Nowadays, the requirement to design highly valuable compounds is undoubtedly one of the major challenges in the field of organic and organometallic chemistry. The use of the versatile and efficient N-heterocyclic carbenes (NHCs) combined with transition metals represents a key feature in modern organometallic chemistry and homogeneous catalysis. In the course of this thesis, the straightforward design and synthesis of a library of Pd(0) bearing NHC ligands was achieved. Their catalytic performances (Chapter 1) and their phosphorescence properties in solution (Chapter 2) were disclosed. Currently, cross-couplings are some of the most important types of reaction in palladium catalysis. The formation of highly hindered biaryls substrates is one of the main requirements in cross-coupling chemistry. The design and synthesis of a palladium dimer bearing a bulky NHC ligand can fulfil this proposal (Chapter 4). The development of new classes of ligands is a topic of interest. For this reason, normal, abnormal, remote and mesoionic N-heterocyclic carbenes copper complexes were investigated and their reactivity compared in the [3+2] cycloaddition of azides and alkynes (Chapter 7). Air and moisture stable Cu(I)-NHC species have also been compared to their silver analogues for the alkynylation of ketones (Chapter 9). The different reactivity of the two latter organometallic species (Cu and Ag) with ethyldiazoacetate reagent via the formation of carbenes or C-H activated product is presented in Chapter 8. Recently, the development of a bimetallic catalytic system is strongly considered and has high impact. For this reason, two dual catalytic transformations (Pd-NHC and Cu-NHC) were studied for the C-H arylation (Chapter 5) and the synthesis of substituted alkenes products via a relay or cooperative mechanisms (Chapter 6). The isolation of intermediates and mechanistic studies were examined in each of these studies.

Thesis (PhD (Chemistry and Polymer Science))--Stellenbosch University, 2008. / Alkylated acetonitrile that forms during the synthesis of the sulfonium salt, [(Me3)2(MeS)S][BF4], is involved in the formation of new , -unsaturated Fischer-type carbene complexes from (CO)5M=C(OMe)CH2Li (M = Cr, W). Metal migration observed when the substitution product obtained from the reaction of the anionic carbene complexes (CO)5M=C(NMe2)CºC¯ (M = Cr, W) with Ph3PAu+ was left in solution, was also kinetically and theoretically investigated. 1H NMR and quantum mechanical (at the B3LYP level of theory) data indicated a complicated mechanism. The a,b-unsaturated Fischer-type carbene complex, (CO)5Cr=C(OMe)CH=C(Me)NH(Me), obtained from the reaction of (CO)5M=C(OMe)CH2¯ with alkylated acetonitrile, was transformed into the new remote one-N, six-membered, carbene ligand (rN1HC6) complex, (CO)5Cr=C(CH=C(Me)N(Me)CH=C(nBu). The carbene ligand unprecedentedly preferred the softer Rh(CO)2Cl moiety to the Cr(CO)5 metal fragment and transferred readily. A new series of remote and abnormal square planar compounds [r/a(NHC)(PPh3)2MCl]CF3SO3 (M = Pd or Ni) was prepared by oxidative substitution. The various positions for metal-carbon bond formation on a pyridine ring to furnish various ligand types i.e. C2 for nN1HC6, C3 for aN1HC6 or C4 for rN1HC6 received attention. The ligands were arranged in increasing order of carbene character, aNHC < nNHC < rNHC and trans influence, nN2HC5 ~ aN1HC6 ~ nN1HC6 < rN1HC6. In competitive situations, oxidative substitution occurred selectively at C4 of the pyridine ring rather than at C2 and on the aromatic ring containing the heteroatom (C4), rather than on an annealed aromatic ring (C7). Crystal and molecular structure determinations confirmed the preferred coordination sites. Quantum mechanical calculations (at the RI-BP86/SV level of theory) indicated that the chosen carbene ligand has a much larger influence than the metal on the BDE of the M-Ccarbene bond; the farther away the N-atom is from the carbene carbon, the stronger the bond. In complexes that also contain additional external nitrogen atoms, e.g. trans-chloro(N-methyl-1,2,4- trihydro-2-dimethylaminepyrid-4-ylidene)bis(triphenylphosphine)palladium(II) triflate and transchloro( N-methyl-1,2,4-trihydro-2-dimethylaminepyrid-4-ylidene)bis(triphenylphosphine)nickel(II) triflate, stabilisation originates from both the nitrogens. 