This study is focused on the use of visible light in photoinitiated cationic polymerization. Photoinitiated polymerization of oxiranes, vinyl ethers, and other vinyl monomers was achieved. In doing so, (2-(2,3 dihydrobenzo [b][1,4]dioxin-6-yl)-3-(2,3-dihydrobenzo[b]-[1,4]dioxin-7-yl)-5-(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)-8-(2,3-dihydrothieno[3,4-b][1,4]dioxin-7yl) quinoxaline) (DBQEd) and poly(2,3,5,8-tetra(thiophen-2-yl)quinoxaline) (TTQ), two dibenzo[a,c]phenazine derivatives / 10,13-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)dibenzo[a,c] phenazine (PHED) and 10,13-bis(4-hexylthiophen-2-yl)dibenzo[a,c]phenazine (PHEHT) were utilized as the photosensitizers for diaryliodonium salt photoinitiators. Novel dyes based on the dibenzo[a,c]phenazine and quinoxaline skeleton were shown to be efficient in carrying out the cationic photopolymerizations of a wide variety of epoxide, oxetane, and vinyl monomers at room temperature upon irradiation with long-wavelength UV and visible light. The polymerizations were initiated at room temperature in the presence of diphenyliodonium hexafluorophosphate (Ph2I+PF-6) and monitored by optical pyrometry (OP). The photopolymerization of an epoxide monomer via solar irradiation was also demonstrated.

Les cellules solaires à colorant (DSSC) sont une alternative sérieuse aux cellules à base de silicium. Le principe de fonctionnement repose sur la photosensibilisation d'un semi-conducteur par un colorant qui est en général un complexe polypyridinique du ruthénium (II). La modulation des propriétés de ces complexes permet d'optimiser les performances des cellules solaires correspondantes. Dans cette thèse, nous avons synthétisé et étudié l'effet de nouveaux ligands bipyridiniques à substituants électro-donneurs [pi]-délocalisés à base de dithiénylpyrroles (DTP). Ces motifs induisent, dans les complexes homoleptiques, bis- et tris-hétéroleptiques du Ru(II), des effets bathochromes (lorsque les motifs DTP sont liés par leur cycle thiophénique à la bipyridine) et d'importantes augmentations des coefficients d'extinction molaires. Les nouveaux composés ont été caractérisés par spectroscopies, électrochimie, photophysique et calcul théorique. Deux complexes hétéroleptiques ont été testés en cellule DSSC. Si la collecte de photons est excellente, les performances restent en dessous de celles de colorants de référence. Comme en attestent les courbes J/V et les courbes IPCE. Ce résultat peut-être dû à une limitation lors de l'injection dans la bande de conduction ou encore à une gêne stéréo-électronique provoquée par le ligand lors de la réduction du colorant oxydé (Ru(III) par le médiateur / Dye-sensitized Solar Cells (DSSC) appear to be promising devices. Operation principle relies on the photosensitization of a wide-gap semiconductor with a dye, the latter typically being a polypyridinyl ruthenium(II) complex. Modulation of the properties of such complexes enables the optimization of the corresponding solar cells' performances. In the present work, we synthesized and investigated the effect of new bipyridine ligands bearing electron-donating dithienylpyrroles (DTP). These moieties induced red-shifts of the absorption spectra in homoleptic, bis- and tris-heteroleptic Ru(II) complexes especially when the DTP was bound by its thiophene unit to the bipyridine ligand. A notable increase of the molar extinction coefficients was also obtained. All new compounds have been characterized by using spectroscopic, electrochemical, photophysical and computational chemistry techniques. Two heteroleptic complexes have been tested in DSSCs. Despite excellent light harvesting properties, performances were found lower than those of standard dyes as revealed by J/V and IPCE curves. Stereoelectronic effects could be involved since the bulky DTP moiety could impede an efficient access of the mediator to Ru(III) centers

