamage, the hybrid material obtained from 1,8-naphthalic anhydride is very efficient to promote tryptophan photooxidation. By using laser flash photolysis it was possible to verify the presence of naphthalene imide transient radical species. The presence of oxygen causes an increase of the yield of radical formation. These results suggest that the mechanism of photodegradation of tryptophan occurs by type I, i.e. the transient radical (TrpH(center dot+)) formed by the direct reaction of the triplet state of the naphthalene imide moiety with tryptophan. Thus a inorganic-organic hybrid material that can be used to promote the oxidation of biomolecules was obtained. (C) 2009 Elsevier B.V. All rights reserved.

Determinação da dose eritematosa mínima como marcador de risco e sensibilidade à radiação ultravioleta

Objetivos: Determinar a dose eritematosa mínima (DEM) medida por exposição controlada à radiação ultravioleta-B (RUV-B), como limiar para dano solar agudo nos diversos fototipos, e medir a cor da pele constitucional pelo sistema colorimétrico CIELAB. Pacientes e Métodos: Um total de 194 voluntários, sadios, com idades acima de 18 anos, distribuídos em um mínimo de 30 participantes por fototipo. Todos foram classificados por fototipos segundo os critérios de Fitzpatrick. As regiões infra-axilar torácica e nádega foram irradiadas em 4 áreas de 1 cm2, assim como foi registrada a cor da pele desses locais pelo sistema CIELAB. Delineamento: Estudo transversal. Resultados: A média de idade dos participantes foi de 38 anos, sendo 68% do sexo feminino. A avaliação da associação entre as medidas das DEMs e dos valores colorimétricos da coordenada L*, mostrou uma correlação de Pearson negativa com r = -0,91 para um valor p<0,05. Para os valores das DEMs e os escores da classificação dos voluntários por fototipos, obteve-se correlação de Spearman (rs) de +0,95 para p<0,05 e, correlacionando os valores colorimétricos com os escores dos fototipos, encontrou-se em tórax um rs de -0,93 e em nádega -0,92 para um p < 0,05. Conclusões – Concluiu-se que: 1)- a mensuração dos valores colorimétricos da coordenada L* nas regiões infra-axilar torácica e nádega mostraram uma forte correlação com os valores das DEMs, sendo de menor poder invasivo e de maior praticidade para mensuração de sensibilidade à radiação ultravioleta; 2)- apesar de os escores de Fitzpatrick terem alta correlação com os valores das DEMs, mostraram superposição de valores nos fototipos adjacentes; 3)- o grau de associação das classes dos fototipos com a cor da pele permite dizer que a categoria numérica do fototipo aumenta à medida que a pele fica mais escura.

Daily dynamics of photosynthesis of the freshwater red alga Sirodotia delicatula (Batrachospermales, Rhodophyta)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Indocyanine green nanoparticles useful for photomedicine

Objective: the aim of this study was to evaluate the potential application of biodegradable nanoparticles (NPs) containing indocyanine green (ICG) in photodynamic therapy (PDT). Methods: Important parameters, such as particle size and external morphology, were established by dynamic light scattering (DLS) and scanning electron microscopy (SEM). Also, drug encapsulation efficiency and in vitro release behavior were evaluated by spectroscopic methods. Results: the particles are spherical in shape, they exhibit an 817-nm diameter, and they have a low tendency to aggregate. The loading efficiency was 65%. ICG photophysical parameters showed a bathocromic shift in ICG-loaded nanoparticles (ICG-NP). Analysis of the cell P388-D1 in the presence of the ICG-NP by SEM showed that the majority of the nanoparticles were uptaken by phagocytic cells after 2 h of incubation. After laser irradiation photodamage was observed in P388-D1 cells where ICG-NPs had been uptaken by phagocytic cells. Conclusion: Polymeric NPs work as an efficient drug delivery system for PDT drugs, and this approach can be used in the administration of amphiphilic photosensitizers in the treatment of neoplasic cells.

Photoprotective and antioxidant effects of Rhubarb: Inhibitory action on tyrosinase and tyrosine kinase activities and TNF-α, IL-1α and α-MSH production in human melanocytes

Background: Exposure to ultraviolet (UV) radiation causes various forms of acute and chronic skin damage, including immunosuppression, inflammation, premature aging and photodamage. Furthermore, it induces the generation of reactive oxygen species, produces proinflammatory cytokines and melanocyte-stimulating hormone (MSH) and increases tyrosinase activity. The aim of this study was to evaluate the potential photoprotective effects of Rheum rhaponticum L. rhizome extract on human UV-stimulated melanocytes.Methods: The effects of Rheum rhaponticum rhizome extract on tyrosine kinase activity, and on interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α), and α-MSH production in human epidermal melanocytes were evaluated under UV-stimulated and non-stimulated conditions. Antioxidant activity was evaluated by lipid peroxidation and 1,1-dyphenyl-2-picryl-hydrazyl (DPPH) assays, while anti-tyrosinase activity was evaluated by the mushroom tyrosinase method.Results: Rheum rhaponticum L. rhizome extract showed in vitro antioxidant properties against lipid peroxidation, free radical scavenging and anti-tyrosinase activities, and inhibited the production of IL-1α, TNF-α, α-MSH, and tyrosine kinase activity in melanocytes subjected to UV radiation.Conclusions: These results support the inclusion of Rheum rhaponticum L. rhizome extract into cosmetic, sunscreen and skin care products for the prevention or reduction of photodamage. © 2013 Silveira et al; licensee BioMed Central Ltd.