2-Chloro-1-methyl-1H-pyrid-4-ylidenephenylammonium triflate afforded complexes with both remote as well as normal nitrogen atoms. New azole complexes of palladium and nickel with remote heteroatoms were also prepared from N-methyl-4',4'-dimethyl-2'-thiophen-3-chloro-2-yl-4,5-dihydro-oxazole. Employing the compound 1,5-dichloroanthraquinone, the product of a double oxidative substitution on two Pd centra could be isolated but not alkylated. The fact that the chemical shift of the metal bonded carbon in the 13C NMR spectrum can not be used as absolute measure of carbene character, was emphasised in a compound where the heteroatom was situated seven bonds away from the carbon donor. In efforts to synthesise a sulphur-bridged complex that contains carbene ligands, crystals of transdi- iodobis(1,3-dimethyl-imidazoline-2-ylidene)palladium were obtained. Bridged thiolato complexes with N1HC6 ligands were unexpectedly found in the attempt to substitute the halogen on chosen square planar carbene complexes of palladium, widening the application possibilities of N1HC6 ligands in organometallic chemistry beyond that of catalysis. A trinuclear cluster, [(PdPPh3)3(μ-SMe)3]BF4 was isolated as a by-product of these reactions. A series normal and abnormal thiazolylidene complexes of nickel and palladium were prepared by oxidative substitution of the respective 2-, 4- and 5-bromothiazolium salts with M(PPh3)4 (M = Pd or Ni), and unequivocally characterised. In a preliminary catalytic investigation, all the thiazolinium and simple pyridinium derived palladium complexes showed activity in the Suzuki-Miyaura coupling reaction. Little variation in activity in the order a (N next to carbon donor) > n > a (S next to carbon donor) was found for the former series, whereas decreased activity was exhibited in the sequence r > a > n of the latter group. The pyridinium derived complexes showed superior activity to the thiazolinium ones. The rNHC complex, trans-chloro(N-methyl-1,2,4-trihydro-2- dimethylaminepyrid-4-ylidene)bis(triphenylphosphine)palladium(II) triflate, showed similar Suzuki-Miyaura activity to the standard N2HC5 carbene complex precatalyst, trans-chloro[(1,3- dimethyl-imidazol-2-ylidene)triphenylphosphine]palladium(II) triflate.

This thesis describes our physical organic and mechanistic investigations into N Heterocyclic Carbene (NHC) mediated organocatalytic transformations, through a collaboration with the research group of Dr AnnMarie O'Donoghue and PhD student Richard Massey at Durham University. Initial research focused upon the determination of kinetic acidities and associated pKₐ values for a range of triazolium salts using C(3) H/D exchange, monitored by ¹H NMR spectroscopy. Estimates for pKₐ values in the range 16.6 17.4 were obtained, which are some ~2 and ~3 5 pK units lower than analogous imidazolium and thiazolium species respectively, with modest N substituent (0.3 pK units) effects observed. At lower pD values, an altered pD dependence indicates a dicationic triazolium species is formed (through N(1) protonation) with an estimated pKₐᴺ¹ of -0.2-0.5 and C(3) H pKₐ values at least 2 units lower than their monocationic analogues. This methodology was subsequently extended to mesoionic NHCs, where pKa values of 23.0 27.1 for a range of triazolium and 30.2 31.0 for a range of imidazolium salts were estimated. A detailed study of the NHC catalysed intramolecular Stetter reaction was also undertaken using ¹H NMR spectroscopy. A range of 3 (hydroxybenzyl)azolium salts (adducts), formed from the addition of NHC to aldehyde were isolated, enabling the generation of reaction profiles and the determination of rate constants. The reaction proceeds via rapid and reversible adduct generation, followed by rate limiting Breslow intermediate formation, with electron withdrawing N aryl substituents increasing the rate of product formation. Consistent with rate limiting deprotonation, deuterium exchange studies of O methylated adduct analogues found electron withdrawing N-aryl units gave faster exchange. Examination of the equilibrium constants for adduct formation revealed that both in the case of NHCs bearing 2,6 disubstituted N aryl units and aldehydes bearing a 2 ether substituent, the equilibrium position is significantly shifted towards adduct. Finally, studies at sub-stoichiometric NHC concentrations, monitored by HPLC, imply the reaction is first order with respect to NHC precursor, but zero order in aldehyde, again indicative of rate limiting deprotonation.