O basidiomiceto Cryptococcus neoformans é um fungo saprófita, com ampla distribuição geográfica, que é normalmente isolado de solos que contêm excretas de pombos e detritos vegetais. Apesar de saprófita, o fungo pode infectar e causar doença em uma grande variedade de hospedeiros animais, como mamíferos, aves e insetos. O número de casos de micoses graves, causadas por C. neoformans e por outros gêneros de fungos, tem aumentado em todo o mundo, principalmente devido ao aumento do número de indivíduos imunocomprometidos. Adicionalmente, a emergência de novas espécies de patógenos e a seleção de linhagens tolerantes aos antifúngicos comumente utilizados fazem com que o desenvolvimento de novas estratégias para o controle de fungos seja extremamente desejável. A inativação fotodinâmica de fungos é um método novo e promissor, que pode ser utilizado tanto para o controle de micoses (em animais e em vegetais), como para a eliminação de fungos do ambiente. A fotoinativação de fungos é baseada no uso de fotossensibilizadores que se acumulam ou que são preferencialmente metabolizados pelas células do microrganismo-alvo. A seguir, o fotossensibilizador é exposto à luz visível, que, na presença de oxigênio, inicia processos fotoquímicos que produzem uma série de espécies reativas de oxigênio capazes de matar as células fúngicas sem provocar danos significativos nos tecidos do hospedeiro. Neste trabalho, foram avaliadas as eficácias do azul de metileno (MB) (em solução e em nanoemulsão), da cloro-alumínio ftalocianina (em nanoemulsão) e do complexo nitrosilo de rutênio (em solução) como fotossensibilizadores para a fotoinativação de células melanizadas e não melanizadas de C. neoformans. C. neoformans foi suscetível à fotoinativação pela cloro-alumínio ftalocianina, com uma inativação próxima de 100% quando foi utilizada uma combinação apropriada da concentração do fotossensibilizador e da dose de luz. A completa fotoinativação do fungo pela ftalocianina, em condições compatíveis com a terapia fotodinâmica, abre a perspectiva da utilização desse fotossensibilizador para o tratamento de micoses causadas por C. neoformans. A fotoinativação pelo MB foi apenas parcial e não ocorreu fotoinativação pelo nitrosilo de rutênio. Nenhum dos fotossensibilizadores matou o fungo na ausência da luz. Também foram feitos experimentos para se verificar a influência do tempo de crescimento e da melanização na tolerância de C. neoformans à fotoinativação pelo MB. Houve diferença significativa na tolerância entre diferentes linhagens de C. neoformans. Para a maioria dos tratamentos [linhagens e tempo de crescimento (4, 6 ou 8 dias)], não houve diferença significativa entre a tolerância de células melanizadas e não melanizadas. Também não foi observada diferença na tolerância entre células com idade de 4 a 8 dias. / The basidiomycete Cryptococcus neoformans is a saprophytic worldwide fungus which is normally isolated from soils contaminated with pigeon excreta and plant detritus. Despite a saprophytic existence, the fungus can infect and cause disease in a wide variety of animal hosts such as mammals, birds and insects. Serious infections caused by C. neoformans and by other genera of fungi have emerged all over the world, primarily due to the increased numbers of immunocompromised individuals. Additionally, the emergence of new species and antimycotic-resistant strains of pathogenic fungi makes the development of new fungus-control techniques highly desirable. Photodynamic inactivation of fungi is a new and promising method that can be used to control localized mycoses or kill fungi in the environment. The photodynamic inactivation of fungi is based on the use of a photosensitizer that accumulates in, or preferentially is metabolized by, cells of the target microorganism. The photosensitizer is then exposed to visible light in the presence of oxygen, and this starts photochemical processes that produce a series of reactive oxygen species (ROS) capable of killing the fungal cells without causing significant damage to host tissues. We report here the efficacy of methylene blue (MB) (in solution and in nanoemulsion), chloroaluminum phthalocyanine (in nanoemulsion) and nitrosyl ruthenium complex (in solution) as photosensitizers in photoinactivation of melanized and nonmelanized Cryptococcus neoformans yeast cells. C. neoformans were susceptible to photoinactivation by chloroaluminum phthalocyanine with inactivation close to 100% when the appropriate combination of photosensitizer concentration and light-exposure dose was used. Photoinactivation by MB was only partial and nitrosyl ruthenium complex was ineffective. In the dark, neither photosensitizers inactivated the fungus. Complementary experiments were performed to estimate the effect of the age of the cells and of melanization in the fungus tolerance to photoinactivation by MB. There was a significative difference in the tolerance among strains of C. neoformans. For most of the treatments (strains and time of growth) there was no difference between the tolerance of melanized and nonmelanized cells. There was no difference in the tolerance among 4 to 8-day cells either.