Respostas de curto período das características fotossintéticas da alga vermelha Batrachospermum delicatulum a temperatura e irradiância

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Five Percent 5-Fluorouracil in a Cream or for Superficial Peels in the Treatment of Advanced Photoaging of the Forearms: A Randomized Comparative Study

BACKGROUND The antimetabolite 5-fluorouracil (5-FU) is used for topical treatment of actinic keratosis. Overall improvement in the skin is also observed. Additionally, 5-FU was reported to be used for superficial peels.OBJECTIVES To evaluate the efficacy and safety of 5% 5-FU cream compared with peels for photodamaged forearms.METHODS This interventional, randomized, comparative, evaluator-blind study included 32 patients with severe photoaging of forearms. The regimens comprised either application of 5% 5-FU cream everyday for 4 weeks on 1 forearm and 4 weekly peels on the other. Efficacy assessment included: clinical photodamage scores, opinion of patients and investigators, and blind photographic evaluation by independent observers. Skin biopsies were performed for histologic and immunohistochemical analysis. Safety evaluation comprised observation of adverse events.RESULTS Clinical and histologic findings confirmed the benefits of topical 5% 5-FU, in cream or peels, which improved skin appearance and decreased the dermal elastotic material. Immunohistochemistry showed reduced levels of epidermal p53 and increase in the level of procollagen I. Results were maintained after 6 months. Predictable adverse events occurred, with no differences between treatments. Patients reported better tolerability to peels.CONCLUSION Five percent 5-FU cream or peels was safe and effective for the treatment of photodamaged forearms. Decreased epidermal p53 levels and new dermal collagen were confirmed.

Mechanism and Efficiency of Cell Death of Type II Photosensitizers: Effect of Zinc Chelation

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment.

Photodynamic Efficiency of Cationic meso-Porphyrins at Lipid Bilayers: Insights from Molecular Dynamics Simulations

Porphyrin derivatives have applications as photoactive drugs in photodynamic therapy. However, little is known about their interactions with phospholipid membranes at the molecular level. We employed molecular dynamics simulations to model the binding between a series of cationic meso-(N-methyl-4-pyridinium)phenylporphyrins and anionic phosphatidylglycerol lipid bilayers. This was done in the presence of molecular oxygen within the membrane. The ability of various porphyrins to cause photodamage was quantified in terms of their immersion depth and degree of exposition to a higher oxygen concentration inside the membrane. Simulations showed that the photodynamic efficiency could be improved as the number of hydrophobic phenyl substituents attached to the porphyrinic ring increased. In the specific case of porphyrins containing two hydrophobic and two charged substituents, the cis isomer was significantly more efficient than the trans. These results correlate well with previous experimental observations. They highlight the importance of both the total charge and amphiphilicity of the photosensitizer for its performance in photodynamic therapy.

Clinical, histopathological and immunohistochemical assessment of human skin field cancerization before and after photodynamic therapy

Background The field cancerization concept in photodamaged patients suggests that the entire sun-exposed surface of the skin has an increased risk for the development of (pre)-malignant lesions, mainly epithelial tumours. Topical photodynamic therapy (PDT) is a noninvasive therapeutic method for multiple actinic keratosis (AK) with excellent outcome. Objectives To evaluate the clinical, histological and immunohistochemical changes in human skin with field cancerization after multiple sessions of PDT with methyl-aminolaevulinate (MAL). Methods Twenty-six patients with photodamaged skin and multiple AK on the face received three consecutive sessions of MAL-PDT with red light (37 J cm(-2)), 1 month apart. Biopsies before and 3 months after the last treatment session were taken from normal-appearing skin on the field-cancerized area. Immunohistochemical stainings were performed for TP-53, procollagen-I, metalloproteinase-1 (MMP-1) and tenascin-C (Tn-C). Results All 26 patients completed the study. The global score for photodamage improved considerably in all patients (P < 0.001). The AK clearance rate was 89.5% at the end of the study. Two treatment sessions were as effective as three MAL-PDT sessions. A significant decrease in atypia grade and extent of keratinocyte atypia was observed histologically (P < 0.001). Also, a significant increase in collagen deposition (P = 0.001) and improvement of solar elastosis (P = 0.002) were noticed after PDT. However, immunohistochemistry showed only a trend for decreased TP-53 expression (not significant), increased procollagen-I and MMP-1 expressions (not significant) and an increased expression of Tn-C (P = 0.024). Conclusions Clinical and histological improvement in field cancerization after multiple sessions of MAL-PDT is proven. The decrease in severity and extent of keratinocyte atypia associated with a decreased expression of TP-53 suggest a reduced carcinogenic potential of the sun-damaged area. The significant increase of new collagen deposition and the reduction of solar elastosis explain the clinical improvement of photodamaged skin.