INTRODUCTION: Le bleu de méthylène est un colorant grandement utilisé dans le domaine médical, notamment pour ses propriétés de coloration histologique. Il est également utilisé comme agent photosensibilisant dans la thérapie photodynamique antimicrobienne, qui une fois photoactivé devient efficace pour l’éradication de plusieurs germes multirésistants. L’objectif de cette étude est d’investiguer le potentiel ototoxique du bleu de méthylène. MÉTHODES: Vingt cochons d’Inde divisés en deux groupes, ont reçu une solution de bleu de méthylène et de gentamicine dans l’oreille testée. L’oreille controlatérale a reçu une solution saline contrôle. Nous avons procédés à des potentiels évoqués auditifs du tronc cérébral avant et une semaine suivant la série d’injections. À la suite des dissections, des analyses histologiques et immunohistochimiques ont été réalisées. RÉSULTATS: La différence moyenne de perte auditive dans le groupe gentamicine comparativement au groupe normal salin était de 66.25 dB (p<0.001). Toutefois, la différence moyenne de perte auditive dans le groupe ayant reçu du bleu de méthylène comparativement à celui ayant reçu des injections de la solution saline était de 1.50 dB, et n’a pas été démontré comme étant statistiquement significative (p=0.688). De plus, la captation de caspase-3 en immunohistochimie (marqueur d’apoptose) n’a pas été significative dans le groupe recevant le bleu de méthylène. CONCLUSION: À la lumière de nos résultats, les injections intratympaniques de bleu de méthylène n’ont pas démontrées de potentiel ototoxique. Nous recommandons des études supplémentaires afin d’en préciser son utilisation sécuritaire dans le domaine otologique. / INTRODUCTION: Methylene blue is widely used in the medical field, especially as a blue dye for staining. It is also used as a photosensitizing agent in antimicrobial photodynamic therapy, which once photoactivated is effective for the eradication of several multiresistant bacteria. The objective of this study is to investigate its ototoxic potential. METHODS: Twenty guinea pigs, forming two groups, received respectively intratympanic methylene blue and gentamicin in one ear. The contralateral ears received a control saline solution. We conducted auditory evoked brainstem response (ABR) before and one week after the injection series. Once completed, the cochleas were dissected and analyzed by histology and immunohistochemistry. RESULTS: The mean difference of hearing loss in the saline group compared to the gentamicin group was 66.25dB (p<0.001). However, the mean difference of hearing loss in the methylene blue group compared to normal saline was 1.50 dB, and it was not shown to be statistically significant (p=0.688). Furthermore, uptake of caspase-3 by immunohistochemistry (apoptotic marker) was negative in the group which received injections of methylene blue. CONCLUSION: In light of our results, intratympanic injections of methylene blue did not demonstrate an ototoxic potential. We recommend further studies to precise its usefulness in the otology field.