Zusammenfassung Transgene Tabakpflanzen, in denen wegen der Expression von Antisense-RNA für zwei Enzyme der Tetrapyrrolbiosynthese die Aktivität der Uroporphyrinogen-Decarboxylase bzw. Coproporphyrinogen-Oxidase vermindert ist, akkumulieren in starkem Ausmaß die Substrate der betroffenen Enzyme. Diese Porphyrinogene werden bei Anwesenheit von Sauerstoff autoxidativ oder durch unspezifische Peroxidasen in ihre photosensibilisierenden Derivate (Porphyrine) umgewandelt, welche die Ursache für zelluläre Schäden darstellen, die sich makroskopisch in Form von Blattläsionen zeigen. Im Verlauf der durch die angehäuften Porphyrine ausgelösten Reaktionen, die u.a. den Folgen der Behandlung von Pflanzen mit photodynamisch wirksamen Herbiziden und auch den Symptomen von Porphyria-Erkrankungen des Menschen ähneln, werden Membranlipide durch Peroxidation geschädigt und alle lichtexponierten Zellen massivem oxidativem Streß ausgesetzt. Dieser läßt sich anhand der kompartiment-übergreifenden und alle Ebenen der Expression umfassenden Aktivierung des antioxidativen Schutzsystems belegen. So können die Transkriptakkumulation und verstärkte Anhäufung der Proteine sowie der damit verbundene Anstieg der Aktivität von Superoxid-Dismutase und Ascorbat-Peroxidase nachgewiesen werden. Die erhöhte Aktivität der Schutzenzyme führt zu beschleunigtem Umsatz und größerem Bedarf an niedermolekularen Antioxidantien wie Ascorbat und Tocopherol. Gleichzeitig kommt es lokal beschränkt auf Blattgewebe in der Umgebung von Nekrosen zur Kreuzinduktion von pathogenese-assoziierten Prozessen, wie der Akkumulation von "pathogenesis related" (PR)-Proteinen, von Salicylsäure und der antimikrobiell wirksamen phenolischen Verbindung Scopolin. Diese Aktivierung der Pathogenabwehr wird durch die verminderte Ausbreitung einer Infektion der Pflanzen mit dem Tabak Mosaikvirus (TMV) widergespiegelt. Der Prozeß der Photosensibilisierung ist stark licht- und sauerstoffabhängig und wird außerdem durch erhöhte Temperaturen beschleunigt. Wird eine bestimmte Lichtdosis überschritten, werden die Porphyrine durch die dann rasch irreversibel geschädigten Plastiden freigesetzt, und es kommt wegen des nachfolgenden Absterbens von Gewebe zur Ausbildung der Blattläsionen. Dieser Effekt wurde ausgenutzt, um die Kinetik von Prozessen der antioxidativen und Pathogenabwehr in den Pflanzen zu untersuchen. Mittels Subtraktiver Suppressions-Hybridisierung (SSH) wurde eine subtrahierte cDNA-Bank angelegt, in der Gene vertreten sind, deren Expression bereits sehr früh als Reaktion auf die Photosensibilisierung induziert wird. Unter diesen Genen sind auch vermutete Komponenten von Signaltransduktionskaskaden einschließlich Transkriptionsfaktoren. Etwas überraschend war die Erkenntnis, daß vor allem pathogenese- und zelltod-assoziierte Prozesse frühzeitig aktiviert werden, die klassischen Enzyme der antioxidativen Streßabwehr aber erst später reagieren. Bei letzteren kommt es vor Veränderungen in der Expression zuerst zu einem Anstieg der Aktivität, der sich in verändertem Gehalt und Redoxstatus der niedermolekularen Antioxidantien niederschlägt. Insgesamt stellt sich die durch Antisense-Expression verursachte Deregulation der Tetrapyrrolbiosynthese und die daraus resultierende Photosensibilisierung als komplexes System dar, in dem antioxidative und Pathogenabwehr kreuzinduziert werden und welches als Modell zur Aufklärung von Mechanismen der Streßabwehr wertvolle Aussagen liefert. / Abstract Transgenic tobacco plants expressing antisense RNA for two enzymes of the tetrapyrrole biosynthetic pathway are characterized by decreased enzyme activity of either uroporphyrinogen decarboxylase or coproporphyrinogen oxidase and accumulate the substrates of these enzymes in large amounts. If oxygen is present the accumulated porphyrinogens are transformed either by autoxidation or unspecific peroxidases into their photosensitizing derivatives (porphyrins). They are the molecular basis for cellular damage which eventually becomes visible as leaf lesions. During photosensitization membrane lipids are damaged due to peroxidation reactions and most of the symptoms observed are similar to the effects caused by the treatment of plants with photodynamic herbicides, or resemble the characteristics of porphyria diseases in human. These plants suffer from oxidative stress which is concluded from the general and intercompartimental activation of the antioxidative stress defense system. Transcripts and proteins of superoxide dismutase and ascorbate peroxidase accumulate and the corresponding enzyme activities are increased. This increase leads to accelerated turnover and enhanced demand of low molecular weight antioxidants like ascorbate and tocopherol. Simultaneously, cross induction of pathogenesis-related responses is observed in the plants, like the accumulation of PR proteins, salicylic acid and the antimicrobial phenolic compound scopolin. The activation of the pathogen defense system