Photo- and redox-active supramolecular systems containing metal complexes

The aim of this Ph.D. project has been the photophysical and photochemical characterization of new photo- and redox-active supramolecular systems. In particular we studied two different classes of compounds: metal complexes and dendrimers. Two different families of bis-cyclometalated neutral Ir(III) complexes are presented and their photophysical properties are discussed. The first family of complexes contains two 2-phenylpyridyl (ppy) or 2-(4,6-difluorophenyl)pyridyl (F2ppy) cyclometalated ligands and an ancillary ligand constituted by a phenol-oxazoline (phox), which can be substituted in the third position with a fluorine group (Fphox). In the second part of this study, we present another family of bis-cyclometalated Ir(III) complexes in which the ancillary ligand could be a chiral or an achiral bis-oxazoline (box). We report on their structural, electrochemical, photophysical, and photochemical properties. Complexes containing phox and Fphox ancillary ligands show blue luminescence with very high quantum yield, while complexes with box ligands do not show particularly interesting photophysical properties. Surprisingly these complexes give an unexpected photoreaction when irradiated with UV light in presence of dioxygen. This photoreaction originates a stable, strong blue emitting and particularly interesting photoproduct. Three successive generations of a family of polyethyleneglycol (PEG)-coated Pd(II) tetrabenzoporphyrin (PdTBP)-based dendritic nanoprobes are presented, and their ability to sensitize singlet oxygen and inflict cellular photodamage are discussed. It was found that the size of the dendrimer has practically no effect on the singlet oxygen sensitization efficiency, that approximate the unity, in spite of the strong attenuation of the triplet quenching rate with an increase in the dendrimer generation. Nevertheless, when compared against a commonly used singlet oxygen sensitizer, as Photofrin, the phosphorescent probes were found to be non-phototoxic. The lack of phototoxicity is presumably due to the inability of PEGylated probes to associate with cell surfaces and/or penetrate cellular membranes. The results suggest that protected phosphorescent probes can be safely used for oxygen measurements in biological systems in vivo. A new family of two photoswitchable (G0(Azo) and G1(Azo)) dendrimers with an azobenzene core, two cyclam units as coordination sites for metal ions, and luminescent naphthalene units at the periphery have been characterized and their coordination abilities have been studied. Because of their proximity, the various functional groups of the dendrimer may interact, so that the properties of the dendrimers are different from those exhibited by the separated functional units. Both the naphthalene fluorescence and the azobenzene photoisomerization can be observed in the dendrimer, but it has been shown that (i) the fluorescent excited state of the naphthalene units is substantially quenched by excimer and exciplex formation and by energy transfer to the azobenzene units, and (ii) in the latter case the fluorescence quenching is accompanied by the photosensitized isomerization of the trans → cis, and, with higher efficiency, the cis → trans reaction. Complexation of these dendrimers, both trans and cis isomers, with Zn(II) ions shows that complexes of 1:1 and 2:1 metal per dendrimer stoichiometry are formed showing different photophysical and photochemical properties compared to the corresponding free ligands. Practically unitary efficiency of the sensitized isomerization of trans → cis and cis → trans reaction is observed, as well as a slight increase in the naphthalene monomer emission. These results are consistent with the coordination of the cyclam amine units with Zn(II), which prevents exciplex formation. No indication of a concomitant coordination of both cyclam to a single metal ion has been obtained both for trans and cis isomer.

Experiments on polar seaweeds

Organisms populating benthic shallow water systems of both polar regions are adapted to a particularly harsh environment. We studied effects of freezing and the combination of high light intensities and low water temperatures on photosynthesis of key macroalgal species from the Arctic intertidal (Fucus distichus) and Antarctic subtidal (Palmaria decipiens). Photosynthetic activity of F. distichus specimens was monitored during the freezing process; there was a marked decrease in quantum yield with decreasing temperatures, and a rapid recovery as soon as temperatures increased again. Thus, under the experimental conditions tested, no indication of photodamage was found. Specimens of Palmaria were exposed to a combination of high light intensities and low water temperatures. A persistent impairment of photosynthetic activity occurred at 0°C at light intensities of 400 µmol photons m-2 s-1. In all treatments, there was a decreasing ratio of phycobiliproteins to chlorophyll a. Overall, the two studies provide baseline data for interpreting physiological responses of two important macroalgal species in an extreme environment, the polar coastal ecosystem.