Neste trabalho investigamos efeitos de fotoirradiação e toxinas sob membranas celulares miméticas. Foram utilizadas, como modelo de membranas lipídicas, vesiculas unilamelares gigantes (GUVs) compostas pro lipídeos oxidados e não oxidados observadas por microscopia ótica de contraste de fase. Inicialmente estudamos a foto-resposta de membranas compostas por POPC e POPG dispersas em solução contendo azul de metileno (MB). Na sequência, estudamos o efeito de toxinas formadoras de poros, Esticolisina I (ST I) e Esticolisina II (ST II), em membranas contendo lipídeos oxidados e não oxidados. Os resultados de MB (10 µM) disperso em solução de membranas compostas por POPC e o lipídeo aniônico POPG indicaram que o aumento da densidade de carga negativa nas membranas das GUVs, que favorece a ligação da moléculas positivamente carregadas como MB nas membranas, tem como consequência um aumento de permeabilidade da membrana muito mais rápído em relação a membranas compostas apenas por POPC. Isto se deve ao fato que a localização preferencial do MB na membrana de POPC:POPG favorece a formação de oxigênio singlete próximo a dupla ligação da cadeia alquílica, dando início a reação de peroxidação lipídica de maneira mais efetiva que em membrana de POPC. Os resultados da ação das toxinas STI e STII (21 nM) em GUVs contendo lipídeos não oxidados PC e esfingomielina evidenciam que apenas STII é capaz de permear estas membranas a esta concentração. Mais ainda, nossos resultados sugerem que a existência de separação de fases fluida-gel na bicamada lipidica composta por PC:SM (razão molar 1:1) favorece a ação da toxina StII. Ao analisarmos membranas contendo lipídeos hidroperoxidados (POPC-OOH) dispersas em solução contendo STII (21 nM) observamos um aumento de permeabilidade na membrana num conjunto de GUVs, associado a formação de poros, apenas em bicamadas lipídicas formadas por misturas de lipídeos oxidados (POPC) e não oxidados (POPC-OOH). Quanto maior a concentração de lipídeos oxidados na membrana mais rapidamente ocorre o aumento de permeabilidade. / In this work we investigate the effects of photoirradiation and toxins on mimetic cell membranes. As a model of lipid membranes, giant unilamellar vesicles (GUVs) composed of oxidized and oxidized pro-lipids were observed by optical phase contrast microscopy. Initially we studied the photo-response of membranes composed of POPC and POPG dispersed in solution containing methylene blue (MB). Following, we studied the effect of pore-forming toxins, Sticolysin I (ST I) and Sticolysin II (ST II), on membranes containing oxidized and non-oxidized lipids. The results of MB (10 M) dispersed in solution of membranes composed of POPC and the anionic lipid POPG indicated that the increase in the negative charge density in the membranes of GUVs, which favors the binding of positively charged molecules as MB in the membranes, consequently increases membrane permeability in regard to membranes composed only of POPC. This is due to the fact that the preferred location of the MB in the POPC: POPG membrane favors the formation of singlet oxygen near the double bond of the alkyl chain, initiating the lipid peroxidation reaction more effectively than in the POPC membrane. The results of the action of the STI and STII toxins (21 nM) on GUVs containing non oxidised lipids PC and sphingomyelin show that only STII is able to permeate these membranes at this concentration. Moreover, our results suggest that the existence of fluid-gel phase separation in the lipid bilayer composed of PC:SM (molar ratio 1:1) favors the action of the StII toxin. When analyzing membranes containing hydroperoxidized lipids (POPC-OOH) dispersed in solution containing STII (21 nM) we observed an increase in membrane permeability in a set of GUVs, associated with pore formation, only in lipid bilayers formed by mixtures of oxidized lipids (POPC-OOH) and non-oxidized ones. The higher the concentration of oxidized lipids in the membrane, the faster the permeability increases.

A terapia fotodinâmica (TFD) é uma modalidade clínica para tratar uma variedade de neoplasias, doenças de pele e representa um promissor tratamento estético. O presente trabalho avaliou os aspectos histopatológicos e moleculares do tratamento pela TFD mediada por azul de metileno (TFD-AM) no modelo experimental in vivo do Carcinoma Espinocelular Cutâneo (CEC) e na pele sadia de camundongos Swiss. O protocolo da TFD foi de uma única sessão, com aplicação da solução de AM a 1% seguido por irradiação com laser diodo na dose total de 24 J/cm2 nos tecidos tumorais e sadios. Os animais foram sacrificados em dois períodos, 24 horas e 15 dias após TFD. Alterações morfológicas foram pouco marcantes nos tecidos tumorais tratados, entretanto, foram mais pronunciadas nos tecidos sadios. Podemos concluir que os efeitos de uma única sessão da TFD mediada pelo AM na dose aplicada não conferiu melhora no tratamento do CEC. Estes resultados motivam novos estudos com ajustes no protocolo para melhorar a eficácia desta terapia. / Photodynamic therapy (PDT) is a clinical method for treating a variety of tumors, skin disorders and represents a promising cosmetic treatment. This study evaluated the histopathological and molecular aspects of the treatment by PDT mediated by methylene blue (PDT-MB) in vivo experimental model of cutaneous squamous cell carcinoma (SCC) and in healthy skin of swiss mice. The PDT protocol was a single session with the application of MB 1% solution followed by irradiation with diode laser at a total dose of 24 J/cm2 in tumor and healthy tissue. The animals were sacrificed at two periods, 24 hours and 15 days after PDT. Morphological changes were less marked in the tumor tissues treated, however, were more pronounced in healthy tissues treated. We can conclude that the effects of a single session of PDT mediated by MB in applied dose conferred no improvement in the treatment of SCC. These results motivate further studies with adjustments in the protocol to improve the effectiveness of this therapy.