leads to restricted spread of Tobacco Mosaic Virus (TMV) infection in the transgenic plants. Photosensitization strongly depends on light and oxygen and is enhanced by elevated temperature. Porphyrins are only released from rapidly damaged plastids into other cellular compartments if a certain light dose is exceeded. Then cell death commences and dead tissue becomes visible as leaf lesions. This light dosage effect was exploited to investigate the kinetics of antioxidative and pathogen defense responses upon light shift. By the use of Suppression Subtractive Hybridization (SSH) a subtracted cDNA library was established that contains genes which are early induced in response to photosensitization. The library also contains putative components of signal transduction chains and transcription factors. Unexpectedly, the expression of genes related to pathogenesis and cell death is rather early induced while the cellular antioxidative stress defense system responds later. The activity of antioxidative enzymes is increased before changes in transcript or protein accumulation occur, resulting in changes of content and redox ratio of low molecular weight antioxidants. In summary, photosensitization caused by the expression of antisense RNA and resulting in the deregulation of tetrapyrrole biosynthesis is a complex model system in which antioxidative and pathogen defense responses are induced. The approach can be used to further elucidate mechanisms of stress response.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-07-17T11:35:24Z No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-18T10:31:42Z (GMT) No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-18T10:31:42Z (GMT). No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The genus Brachiaria has provided important forage species to the countries located in tropical regions. However, this genus of grass is associated with photosensitization and poisoning in animal production. Thus, the aim of this study was to establish the standard for collection, processing, and conservation of Brachiaria brizantha for protodioscin quantification and spore count from Pithomyces chartarum and the evaluation of guinea pig (Cavia porcellus) as an experimental model of poisoning by diosgenin. For Brachiaria brizantha evaluation, samples were collected from eight pastures, which were subdivided into young leaves, mature leaves, old leaves, and whole plant. After that, the samples were submitted to nine different drying and conservation treatments. After separation and treatment of the samples, saponins were quantified and spores were counted. In most of treatments, the young leaves presented larger amounts of protodioscin than the old leaves and the whole plant. The treatments that maintained the highest amounts of protodioscin were room temperature, oven, and oven with forced air circulation. No fungal spores were found in the pasture samples. Thus, we concluded that the concentration of protodioscin varies with the maturation stage of the leaves; drying at room temperature, in an oven, or in an oven with forced air circulation are the best methods, and freezing plants for preservation of protodioscin is not recommended. In the guinea pigs evaluation, 14 guinea pigs were divided into two groups, the treatment group (TG) and the control group (CG). The TG received 480 mg/kg of diosgenin and the CG received only vegetable oil. Both groups received the treatment for 30 days. Blood samples were collected on day 0 (M0) and day 30 (M30) for blood cell count and biochemical analyzes. In clinical biochemistry, the analyzes were performed for alanine aminotransferase, aspartate aminotransferase, gamma glutamyl trasferase, alkaline phosphatase, urea, creatinine, glucose, cholesterol, HDL, triglycerides, lactate, cholinesterase, and calcium. Hematology and biochemistry did not present alterations, which could not be related to hepatic disease caused by saponins. In histology, only one animal had bile duct hyperplasia, the other alterations observed were generally nonspecific. Therefore, we concluded that the administration of 480 mg/kg of diosgenin to guinea pigs did not produce significant hepatic lesions or hematological and biochemical alterations related to liver disease. / O gênero Brachiaria possui importantes espécies de forrageiras utilizadas em países localizados em regiões tropicais. No entanto, esse gênero de gramíneas está associado à fotossensibilização e intoxicações em animais de produção. Deste modo, objetivou-se com este estudo estabelecer o padrão para colheita, processamento e conservação de amostras de Brachiaria brizantha para quantificação de protodioscina e contagem de esporos de Pithomyces chartarum e a avaliação da Cavia porcellus (cobaias) como modelo experimental na intoxicação por diosgenina. Para a avaliação da Brachiaria brizantha, amostras foram colhidas de oito pastagens as quais foram subdivididas em folhas jovens, folhas maduras, folhas