A nanotecnologia tem sido aplicada para o desenvolvimento de materiais para diversas aplicações inclusive na inativação de patógenos. As nanopartículas de sílica (npSi) destacam-se pela alta área superficial, facilidade na alteração da superfície para aumento da eficiência adsortiva, penetrabilidade e toxicidade para bactérias gram-negativas sendo biocompatíveis para células de mamíferos e mais foto-estáveis que a maioria dos compostos orgânicos. Devido as suas vantagens, as npSi podem ser usadas para veicular fotossensibilizadores (FSs) uma vez que permitem sua utilização em solução aquosa em que os FSs geralmente são insolúveis. Além disso, o uso de FSs em vez de antibióticos, permite a inativação microbiológica pela Terapia Fotodinâmica sem que as bactérias adquiram resistência por mecanismos genéticos. Esse processo ocorre pela interação entre um FS, luz e oxigênio molecular produzindo oxigênio singleto que é extremamente reativo danificando estruturas celulares. O objetivo desse estudo foi otimizar a fotoinativação dinâmica de E .coli utilizando Azul de Metileno (AM) e Azul de Toluidina O (ATO) veiculados por npSi. As npSi foram preparadas pela metodologia sol-gel, caracterizadas por microscopia eletrônica de varredura (MEV) e submetidas à adsorção de AM e ATO em sua superfície. A presença de AM e ATO na superfície das npSi foram analisadas por espectroscopia no infravermelho; espectroscopia de fluorescência por raio-X e análise termogravimétrica. O planejamento experimental, iniciado pelo fatorial 23 e modelado ao composto central em busca das condições ótimas foi adotado pela primeira vez nessa aplicabilidade, visando a fotoinativação de E. coli empregando AM e ATO em solução e em seguida com npSi. AM e ATO veiculados por npSi permitem a fotoinativação em concentrações mais baixas de FS (20 e 51% respectivamente), causando desestruturação da integridade bacteriana demonstrada por MEV. Os resultados sugerem que a veiculação de AM e ATO por npSi é extremamente efetiva para a fotoinativação dinâmica de E. coli e que o planejamento composto central pode levar à completa inativação das bactérias. / Nanotechnology has been applied to the development of materials for several apllications inclusive inactivation of pathogens. The silica nanoparticles (npSi) are distinguished by high surface area, ease of change the surface in order to increase the adsorption efficiency, penetrability and toxicity in gram-negative bacteria being biocompatible with mammalian cells and more photo-stable than most the organic compounds. Due to its advantages, npSi can be used to carry photosensitizers (PSs) since they allow its use in aquous solution in which PSs are frequently insoluble. Furthermore, the use of PSs instead of antibiotics, allows the microbiological inactivation by Photodynamic Therapy without bacteria to develop resistance by genetic mechanisms. This process occurs by the interaction among a PS, light and molecular oxygen producing singlet oxygen, which is extremely reactive, causing damage to cellular structures. The aim of this study was to optimize the photoinactivation of E. coli using Methylene Blue (MB) and Toluidine Blue O (TBO) carried by npSi using the central composite design. The npSi were prepared by sol-gel method, characterized by scanning electron microscopy (SEM) and subjected to adsorption of MB and TBO on its surface. The presence of FSs on the surface of npSi were analyzed by infrared spectroscopy, fluorescence X-ray spectroscopy and thermogravimetric analysis. The experimental design, initiated by the factorial 23 and modeled by the central composite in search of the optimal conditions was adopted for the first-time for this applicability, aiming the E. coli photoinactivation employing MB and TBO in solution and then with npSi. MB and TBO carried by npSi allowed the photoinactivation in lower concentrations of PS (20 and 51% respectively), causing disruption of bacterial integrity demonstrated by SEM. The results suggest that MB and TBO carried by npSi are extremely effective for dynamic photoinactivation of E. coli and the central composite design can lead to complete inactivation of bacteria.

Neste trabalho são apresentados estudos do efeito de interfaces nas propriedades fotofísicas e fotoquímicas do azul de metileno (AM) e de derivados fenotiazínicos com o intuito de avaliar o potencial destes compostos como fotossensibilizadores (FS) em terapia fotodinâmica. As propriedades físico-químicas do AM foram estudadas em soluções de SOS e observou-se que a presença do AM em solução altera o equilíbrio entre as micelas de SOS, diminuindo o valor da concentração micelar crítica de 7mmolL-1 para 70&#181;moIL-1. A presença das micelas em solução também interfere nas propriedades do AM. Em baixas concentrações de SOS há formação de dímeros de AM, constatados pelo aumento da absorbância em 580nm e diminuição da emissão de fluorescência. A caracterização das espécies transientes mostrou a existência de moléculas de azul de metileno no estado triplete (3AM) e de oxigênio singlete em soluções com altas concentrações de SOS e a formação de espécies radicalares do AM em baixas concentrações do tensoativo. Esta observação sugere que o mecanismo fotoquímico do AM é dependente da sua concentração local próxima de interfaces carregadas. As interações do AM e de alguns de seus derivados fenotiazínicos (tionina, azure A e azure B) com vesículas e com células HeLa foram estudadas e em ambos os casos observou-se que as moléculas com estruturas assimétricas são incorporadas em maior extensão. Em estudos de fototoxicidade, os compostos assimétricos apresentaram maior nível de morte celular do que o verificado para os compostos simétricos. Entretanto, ao se considerar a incorporação em células, os compostos simétricos se mostraram mais eficientes por molécula. Foi desenvolvido um método para determinação do 10gP dos fotossensibilizadores (FS) por voltametria com microeletrodos que se mostrou reprodutível. Novos FSs assimétricos derivados do Azure A com duas caudas hidrofóbicas de 4, 6 e 8 carbonos (AzC4, AzC6 e AzC8) foram sintetizados. Esses novos compostos possuem eficiências de fluorescência semelhantes ao AM. O composto AzC4 apresenta eficiência de geração de oxigênio singlete bem próxima à do AM (0,56), enquanto os outros dois compostos têm uma eficiência de geração de oxigênio singlete cerca de duas vezes menor. Este comportamento se deve ao fato dos FSs com cadeias carbônicas maiores interagirem mais fortemente entre si, apresentando maior grau de agregação. Os compostos sintetizados apresentam maior incorporação em vesículas que os compostos anteriormente estudados, indicando que a assimetria da molécula favorece a incorporação, assim como, o comprimento da cadeia hidrofóbica. Resultados de incorporação em células HeLa, mostraram que o composto AzC4 interage mais facilmente com a membrana celulare apresenta maior nível de morte celular devido à sua maior incorporação. / The effect of interfaces on photophysical and photochemical properties of methylene blue (MB) and its derivatives was studied in this work, aiming to emploit their potencial as photosensitizers (PS) in photodynamic therapy.The presence of MB in SDS solutions affect the micelle equilibrium decreasing the apparent critical micelle concentration of SDS from 7mmolL-1 to 70&#181;moIL-1. The properties of MB were also affected. At low SDS concentrations the formation of MB dimers was detected by the increase in the absorption in 580 nm and decrease of fluorescence emission. The characterization of transient species of MB showed the existence of MB molecules in the triplet state and emission of singlete oxygen at large SDS concentration and the formation of MB radicals in small surfactant concentration. These observations suggest that the photochemical mechanism of MB depends on its local concentration close to charged interfaces.The interactions of MB as well as other phenotiazine derivatives with synthetic vesicles and HeLa cells were studied. In both cases, the PS with asymmetrical structure presented higher degrees of incorporation. Studies of phototoxicity showed that the PS with asymmetrical structures present higher degree of cell death than the symmetrical compounds. However, if one consider the degree of incorporation the symmetrical compounds are more efficient per molecule. A method to determinate logP by voltammetry with microelectrodes was developed, showing good repeatability. New asymmetric photosensitizers derived from Azure A with two hydrophobic chains of 4, 6 or 8 carbons (AZC4, AZC6 e AzC8) were synthesized in order to study structure/activity relationship. These new compounds presented similar fluorescence efficiency to MB. The oxygen singlet generation efficiency of AZC4 is similar to MB, while the efficiency of the other PSs was twice smaller. This behavior was explained in terms PS aggregation, due to their longer hydrophobic chains. The synthesized PSs presented larger degree of incorporation than the commercial PSs, showing the role of asymmetry and hydrophobicity in incorporation yields. Incorporation in HeLa cells showed that AZC4 interacts more strongly with cells than the other synthesized PSs due to the rigidity of plasmatic membrane. Under the same illumination conditions, AzC4 was the PS that presented higher degree of cell death as a result of its larger incorporation in cells.