velhas e planta inteira e submetidas a nove diferentes métodos de processamento. Após separação e tratamento das amostras foi realizada a quantificação de saponinas e a contagem de esporos. Observou-se que na maioria dos tratamentos as folhas jovens apresentaram quantidades maiores de protodioscina que as folhas velhas e a planta inteira. Os tratamentos que mantiveram as maiores quantidades de protodioscina foram temperatura ambiente, estufa e estufa com ventilação forçada. Nas amostras das pastagens não foram encontrados esporos do fungo. Desta forma, conclui-se que a concentração de protodioscina é maior nas folhas jovens; a secagem em temperatura ambiente, em estufa ou em estufa de ventilação forçada são os melhores métodos para preservação de protodioscina e o congelamento de plantas para a quantificação de protodioscina não é recomendado. Para a avaliação das cobaias como modelos experimentais na intoxicação por diosgenina foram utilizadas 14 cobaias, divididas em dois grupos, o grupo tratado (GT) e o grupo controle (GC). O GT recebeu 480 mg/kg de diosgenina diluída em óleo vegetal e o GC recebeu somente o óleo vegetal, ambos por 30 dias. Para avaliação do hemograma e análises bioquímicas foram colhidas amostras sanguíneas no dia 0 (M0) e no dia 30 (M30). Na bioquímica plasmática foram realizadas análises para ALT, AST, GGT, ALP, ureia, creatinina, glicose, colesterol, HDL, triglicerídeos, lactato, colinesterase e cálcio. A hematologia e a bioquímica não apresentaram alterações relacionadas à doença hepática causada por saponinas. Na histologia apenas um animal apresentou hiperplasia de ductos biliares, as outras alterações observadas foram de forma geral inespecíficas. Conclui-se que, na dose utilizada, a administração da diosgenina a cobaias não produz lesões hepáticas expressivas nem alterações hematológicas e bioquímicas relacionadas a hepatopatia.

Made available in DSpace on 2016-04-28T20:16:32Z (GMT). No. of bitstreams: 1 2009 - Josilene Nascimento Seixas.pdf: 4044215 bytes, checksum: dfe1223a8c9106028077731ca7dd2fe5 (MD5) Previous issue date: 2009-02-18 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Numerous outbreaks of photosensitization in herbivorous animals maintained on Brachiaria pastures occur in Brazil, and the economic losses caused are of great concern due to the large areas in the country planted with this grass. The vast majority of outbreaks has been attributed to the sporidesmin containing spores of Pithomyces chartarum, a fungus which occurs in many countries of temperate climate. However, there are differences between the liver lesions in animals that develop photosensitization on pastures of Brachiaria spp and the ones described in P. chartarum poisoning. In several outbreaks reported from Brazil, no evidence for toxicity of the spores was revealed. As the isolation of toxic saponins from Brachiaria grasses has been reported in the literature, the real cause of the photosensitization in Brazil needs to be clarified, in order to be able to adopt correct preventive measures to avoid the condition. The main objective of this study was to show the different epidemiological, clinical, pathological and toxicological aspects of the two poisonings, based on data from the literature and our own observations of pithomycotoxicosis in Portugal and on the data of the photosensitization which occurs on Brachiaria pastures in Brazil. We concluded that the saponin content of the grass is be responsible for the outbreaks of photosensitizing disease which occur annually on Brazilian pastures. / Numerosos surtos de fotossensibiliza??o em animais mantidos em pastos de Brachiaria spp v?m ocorrendo no Brasil e as perdas econ?micas resultantes t?m sido foco de preocupa??o, em especial devido ?s grandes ?reas cultivadas dessa gram?nea no pa?s. A maioria dessas ocorr?ncias vem sendo atribu?das ? esporidesmina, presente em esporos do fungo Pithomyces chartarum, que ocorre em muitos pa?ses de clima temperado. No entanto, h? diferen?as entre o aspecto an?tomo-histopatol?gico verificado no f?gado de animais que desenvolvem fotossensibiliza??o em pastos de Brachiaria spp e o descrito na intoxica??o por P. chartarum. Nos diversos surtos relatados no Brasil n?o se conseguiu demonstrar a toxidez dos esporos. Como o isolamento de saponinas t?xicas tem sido reportado em pastagens de Brachiaria, a causa dos surtos de fotossensibiliza??o no Brasil precisa ser esclarecida, para que corretas medidas preventivas sejam adotadas e, a doen?a evitada. Este trabalho tem por objetivo principal demonstrar, atrav?s das diferen?as epidemiol?gicas, cl?nico-patol?gicas e toxicol?gicas peculiares ?s duas condi??es, colhidas na literatura e confrontadas com nossas pr?prias observa??es sobre pitomicotoxicose em Portugal e intoxica??o pela Brachiaria sp no Brasil, que as saponinas contidas na Brachiaria sp s?o respons?veis pelos surtos de fotossensibiliza??o que ocorrem anualmente nas pastagens brasileiras.