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.

O objetivo deste trabalho foi observar a ação da terapia fotodinâmica (PDT) antimicrobiana do azul de metileno (AM) frente ao Aggregatibacter actinomycetemcomitans em estudo in vitro e em periodontite induzida em ratos. No estudo in vitro foram utilizadas suspensões de cultura de A. Actinomycetemcomitans selvagens na avaliação de diferentes parâmetros de irradiação na busca de otimizar a redução bacteriana. No estudo in vivo foram selecionados 20 ratos Wistar machos, adultos, com massa corpórea semelhante de aproximadamente 300 g. Os animais foram submetidos a uma antibioticoterapia durante cinco dias. A indução foi elaborada através da colocação de ligaduras embebidas em A. actinomycetemcomitans por um período de 15 dias. Após este período, observou-se clinicamente e por sondagem a presença da doença sendo os animais divididos em dois grupos: Grupo RM (n=10) - remoção mecânica de placa e Grupo RM + PDT (n=10) - remoção mecânica de placa + terapia fotodinâmica. Para a PDT, o azul de metileno a 0,01% foi aplicado topicamente diretamente nas bolsas periodontais que foram então irradiadas com um laser de emissão vermelha, A = 660nm, P= 100 mW, t= 54s e E= 5,4 J. Foram realizadas coletas microbiológicas antes, imediatamente após tratamento, 3 dias e sete dias para análises por cultura e reação de polimerase em cadeia (PCR). Os resultados microbiológicos obtidos no experimento in vitro apresentaram uma eficácia da terapia fotodinamica frente ao A. actinomycetemcomitans, 98,4% quando irradiado com uma energia de 5,4J e 99,9% com energia de 9J. No estudo in vivo, foi obtida uma redução microbiana de 93,5% no grupo RM+PDT, frente a uma redução de 87,7% no grupo RM. No controle do dia 7 foi observado uma recolonização em ambos os grupos. As análises por PCR confirmaram a presença do microorganismo em todas as coletas. Estes estudos sugerem que a terapia fotodinâmica pode ser uma alternativa promissora para sua utilização coadjuvante em periodontia. / The aim of this study was to evaluate the antimicrobial effects of photodynamic action of methylene blue (MB) against the Aggregatibacter actinomycetemcomitans in vitro and on periodontitis-induced in rats. The in vitro study used suspensions of A. actinomycetemcomitans to evaluate the different irradiation parameters to optimize the bacterial reduction. Twenty four male adult rats, body mass of 300 g were selected for in vivo study. The animals were submitted to an antibiotic therapy during five days. The periodontitis induction was performed by the ligature insertion imbibed in A. actinomycetemcomitans for 15 days. After this period, the disease was confirmed by clinical and probing aspects. The animals were divided into two groups: Group MR (n=10) - mechanical plaque removal and Group MR+PDT (n=10) - mechanical plaque removal + photodynamic therapy. The MB solution 0.01% (m/w) was topically applied into the periodontal pockets which were irradiated by a laser in red emission, A = 660nm, P= 100mW, t= 54s e E= 5.4 J. Microbiological samples were collected before, immediately after treatment, 3 and 7 days to cultural and Polymerase Chain Reaction (PCR) analysis. The results of in vitro study showed an antimicrobial efficacy of PDT against A. actinomycetemcomitans: reduction of 98.4% when the samples were irradiated with 5.4J and 99.9%, with 9J. The in vivo results showed reduction of 93.5% for Group MR+PDT and 87.7% for Group MR. The analysis on day 7 showed a recolonization in both groups. The PCR analysis confirmed the presence of the microorganism in all samples. This study suggests that the photodymnamic therapy could be a promising adjunctive therapy to periodontal treatment.