O basidiomiceto Cryptococcus neoformans é um fungo saprófita, com ampla distribuição geográfica, que é normalmente isolado de solos que contêm excretas de pombos e detritos vegetais. Apesar de saprófita, o fungo pode infectar e causar doença em uma grande variedade de hospedeiros animais, como mamíferos, aves e insetos. O número de casos de micoses graves, causadas por C. neoformans e por outros gêneros de fungos, tem aumentado em todo o mundo, principalmente devido ao aumento do número de indivíduos imunocomprometidos. Adicionalmente, a emergência de novas espécies de patógenos e a seleção de linhagens tolerantes aos antifúngicos comumente utilizados fazem com que o desenvolvimento de novas estratégias para o controle de fungos seja extremamente desejável. A inativação fotodinâmica de fungos é um método novo e promissor, que pode ser utilizado tanto para o controle de micoses (em animais e em vegetais), como para a eliminação de fungos do ambiente. A fotoinativação de fungos é baseada no uso de fotossensibilizadores que se acumulam ou que são preferencialmente metabolizados pelas células do microrganismo-alvo. A seguir, o fotossensibilizador é exposto à luz visível, que, na presença de oxigênio, inicia processos fotoquímicos que produzem uma série de espécies reativas de oxigênio capazes de matar as células fúngicas sem provocar danos significativos nos tecidos do hospedeiro. Neste trabalho, foram avaliadas as eficácias do azul de metileno (MB) (em solução e em nanoemulsão), da cloro-alumínio ftalocianina (em nanoemulsão) e do complexo nitrosilo de rutênio (em solução) como fotossensibilizadores para a fotoinativação de células melanizadas e não melanizadas de C. neoformans. C. neoformans foi suscetível à fotoinativação pela cloro-alumínio ftalocianina, com uma inativação próxima de 100% quando foi utilizada uma combinação apropriada da concentração do fotossensibilizador e da dose de luz. A completa fotoinativação do fungo pela ftalocianina, em condições compatíveis com a terapia fotodinâmica, abre a perspectiva da utilização desse fotossensibilizador para o tratamento de micoses causadas por C. neoformans. A fotoinativação pelo MB foi apenas parcial e não ocorreu fotoinativação pelo nitrosilo de rutênio. Nenhum dos fotossensibilizadores matou o fungo na ausência da luz. Também foram feitos experimentos para se verificar a influência do tempo de crescimento e da melanização na tolerância de C. neoformans à fotoinativação pelo MB. Houve diferença significativa na tolerância entre diferentes linhagens de C. neoformans. Para a maioria dos tratamentos [linhagens e tempo de crescimento (4, 6 ou 8 dias)], não houve diferença significativa entre a tolerância de células melanizadas e não melanizadas. Também não foi observada diferença na tolerância entre células com idade de 4 a 8 dias. / The basidiomycete Cryptococcus neoformans is a saprophytic worldwide fungus which is normally isolated from soils contaminated with pigeon excreta and plant detritus. Despite a saprophytic existence, the fungus can infect and cause disease in a wide variety of animal hosts such as mammals, birds and insects. Serious infections caused by C. neoformans and by other genera of fungi have emerged all over the world, primarily due to the increased numbers of immunocompromised individuals. Additionally, the emergence of new species and antimycotic-resistant strains of pathogenic fungi makes the development of new fungus-control techniques highly desirable. Photodynamic inactivation of fungi is a new and promising method that can be used to control localized mycoses or kill fungi in the environment. The photodynamic inactivation of fungi is based on the use of a photosensitizer that accumulates in, or preferentially is metabolized by, cells of the target microorganism. The photosensitizer is then exposed to visible light in the presence of oxygen, and this starts photochemical processes that produce a series of reactive oxygen species (ROS) capable of killing the fungal cells without causing significant damage to host tissues. We report here the efficacy of methylene blue (MB) (in solution and in nanoemulsion), chloroaluminum phthalocyanine (in nanoemulsion) and nitrosyl ruthenium complex (in solution) as photosensitizers in photoinactivation of melanized and nonmelanized Cryptococcus neoformans yeast cells. C. neoformans were susceptible to photoinactivation by chloroaluminum phthalocyanine with inactivation close to 100% when the appropriate combination of photosensitizer concentration and light-exposure dose was used. Photoinactivation by MB was only partial and nitrosyl ruthenium complex was ineffective. In the dark, neither photosensitizers inactivated the fungus. Complementary experiments were performed to estimate the effect of the age of the cells and of melanization in the fungus tolerance to photoinactivation by MB. There was a significative difference in the tolerance among strains of C. neoformans. For most of the treatments (strains and time of growth) there was no difference between the tolerance of melanized and nonmelanized cells. There was no difference in the tolerance among 4 to 8-day cells either.

Le travail présenté dans ce manuscrit est basé sur l’utilisation de la modélisation moléculaire, de la simulation et de la chimie théorique pour l’étude de la photosensibilisation de l’ADN ; c’est à dire l’augmentation de la sensibilité de l’ADN vis-à-vis de la lumière au travers de l’action d’agents photosensibilisants. Premièrement, les voies photophysiques et photochimiques de plusieurs molécules connues telles que nile bleu, nile rouge, BMEMC ou une base modifiée endogène, Pyo, ont été étudiés dans le but de comprendre leurs mécanismes de photosensibilisation. Les phénomènes associés qui ont été mis en évidence sont des transferts d’électrons et d’énergie, la production d’électrons solvatés et l’activation à deux photons. De plus, deux outils pour l’étude des interactions entre les molécules et l’ADN ont été dévellopés; i) un protocole calculatoire capable de fournir l’énergie libre d’interaction de drogues dans leurs poches; ii) un outil basé sur l’hamiltonien semi-empirique de Frenkel qui permet de modéliser le spectre de dichroïsme circulaire électronique de biomacromolécules. Ensuite, les effets de photolésions sur la structure et la flexibilité de l’ADN ont été étudiés ; i.e. les dimères de pyrimidines, la pyrimidine(6-4)pyrimidone (6-4PP) et les clusters de sites abasiques. Finalement, la reconnaissance de brins d’ADN lésés par des protéines de réparation et le rapport avec leurs activités enzymatiques a été analysé. Le lecteur peut se référer à la première partie de ce manuscrit pour une présentation vulgarisée du contexte de ce projet / The work presented in this manuscript is based on the use of molecular modeling, simulation and theoretical chemistry in order to study the photosensitization of DNA; i.e. the enhancement of the sensitivity of DNA to light through the action of a photosensitizing agent. A first aspect has been to study the photophysical and photochemical pathways of several known sensitizers such as nileblue, nilered, BMEMC or an endogenous modified nucleobase, Pyo, in order to understand their mechanisms of photosensitization. The related phenomena that have been observed are electron transfers, triplet-triplet energy transfers, production of solvated electrons and two-photons activations. Moreover, two tools have been developed to study the interaction between photosensitizing agents and DNA; i) a protocol able to provide the binding free energy of drugs in their interaction pockets; ii) a tool based on the semi-empirical Frenkel Hamiltonian to model the electronic circular dichroism of biomacromolecular systems in a straightforward way. Then the effects of photoinduced lesions on the DNA structure and flexibility have been investigated; i.e. cylcopyrimidine dimers (CPD), pyrimidine(6-4)pyrimidone (6-4PP) and cluster abasic sites. Finally the recognition of damaged DNA strands by repair enzymes is presented and the implication on enzymatic activities has been highlighted. The reader can refer to the first section of the manuscript for a popularized presentation of the project context

Estudos envolvendo o oxigênio molecular singlete (1O2) tem uma relevância biológica, uma vez que esta espécie, devido ao caráter eletrofílico, reage com moléculas ricas em elétrons como proteínas, lipídeos e DNA provocando danos que resultam em perdas de função e integridade celular. Em sistemas biológicos, a presença de outras espécies reativas de oxigênio e nitrogênio, dificultam a identificação de lesões específicas causadas por 1O2 .Neste contexto, este trabalho foi desenvolvido objetivando a síntese de endoperóxidos isotopicamente marcados com 17O para serem utilizados como fonte geradora limpa de 17[1O2] em estudos mecanísticos. A capacidade de geração de 17[1O2] pelo endoperóxido N,N\'-di(2,3-dihidroxipropil)-3,3\'-(1,4 naftilideno) dipropanamida 17O (DHPN17O2) foi confirmada utilizando o captador químico sulfato mono-{2-[10-(2-sulfoxi-etil)-antracen-9-il]-etil}éster de sódio e o nucleosídeo 2\'- desoxiguanosina. Os produtos isotopicamente marcados com 17O formados foram analisados por espectrometria de ressonância magnética nuclear e cromatografia líquida de alta eficiência acoplado ao espectrômetro de massa. Os lipídeos, em especial o colesterol ao reagir com o 1O2 geram hidroperóxidos de colesterol como produtos de oxidação primária e na presença de metais resulta em compostos de maior reatividade e toxicidade, como os radicais peroxila, que contribuem para a propagação da peroxidação lipídica. Neste trabalho, demonstramos que os hidroperóxidos de colesterol são capazes de gerar 1O2 na presença de metal através de medidas de luminescência, utilização de supressores e captador químico de 1O2. Os mecanismos de reação envolvidos foram estudados e determinados por espectrometria de massa acoplada a cromatografia líquida de alta eficiência. Por fim, a caracterização detalhada dos produtos formados por espectrometria de ressonância magnética nuclear e massa na reação do colesterol com 1O2 mostrou que além dos hidroperóxidos a reação também produz um aldeído, o 3&#946; -hidroxi-5&#946;-hidroxi-B-norcolestano-6&#946;-carboxialdeído. Até o momento, este composto havia sido identificado como um produto específico da ozonização do colesterol. Neste estudo, baseado nos estudos por reações de quimiluminescência, é proposto o mecanismo de formação deste aldeído em reações de oxidação de colesterol por 1O2 envolvendo intermediário dioxetano. / Studies involving singlet molecular oxygen (1O2) has biological relevance, once this species, due to its eletrophylic character, reacts with rich electron molecules such as proteins, lipids and DNA causing damages that result in loss of function and cellular integrity. In biological system, the presence of other reactive species of oxygen and nitrogen impair the identification of lesions caused by 1O2. In this context, this work was developed with the aim of synthesizing <SUP17O-labeleded endoperoxides to be used as a clean source of (1O2) in mechanistic studies. The ability of 17[1O2]generation by N,N\'-di(2,4-dihydroxypropyl)-1,4-naphthalene-dipropanamide labeled with 17O(DHNP17O2) was observed using the disodium salt of anthracene-9,10-diyldiethyl disulfate as a chemical trap and the nucleoside 2\'-deoxyguanosine. The products isotopically labeled with 17O were analyzed by nuclear magnetic resonance spectroscopy and high performance liquid chromatography coupled to a mass spectrometer. Lipds, in special the cholesterol, when reacting with singlet molecular oxygen generate cholesterol hydroperoxides as primary products and in the presence of metals result in compounds of higher reactivity and toxicity, such as peroxyl radicals which contribute to the propagation of lipid peroxidation. In this work, we demonstrated that cholesterol hydroperoxides are able to generate singlet molecular oxygen in the presence of metal by chemiluminescence measurements by testing the effect of singlet molecular oxygen quencher and by chemical trap. The involved reaction mechanisms were studied and determined by mass spectrometry coupled to the high performance liquid chromatography. Finally, we detailed characterization of the products formed in the reaction of cholesterol with 1O2 by nuclear magnetic resonance and mass spectroscopy showed that besides cholesterol hydroperoxides, the reaction also produces an aldehyde, 3&#946;hydroxy-5&#946;-hydroxy-B-norcholestan-6&#946;-carboxyaldehyde which had been identified as a specific product of cholesterol ozonization. In this study, based on the studies of chemiluminescence reactions, the mechanism of formation of this aldehyde in reaction of oxidation of cholesterol by 1O2 involving a dioxetane intermediate has been proposed.

L'objectif principal du travail effectué au cours de cette thèse était de développer, caractériser et analyser des matériaux originaux pour des réactions d'oxydation photosensibilisée. Une attention particulière a été accordée à la détermination des propriétés photophysiques des sensibilisateurs (PSs) et à l'effet induit par l'immobilisation sur un support inerte (silice). Les espèces réactives formées lors de l'irradiation ont été identifiées et analysées. La production d'oxygène singulet a été suivie par deux méthodes complémentaires. Les sensibilisateurs supportés ont été employés, sous la forme de monolithes ou de poudres, pour la photo-oxydation du diméthyl sulfure à l'interface gaz-solide et de l’-terpinène à l'interface liquide-solide. L'oxygène singulet est la principale espèce réactive de l’oxygène formée par les PSs sélectionnés; néanmoins, les produits de réaction ont été analysés afin de mettre en évidence d’autres mécanismes. / The main aim of the work carried out during this PhD project was to develop, characterize and analyze original materials for photosensitized oxidation reactions. Particular attention was paid to the determination of the photophysical properties of the selected photosensitizers (PSs) and the effect induced by the immobilization on an inert support (silica). The reactive species formed upon irradiation were identified and analyzed. Singlet oxygen production was monitored by two complementary methods. The solid-supported sensitizers were employed, in the form of either monoliths or powders, for the photooxidation of dimethyl sulfide at the gas-solid interface and of -terpinene at the liquid-solid interface. Singlet oxygen was the main reactive oxygen species formed by the selected PSs; nonetheless, the reaction products were analyzed and other possible mechanistic scenarios investigated.